Microvesicles与非瓣膜性心房颤动血栓前状态关系的研究
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摘要
研究背景
非瓣膜性心房颤动(房颤)是脑卒中及血栓栓塞事件强烈的独立危险因素。抗凝治疗是预防房颤卒中最为有效的手段,但仍然有33%-38%的卒中不能被防治,提示非瓣膜性房颤患者血栓形成的机制复杂,可能与房颤的基础病因相关。晚近,人们认为,血小板活化是高凝状态及血栓形成的始动因素。一系列凝血因子借助活化血小板的磷脂表面为反应平台,通过级联放大反应产生了数量庞大的凝血酶,最终形成血栓。
但是以抗血小板作为首选的治疗方案同样受到以往研究的挑战。与安慰剂相比,阿司匹林可使房颤患者血栓栓塞的相对危险降低21%,其效果显著低于华法林。其中最重要的原因之一就是与血小板激活途径的多样性有关。事实上非瓣膜性房颤中血小板被激活的机制尤其是被房颤基础病因激活的机制复杂,至今仍未完全阐明。
近期的研究强调了炎症、氧化应激以及代谢紊乱在非瓣膜性房颤血栓前状态形成中的潜在作用。上述研究分别提供了各自独立的证据,却无法将这些导致高凝状态的各种机制作为一个整体进行系统性研究,使治疗缺乏特异性。因此,寻找一种能够同时承载上述启动血小板活化的各种机制的载体,以此为工具深入研究房颤时血栓前状态的发生机制,将为探索房颤时血栓前状态综合防治的新的有效靶点提供依据。晚近有关microvesicles(微囊泡)的研究为我们提供了线索。
Microvesicles是由激活或凋亡的多种细胞通过分泌或脱落的形式释放到细胞外的小囊泡体。其中,与高凝状态和血栓形成关系最为密切的是血小板源的microvesicles,其在促凝的同时携带了多种血小板活化的信息。由此,我们推测,血小板来源的]microvesicles是房颤时传递血小板活化信息的重要载体,是启动凝血过程的关键环节。已有研究证实,microvesicles的强促凝活性主要依赖于其表达的PS(与Annexin V有很强的亲和力),而血小板膜糖蛋白CD36是PS的主要受体。CD36属于B类清道夫受体,是一种细胞膜表面单链糖蛋白,广泛存在于单核细胞、血小板、内皮细胞及脂肪细胞中。CD36可与多种配体结合,通过与不同配体的结合,在多种病理生理过程中发挥重要作用。因此,我们推测,在非瓣膜性房颤各组基础病因环境下,microvesicles是通过与血小板膜CD36结合途径活化血小板,并以级联放大的方式启动血栓前状态或高凝状态。迄今为止,国内外尚缺乏这方面的研究。因此,本课题拟采用病例对照研究,以正常对照组、非瓣膜性房颤脑卒中中低危组及非瓣膜性房颤脑卒中高危组的临床患者为研究对象,筛选出调控microvesicles特别是血小板源的microvesicles的各种危险因素；研究血小板源microvesicles和Annexin V阳性的microvesicles的表达及其与血小板活化指标的相关性；探讨血小板膜CD36的表达情况及其与microvesicles、血小板活化指标的相关性。
研究目的
1.非瓣膜性房颤状态下,筛选出调控血小板源microvesicles释放的各种危险因素；
2.非瓣膜性房颤患者血小板源microvesicles和Annexin V阳性的microvesicles的表达及其与房颤脑卒中风险的关系；
3.非瓣膜性房颤患者血小板膜糖蛋白CD36的表达水平及其与房颤脑卒中风险的关系；
4.探讨在非瓣膜性房颤状态下,血小板来源的microvesicles和Annexin V阳性的microvesicles的水平,血小板膜糖蛋白CD36的表达,血小板活化指标等各因素之间的相关性及内在联系。
研究对象与方法
选择阵发性或持续性非瓣膜性房颤患者210例进行病例—对照研究,平均年龄62.23±11.47岁,男性127例,女性83例。正常对照组35例,平均年龄55.71±7.43岁,男性17例,女性18例,均来自健康人群志愿者。对所有参与者进行体格检查,记录年龄、性别,测量身高、体重、腰围、臀围和血压等,并询问现病史、既往史、烟酒史、用药史和家族史等。部分参与者进行了超声心动图和颈动脉超声检查。所有参与者均经隔夜禁食12～14小时,次日清晨抽取空腹肘静脉血,测定血常规、葡萄糖(GLU)、总胆固醇(cholesterol, Cho)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-c)、低密度脂蛋白胆固醇(LDL-c)、尿酸(UA)等指标；采用ELISA法检测血浆中8-表氧-前列腺素F2a (8-iso-prostaglandinF2a)、氧化型低密度脂蛋白(ox-LDL)、白介素6(IL-6)、晚期糖基化终术产物(AGEs)及可溶性P选择素(P-selectin)的水平；固相免疫吸附法检测血小板源的microvesicles及Annexin V阳性的microvesicles;采用流式细胞术测定血小板血小板膜CD36和血小板糖蛋白(GP)Ⅱb/Ⅲa的表达。
结果
1.非瓣膜性房颤患者卒中风险分层：
所有入选的非瓣膜性房颤患者根据CHADS2卒中危险评分标准分为脑卒中高危组和中低危组。CHADS2评分参数包括：充血性心力衰竭、高血压、年龄>75岁、糖尿病、既往卒中或TIA史,前四项危险因素各1分,最后一项危险因素为2分。CHADS2评分总分为6分,0-1分为中低危组,2-6分为高危组。所有房颤患者中,113例(53.81%)属于卒中高危组(平均年龄：66.68±10.46岁,男性62例,女性51例),97例(46.19%)属于卒中中低危组(平均年龄：57.05±10.41岁,男性65例,女性32例)。
2.正常对照组、非瓣膜性房颤卒中中低危组及高危组临床特征的比较：
(1)正常对照组、非瓣膜性房颤脑卒中中低危组及高危组间性别、体重指数、舒张压、胆固醇、甘油三酯、LDL-C、HDL-C和血小板计数均无显著性差异(P>0.05)。
(2)正常对照组和非瓣膜性房颤脑卒中中低危组之间年龄、性别、体重指数、舒张压、空腹血糖、胆固醇、甘油三酯、LDL-C、HDL-C、尿酸、血肌酐和血小板计数差别均无统计学意义(P>0.05)；与对照相比,中低危组患者收缩压、腰臀比显著增高(P<0.01)。
(3)按照CHADS2卒中危险评分标准,非瓣膜性房颤卒中高危组除年龄较大外,其心衰、高血压、糖尿病及卒中史的发生率也远高于正常对照组和非瓣膜性房颤脑卒中中低危组(P<0.001)；此外,高危组患者的收缩压、空腹血糖、血肌酐、白细胞计数等指标较对照组和中低危组显著增高(P<0.01)；高危组患者腰臀比较对照组显著增高(P<0.001),较中低危组无显著性差异(P>0.05)。
3.正常对照组、非瓣膜性房颤卒中中低危组及高危组临床用药情况的比较：
与中低危组相比,非瓣膜性房颤卒中高危组华法林、钙通道阻滞剂、p受体阻断剂和它汀类药物的应用比例无显著差异(P>0.05),但是阿司匹林、ACEI/ARB的应用比例显著增高(P<0.01)。正常对照组未用任何上述心血管疾病治疗相关药物。
4.正常对照组、非瓣膜性房颤卒中中低危组及高危组超声指标的比较：
(1)超声心动图结果显示：非瓣膜性房颤卒中中低危组患者与正常对照组之间IVST、LVPWT及LVEF差别无统计学意义,但是中低危组患者LAD、RAD、E/E’及PCWP均有明显升高(P<0.01)；非瓣膜性房颤卒中高危组患者与正常对照组相比LVEF显著降低(P<0.01), LAD、RAD、IVST、LVPWT、E/E'及PCWP显著升高(P<0.01)；与中低危组相比,高危组LVEF降低(P<0.05)、E/E’升高(P<0.05), LAD、RAD、IVST、LVPWT及PCWP均无显著差异。
(2)颈动脉超声发现,非瓣膜性房颤卒中中低危组患者IMT及斑块指数较正常对照明显增高(P<0.01)；与正常对照相比,高危组患者IMT及斑块指数显著增高(P<0.001)；与中低危组相比,高危组患者IMT无显著差异,但是斑块指数显著增高(P<0.05)。
5.正常对照组、非瓣膜性房颤卒中中低危组及高危组氧化应激、炎症等指标的比较：
(1)非瓣膜性房颤卒中中低危组患者血浆中氧化应激指标oxLDL、8-iso-PGF2α较正常对照组显著升高(P<0.01)；房颤卒中高危组患者血浆oxLDL、8-iso-PGF2α水平较正常对照及中低危组房颤患者均明显升高(P<0.05)。
(2)房颤卒中中低危组患者血浆中IL-6水平较正常对照组明显升高(P<0.05)；高危组患者血浆IL-6水平较正常对照及中低危组均显著升高(P<0.001)。
(3)AGEs是糖代谢紊乱的产物及指标。房颤卒中中低危组患者血浆中AGEs水平较正常对照组明显升高(P<0.001)；高危组患者血浆AGEs水平较正常对照组及中低危组均显著升高(P<0.01)。
6.正常对照组、非瓣膜性房颤卒中中低危组及高危组microvesicles水平的比较：
与正常对照组相比,非瓣膜性房颤脑卒中中低危组患者血小板来源的microvesicles (PMVs)及Annexin V阳性的PMVs含量均增高(P<0.01)；与对照组相比,房颤卒中高危组患者PMVs及Annexin V阳性的PMVs含量显著增高(P<0.001)；此外,高危组患者PMVs及Annexin V阳性的PMVs含量较中低危组有所增高(P<0.01)。
7.正常对照组、非瓣膜性房颤卒中中低危组及高危组血小板相关指标的比较：
(1)与正常对照组相比,非瓣膜性房颤房颤卒中中低危组患者血小板膜蛋白CD36的表达显著增强(P<0.001)；房颤卒中高危组患者血小板膜蛋白CD36的表达较正常对照及中低危组明显增强(P<0.01)。
(2)血小板活化状态的检测包括流式细胞术测定血小板GPⅡb/Ⅲa的表达及ELISA测定血浆中可溶性P-selectin的水平。与对照组相比,中低危组患者血小板GPⅡb/Ⅲa的表达及可溶性P-selectin水平均显著增高(P<0.001)；高危组患者血小板GPⅡb/Ⅲa的表达及可溶性P-selectin水平较正常对照组(P<0.001)或中低危组患者均显著增高(P<0.05)。
8. CHADS2评分与血小板活化指标的相关分析
CHADS2评分与血小板活化指标GPⅡb/Ⅲa相关(r=0.264,P<0.001)；CHADS2评分与血小板可溶性P-selectin的分泌亦密切相关(r=0.448,P<0.001),提示非瓣膜性房颤患者脑卒中高风险与血小板激活密切相关。
9. CHADS2评分与microvesicles的相关分析
CHADS2评分与血浆中PMVs水平相关(r=0.213, P<0.001); CHADS2评分与Annexin V阳性的PMVs亦密切相关(r=0.449,P<0.001),提示血浆中PMVs,尤其是Annexin V阳性的PMVs有可能参与了房颤患者血栓前状态的发生。
10. CHADS2评分与氧化应激、炎症、糖代谢紊乱等指标的相关性分析
CHADS2评分与8-iso-PGF2α、ox-LDL(反映氧化应激指标)相关(r=0.353,P<0.001; r=0.338, P<0.001); CHADS2评分与炎症指标IL-6密切相关(r=0.410,P<0.001); CHADS2评分与AGEs(糖代谢紊乱相关指标)显著相关(r=0.511,P<0.001),提示氧化应激、炎症及糖代谢紊乱有可能是非瓣膜性房颤患者血栓前状态发生的危险因素。
11. PMVs与氧化应激、炎症、糖代谢紊乱等指标的相关性分析：
PMVs与8-iso-PGF2a显著相关(r=0.320, P<0.001); PMVs与IL-6显著相关(r=0.150,P=0.027); PMVs与AGEs相关(r=0.226,P=0.001)。
12. Annexin V阳性的PMVs与氧化应激、炎症、糖代谢紊乱等指标的相关性分析：
Annexin V阳性的PMVs与8-iso-PGF2a相关(r=0.228, P=0.004); Annexin V阳性的PMVs与ox-LDL显著相关(r=0.321, P<0.001); Annexin V阳性的PMVs与IL-6相关(r=0.176,P=0.008); Annexin V阳性的PMVs与AGEs相关(r=0.228,P=0.001)。
13.血浆中可溶性P-selectin与microvesicles的相关分析：
PMVs与可溶性P-selectin(反映血小板活化指标)显著相关(r=0.184, P=0.007); Annexin V阳性的PMVs与可溶性P-selectin相关(r=0.173,P=0.009),提示PMVs及Annexin V阳性的PMVs可能参与了非瓣膜性房颤状态下血小板的活化。
14.血小板CD36与血小板活化指标的相关分析：
血小板CD36的表达与血小板活化指标GPⅡ b/Ⅲa显著相关(r=0.296,P<0.001)；血小板CD36与可溶性P-selectin亦密切相关(r=0.248,P<0.001)。以年龄、收缩压、BMI、WHR、空腹血糖、胆固醇、血小板计数、血小板CD36表达为自变量,以血小板GPⅡb/Ⅲa为因变量,进行逐步回归多元线性回归分析,血小板CD36(β=0.314,P=0.011)和BMI (β=0.474,P<0.001)进入方程；以年龄、收缩压、BMI、WHR、空腹血糖、胆固醇、血小板计数、血小板CD36表达为自变量,以血浆可溶性P-selectin为因变量,进行多元线性回归分析,血小板CD36(β=0.114,P=0.045)、年龄(β=0.360,P=0.004)和WHR(β=0.501,P<0.001)进入方程。
结论：
(1)与正常对照组及脑卒中中低危组患者相比,非瓣膜性房颤卒中高危组具有较高的氧化应激、炎症及AGEs水平；
(2)与正常对照组及脑卒中中低危组患者相比,非瓣膜性房颤卒中高危组具有较高的PMVs及Annexin V阳性的PMVs水平；
(3)与正常对照组及脑卒中中低危组患者相比,非瓣膜性房颤卒中高危组具有较高的血小板膜CD36水平及血小板活化水平；
(4) PMVs及Annexin V阳性的PMVs与氧化应激、炎症、AGEs等指标密切相关,提示炎症、氧化应激及AGEs有可能是调控非瓣膜性房颤状态下PMVs释放的重要因素；
(5) PMVs及Annexin V阳性的PMVs分别与血小板活化指标密切相关,提示PMVs可能是传递血小板活化信息的重要载体；
(6)血小板膜CD36与血小板活化指标密切相关,提示血小板CD36作为信号分子有可能介导非瓣膜性房颤状态下血小板激活。
研究背景
非瓣膜性心房颤动(房颤)是脑卒中及血栓栓塞事件最强烈的独立危险因素。房颤时血栓前状态及血栓的形成是由房颤本身引起的还是由房颤伴随的基础病因引起的,目前仍存在争议。近年来相继揭晓的有关房颤治疗的临床研究(AFFIRM、RACE及AF/CHF)证实,“节律”控制治疗并不优于“室率”控制治疗,2种方法的卒中发生率无显著性差异。此外,孤立性房颤的卒中发生率不比正常人高。这提示我们,房颤时血栓前状态及血栓的形成并不完全依赖于房颤本身,推测可能是与房颤伴随的基础病因有关。
非瓣膜性房颤的上游疾病如冠心病、高血压、糖尿病、老龄化等使房颤患者处于较高的炎症、氧化应激水平。高水平的炎症、氧化应激状态可显著加速蛋白质、核酸或脂质等大分子物质的糖基化反应,得到的产物即晚期糖基化终产物(advanced glycation end products, AGEs)。AGEs的形成参与了房颤的发病机制,如动脉粥样硬化、心房纤维化等。我们在论文Ⅰ中证实,脑卒中高危的非瓣膜性房颤患者血浆中的氧化应激、炎症指标及AGEs水平远远高于中低危患者。综上,氧化应激、炎症及聚积的AGEs不仅参与了房颤的发生机制,而且很可能与房颤时血栓前状态及血栓的形成密切相关。
血小板粘集堆的形成是血栓形成的第一步,活化血小板的磷脂表面为凝血瀑布的激活及纤维蛋白的形成提供了一个反应平台。可见,血小板在非瓣膜性房颤血栓前状态及血栓形成中发挥着重要作用。房颤发生发展的基础病因,如氧化应激、炎症及聚积的AGEs是如何激活血小板导致血栓前状态的?至今仍未完全阐明。晚近有关microvesicles(微囊泡)的研究为我们提供了线索。
Microvesicles是细胞在活化或凋亡时从胞膜表面以生芽方式脱落的一些磷脂膜包裹的膜性小囊泡结构,其表面携带大量的信息蛋白等,参与多种病理生理过程。血小板源microvesicles (PMVs)是循环中微囊泡的主要来源,在血栓形成和止血的过程中发挥重要作用。脑卒中高危的非瓣膜性房颤患者血浆中的PMVs的总量及具有促凝活性的Annexin V阳性的PMVs的数量远远高于中低危患者。这提示我们,PMVs有可能是承载上述启动血小板活化的各种机制的载体,将有利于我们研究房颤时血栓前状态的发生机制,从而为探索房颤时高凝状态综合防治的新的有效靶点提供依据。
PMVs依靠何种途径传递血小板活化信息?PMVs的强促凝活性主要依赖于其表达的PS(与Annexin V有很强的亲和力),而血小板膜糖蛋白CD36是PS的主要候选受体之一。因此,PMV-CD36结合体可能是活化血小板的关键环节。CD36是一种多功能膜受体,广泛存在于血小板、单核细胞、内皮细胞及脂肪细胞中。它能够与多种配体结合,参与多种病理生理过程。PMV-CD36结合体如何启动血小板的活化过程?Chen等研究发现,MKK4/JNK2信号转导通路介导了ox-LDL与血小板表面CD36结合后激活血小板的过程。深入研究发现,CD36与oxLDL的结合是由与oxLDL上的脂质成分介导的。蛋白质组学证实microvesicles表面含有大量脂质成分。因此,PMV-CD36结合体活化血小板的信号转导途径与ox-LDL激活血小板的过程可能完全相同。由此我们推断,MKK4/JNK2信号转导通路同样介导了PMV-CD36结合体激活血小板的过程。
在非瓣膜性房颤状态下,PMVs能否介导房颤基础病因诱导的血小板激活,从而启动高凝状态尚不清楚。因此,我们提出如下假说：非瓣膜性房颤的基础病因,如氧化应激、炎症或聚集的AGEs,使体内PMVs总量增多,通过与血小板膜CD36结合,启动了PMV-CD36结合体介导的MKK4/JNK2信号转导途径活化血小板,从而参与房颤的血栓前状态或高凝状态的形成。
研究目的
1.氧化应激、炎症或聚积的AGEs对PMVs产生的影响；
2.携带氧化应激、炎症或AGEs刺激信息的PMVs对血小板活化的作用及信号转导机制；
3. PMV-CD36结合体及其介导的信号通路作为血栓前状态综合防治的新的有效靶点的可能性。
方法
1.正常人血小板的分离：健康献血志愿者均来自本实验室,近两周未服用任何抗血小板药物。志愿者隔夜禁食12～14小时,次日清晨在轻扎压脉带或不扎压脉带的情况下,抽取空腹肘静脉血20mL于0.109mol/L枸橼酸钠抗凝(1：9)的塑料采血管中。常温下,抗凝血静置10min后,经过120g离心10分钟后获得富含血小板血浆(PRP),后者加入100nmol/L PG-E1后经800g离心后得到血小板沉淀。血小板沉淀经改良台式液(137mmol/L NaCl,2.7mmol/L KCl,12mmol/L NaHCO3,0.4mmol/L NaH2PO4,5mmol/L HEPES,0.1%Glucose,0.35%BSA,100nmol/L PG-E1, pH7.2)洗涤、重悬。血小板悬液的浓度经细胞计数仪调整为1×106/mL,且立即应用于各项实验。
2.氧化应激、炎症及AGEs刺激对PMVs生成的影响：于37℃条件下,分别采用oxLDL (oxidized LDL,50μg/mL)、IL-6(1μg/mL)或AGEs (200μg/mL)孵育刺激洗涤血小板悬液(1×106/mL),同时各刺激组均设空白对照(等量缓冲液)、同型对照或阳性对照——二磷酸腺苷刺激(ADP,10μmol/L)。分别采用流式细胞术和固相免疫吸附法检测microvesicles的生成。
3.流式细胞术观察PMVs的释放：
PMVs免疫荧光标记：于BD流式管中先加入5μL PE-cyTM5标记抗人CD41a抗体,再加入2.5μL刺激后或未刺激的血小板悬液(1×106/mL),轻轻混匀后,于常温下避光孵育15min后,用1mL PBS悬浮细胞立即上机检测。
流式细胞仪测定及检测原理：开机校准流式细胞仪后,在Cell Quest软件环境下,将流式细胞仪的前向角散射(FSC),侧向角散射(SSC)及荧光(FL1-FL3)检测信号均设置为对数放大,以PE-cyTM5-CD41a阳性为获取条件,在CD41a vs. SSC点图中设门找出血小板群圈门,以血小板的位置定位设门找出PMVs群圈门,计数门内10,000个血小板,每管均在同一检测条件及程序设定下检测,数据分析采用Cell Quest软件。
4.固相免疫吸附法检测PMVs的生成
OxLDL、IL-6、AGEs及各组对照试剂孵育刺激后的血小板悬液,经过3,000g离心10min后,上清液再经12,000g离心3min以去除血小板的干扰,采用固相免疫吸附法检测最终上清液的PMVs,总量及Annexin V阳性的PMVs含量。具体步骤同论文Ⅰ。
5.氧化应激、炎症及AGEs刺激条件下PMVs的制备：分别将经过oxLDL (50μg/mL)、IL-6(1μg/mL)或AGEs (200μg/mL)孵育刺激后的血小板悬液(1×106/mL)3,000g离心10min后,取上清。上清中加入2.0mmol/L苯甲基黄酰化氟(PMSF)后,于4℃条件下15,000g高速离心60min,此时获得的microvesicles沉淀经过两次充分洗涤以去除oxLDL、IL-6或者AGEs的污染,采用BCA法检测提取的PMVs的蛋白浓度,-80℃保存备用。
6.双色流式细胞术检测血小板膜蛋白CD36的表达：在BD流式管内加入血小板悬液(2.5μL)与5μL小鼠抗人PEcy5-CD41a,5μL小鼠抗人PE-CD36轻轻混匀；同型对照管内加入等量样本及小鼠抗人PEcy5-CD41a,5μL小鼠抗人PE-IgM。样本与抗体在室温下避光孵育15min后,用1mL PBS重悬细胞,即刻上机分析。
7.双色流式细胞术检测血小板膜糖蛋白(GP)Ⅱb/Ⅲa:分别将步骤5中提取的各种来源的PMVs (30μg/mL)与洗涤血小板悬液(1×106/mL)于常温下孵育刺激30min。于BD流式管内加入处理后的血小板悬液(2.5μL)与5μL小鼠抗人PEcy5-CD41a,5μL FITC结合PAC-1抗体(能够识别与结合因血小板激活而异构的GPⅡb/Ⅲa),轻轻混匀后,室温下避光孵育15min后,用1mL PBS重悬细胞,即刻上机分析。
8.比浊法测定血小板聚集：将正常人空腹静脉血以120g离心10min,小心取出上层血浆为PRP,将取出PRP后的标本以3,000g离心10min,其上层较为透明的液体即为PFP。分别计数PRP与PFP中的血小板数,用PFP将PRP中的血小板数调节至25万/mm3。分别将步骤5中提取的各种来源的PMVs(30μg/mL)与PRP常温下孵育30min。取225μL的PFP于比浊管中作为空白对照,取等量的经PMVs刺激后的PRP于比浊管内,放入一磁棒,在37℃温育3min后,放入测定仪。用对照管内的PFP调零后,加入25μL ADP的同时开始记录,测定时间为5min,根据悬液透光度变化的程度及速度即可确定血小板聚集的程度与速度,并同时记录下血小板聚集曲线。
9.酶联免疫法测定细胞上清中可溶性P-selectin含量：分别将步骤5中提取的各种来源的PMVs (30μg/mL)与洗涤血小板悬液(1×106/mL)于常温下孵育刺激30min后,将PMV-血小板混合液3,000g离心10min后取上清,后者再次12,000g离心3min以去除血小板的污染。ELISA法检测最终细胞上清液中可溶性P-selectin含量,操作方法及步骤严格按照说明书的要求进行。
10. Western blot检测：裂解经各种来源PMVs刺激的血小板悬液(1×106/mL),提取蛋白,western blot检测CD36阳性或CD36阴性的血小板内磷酸化及非磷酸化的MKK4和JNK的表达量。
11.丹参酮ⅡA对PMVs活化血小板的干预试验：在加入各种刺激来源的PMVs (30μg/mL)前,给予洗涤血小板(1×106/mL)不同浓度的丹参酮ⅡA(5μg/mL、10μg/mL、20μg/mL、50μg/mL、100μg/mL)预处理15min,然后采用流式细胞术检测处理后的血小板表面GPⅡb/Ⅲa及CD36的表达；采用western blot的方法检测MKK4/JNK信号分子的磷酸化水平。
研究结果
1. PMVs介导氧化应激对血小板功能影响的研究
(1) OxLDL能够促进PMVs及Annexin V阳性的PMVs的释放
OxLDL (50μg/mL)及经典的血小板激活剂ADP(10μmol/L)在与洗涤血小板(1×106/mL)作用15min后,均能显著促进PMVs的释放,表现为M门内PE-cyTM5-CD41a阳性的微粒数显著增多(P<0.05),但是,天然的未经氧化的低密度脂蛋白(nLDL)却对PMVs的生成基本无影响。ELISA结果显示,与对照组及nLDL(?)相比,oxLDL能够显著促进PMVs总量及Annexin V阳性的PMVs的释放,其作用可与ADP相比(P<0.05)。
(2) OxLDL依赖的PMVs对血小板活性的影响
为了验证oxLDL刺激产生的PMVs (oxLDL-PMVs)对血小板活性的影响,我们将洗涤血小板(1×106/mL)与oxLDL-PMVs(30μg/mL)于常温下孵育30min。血小板激活以血小板膜糖蛋白Ⅱb/Ⅲa (GPⅡb/Ⅲa)的空间构象改变为特点,从而显露出纤维蛋白原等粘附分子的识别与结合部位,由此导致血小板聚集。抗体PAC-1能够识别并结合血小板因激活而异构的GPⅡb/Ⅲa,是检测血小板活化最常用的指标之一。本实验发现,与对照组相比,oxLDL-PMVs能够显著促进血小板PAC-1的平均荧光强度(MFI)增强(P<0.05), PAC-1阳性的血小板百分率增加(P<0.05)；此外,oxLDL-PMVs能够明显促进ADP诱导下的血小板的聚集。
(3) OxLDL-PMVs对CD36阴性的血小板活性的影响
OxLDL-PMVs (30μg/mL)与CD36阴性的血小板(1×106/mL)在常温下相互作用30min,血小板PAC-1的MFI及阳性百分率都未见明显变化；血小板聚集率未见明显增强。
(4) OxLDL-PMVs诱导的血小板活化呈CD36、磷脂酰丝氨酸(PS)依赖性为了进一步验证PMVs表面的PS及血小板表面的CD36对PMV-血小板相互作用的影响,在oxLDL-PMVs与血小板相互作用之前,我们分别采用CD36中和抗体预处理洗涤血小板和Annexin V预处理oxLDL-PMVs,并检测了血小板活化指标——GP Ⅱb/Ⅲ a的表达及可溶性P-selectin的分泌。结果发现,oxLDL-PMVs (30μg/mL)能够显著增加CD36阳性血小板GPⅡb/Ⅲa的表达及可溶性P-selectin的分泌(P<0.05)；阻断血小板膜CD36后,血小板活化指标均明显降低(P<0.05),但是相同条件下经CD36中和抗体的同型对照IgG预处理却不能抑制oxLDL-PMVs对血小板的活化作用；采用Annexin V阻断PMVs表面的PS同样能够抑制oxLDL-PMVs诱导的血小板活化(P<0.05)。同时,我们检测了经过上述各种处理后血小板表面CD36的表达情况,发现中和CD36后血小板膜CD36表达有所降低,其它各组并无显著变化。
(5) OxLDL-PMVs诱导血小板的活化呈JNK信号通路依赖性为了明确JNK信号传导通路对oxLDL-PMVs诱导血小板的活化的潜在作用,我们用JNK抑制剂SP600125预处理血小板后加入oxLDL-PMVs刺激。结果显示,阻断JNK信号通路能够显著抑制oxLDL-PMVs对血小板的激活作用,包括GPⅡb/Ⅲa的表达及可溶性P-selectin的分泌(P<0.05)；经SP600125预处理后,血小板表面的CD36表达并无显著变化。
(6) OxLDL-PMVs能够上调血小板JNK2及其上游MKK4的磷酸化水平对于CD36阳性血小板,oxLDL-PMVs刺激能够显著促进JNK2及其上游信号分子MKK4的磷酸化水平升高(P<0.05)；当oxLDL-PMVs (30μg/mL)分别作用Omin、5min、15min、30min时,JNK2磷酸化水平呈时间依赖性提高；分别用不同浓度(0.μg/mL、10μg/mL、30μg/mL、50μg/mL) oxLDL-PMVs作用于CD36阳性血小板30min,发现随着oxLDL-PMVs浓度的升高,JNK2磷酸化水平呈剂量依赖性提高。但是对于CD36阴性的血小板,oxLDL-PMVs刺激对MKK4/JNK2的磷酸化水平基本无影响。
2. PMVs介导炎症对血小板功能影响的研究
(1)IL-6刺激能够促进PMVs及Annexin V阳性的PMVs的释放
与对照组相比,IL-6能够显著促进PMVs总量及Annexin V阳性的PMVs的生成,其作用可与血小板激活剂ADP相比(P<0.05)。
(2)IL-6依赖的PMVs(IL-6-PMVs)对血小板活性的影响
与对照组相比,IL-6-PMVs刺激组血小板PAC-1的MFI及阳性百分率均显著增加(P<0.05)；此外,IL-6-PMVs能够明显促进ADP诱导下的血小板的聚集。
(3) IL-6-PMVs诱导血小板的活化呈CD36、PS依赖性
IL-6-PMVs (30μg/mL)与CD36阴性血小板(1×106/mL)在常温下相互作用30min后,血小板PAC-1的MFI及阳性百分率都未见明显变化；ADP诱导的.血小板聚集率未见明显增强。
IL-6-PMVs能够显著增加CD36阳性血小板GPⅡ b/Ⅲa的表达及可溶性P-selectin的分泌(P<0.05)；阻断血小板膜CD36后,血小板活化指标均明显降低(P<0.05)；采用Annexin V阻断PMVs表面的PS同样能够抑制IL-6-PMVs诱导的血小板活化(P<0.05)。
(4) MKK4/JNK2信号通路对IL-6-PMVs诱导血小板活化的影响
阻断JNK信号通路能够显著抑制IL-6-PMVs对血小板的激活作用,包括GP Ⅱb/Ⅲa的表达及可溶性P-selectin的分泌(P<0.05)。
Western blot结果显示,对于CD36阳性血小板,IL-6-PMVs (30μg/mL)能够显著上调JNK2及其上游信号分子MKK4的磷酸化水平(P<0.05)；当IL-6-PMVs (30μg/mL)作用0min、5min、15min、30min, JNK2磷酸化水平呈时间依赖性提高；分别用不同浓度(Oμg/mL、10μg/mL、30μg/mL、50μg/mL) IL-6-PMVs作用CD36阳性血小板30min,发现随着IL-6-PMVs浓度的升高,JNK2磷酸化水平呈剂量依赖性提高。但是对于CD36阴性的血小板,IL-6-PMVs刺激对MKK4/JNK2的磷酸化水平基本无影响。
(5)丹参酮ⅡA能够抑制IL-6-PMVs诱导的血小板活化
为了验证血小板PMV-CD36结合体及其介导的信号通路能否成为高水平炎症状态下血小板过度激活的干预靶点,我们进一步探讨其被药物干预的可能性。在加入IL-6-PMVs(30μg/mL)刺激前给予血小板不同浓度的丹参酮ⅡA(5μg/mL、10μg/mL、20μg/mL、50μg/mL、100μg/mL)预处理15min。结果发现,丹参酮ⅡA在体外能够呈剂量依赖性的抑制IL-6-PMVs诱导的血小板活化：5μg/mL丹参酮ⅡA使血小板PAC-1百分率降低,但效果不显著；10μg/mL丹参酮ⅡA对血小板活化指标的抑制作用达到统计学意义(P<0.05)；随着丹参酮ⅡA浓度的增加,其对血小板活化的抑制作用也逐渐增强。同时,我们检测了丹参酮ⅡA对血小板膜CD36表达的影响。结果显示：丹参酮ⅡA在体外能够呈剂量依赖性的抑制血小板CD36的表达；当丹参酮ⅡA浓度达到20μg/mL时,其抑制作用达到统计学意义(P<0.05)；随着丹参酮ⅡA浓度的增加,其对血小板CD36表达的抑制作用也逐渐增强。Western blot结果显示,丹参酮ⅡA能够有效下调IL-6-PMVs诱导的JNK2及其上游信号分子MKK4磷酸化水平(P<0.05)。
3. PMVs介导AGEs对血小板功能影响的研究
(1) AGEs刺激能够促进PMVs及Annexin V阳性的PMVs的释放
AGEs (200μg/mL)及ADP (10μmol/mL)刺激组M门内的PMVs数量显著增多,BSA (200μg/mL)刺激组较对照组未见明显变化。定量分析流式细胞术和ELISA的结果显示：与对照组及BSA处理组相比,AGEs能够显著促进PMVs总量及Annexin V阳性的PMVs的生成(P<0.05),其作用可与血小板激活剂ADP相比。
(2) AGEs依赖的PMVs (AGE-PMVs)对血小板活性的影响
与对照组相比,AGE-PMVs刺激组及ADP刺激组的血小板被明显活化,表现为GPⅡb/Ⅲa的MFI及阳性百分率均明显升高(P<0.05)；此外,AGE-PMVs能够显著增强ADP诱导的血小板的聚集。
(3) AGE-PMVs诱导血小板的活化呈CD36、PS依赖性
AGE-PMVs (30μg/mL与CD36阴性血小板(1×106/mL)在常温下相互作用30min后,流式细胞术结果显示,血小板PAC-1的MFI及阳性百分率都未见明显变化；ADP诱导的血小板聚集率未见明显增强。
AGE-PMVs能够显著增加CD36阳性的血小板GPⅡb/Ⅲa的表达及可溶性P-selectin的分泌(P<0.05)；阻断血小板膜CD36后,血小板活化指标均明显降低(P<0.05)；采用Annexin V阻断PMVs表面的PS同样能够抑制AGE-PMVs诱导的血小板活化(P<0.05)。
(4) MKK4/JNK2信号通路对AGE-PMVs诱导的血小板活化的影响
阻断JNK信号通路能够显著抑制AGE-PMVs对血小板的激活作用,包括GP Ⅱ b/Ⅲa的表达及可溶性P-selectin的分泌(P<0.05)。
Western blot结果显示,对于CD36阳性血小板,AGE-PMVs (30μg/mL)能够显著上调JNK2及其上游信号分子MKK4的磷酸化水平(P<0.05)；当AGE-PMVs (30μg/mL)分别作用0min、5min、15min、30min时,JNK2磷酸化水平呈时间依赖性提高,15min达到峰值水平；分别用不同浓度(Oμg/mL、10μng/mL)、30μg/mL、50μg/mL) AGE-PMVs作用CD36阳性血小板30min,发现随着AGE-PMVs浓度的升高,JNK2磷酸化水平呈剂量依赖性提高。但是对于CD36阴性的血小板,AGE-PMVs刺激对MKK4/JNK2的磷酸化水平基本无影响。
(5)丹参酮ⅡA能够抑制AGE-PMVs诱导的血小板活化
在加入AGE-PMVs (30μg/mL)刺激前,用不同浓度的丹参酮ⅡA (5μg/mL、10μg/mL、20μg/mL、50μg/mL)预处理洗涤血小板15min,然后检测血小板活化指标GPⅡb/Ⅲa。流式细胞术结果显示,丹参酮ⅡA在体外能够呈剂量依赖性的抑制AGE-PMVs诱导的血小板活化；当丹参酮ⅡA浓度达到10μg/mL时,其对血小板活化的抑制作用达到统计学意义(P<0.05)；随着丹参酮ⅡA浓度的增加,其对血小板活化的抑制作用也逐渐增强。同时,我们检测了丹参酮ⅡA对血小板膜CD36表达的影响。流式细胞术结果显示：丹参酮ⅡA在体外能够呈剂量依赖性的抑制血小板CD36的表达；当丹参酮ⅡA浓度达到20μg/mL时,其抑制作用达到统计学意义(P<0.05)；随着丹参酮ⅡA浓度的增加,其对血小板CD36表达的抑制作用也逐渐增强。Western blot结果显示,丹参酮ⅡA和SP600125(JNK抑制剂,阳性对照)均能够显著下调AGE-PMVs诱导的JNK2磷酸化水平升高(P<0.05)；丹参酮ⅡA还能够有效抑制AGE-PMVs诱导的MKK4的磷酸化水平(P<0.05)。
结论
(1)OxLDL、IL-6及AGEs刺激均能够促进PMVs的释放；
(2)各种刺激来源的PMVs均能够促进CD36阳性的血小板活化,但对CD36阴性的血小板几乎无影响；
(3)各种刺激来源的PMVs对血小板的活化作用呈CD36、PS依赖性；
(4) MKK4/JNK2信号转导通路参与各种刺激来源的PMVs对血小板的激活；
(5)丹参酮ⅡA能够抑制各种刺激来源的PMVs对血小板的激活；
(6)丹参酮ⅡA对PMVs作用的抑制是通过对CD36/MKK4/JNK2信号通路的抑制而发挥作用的,提示PMV-CD36及其介导的信号通路可以作为高氧化应激、高炎症及AGEs刺激条件下防治血小板过度激活的有效靶点。
研究背景
非瓣膜性心房颤动(房颤)是最常见的快速性心律失常,导致房颤患者致死、致残的最主要原因是血栓栓塞所致的脑卒中。血小板活化在血栓形成中起着非常重要的作用。脑卒中高危的非瓣膜性房颤患者血小板活化水平远远高于中低危患者。但是,非瓣膜性房颤中血小板被激活的机制复杂,至今仍未完全阐明。
药物维持窦性心律和控制心室率的循证医学(AFFIRM、RACE及AF/CHF)结果表明,“节律”控制治疗并不优于“心率”控制治疗,2种方法在卒中发生率方面并无显著性差异。这提示我们,房颤时血栓前状态及血栓的形成并不完全依赖于房颤本身,推测可能是与房颤伴随的基础病因有关。非瓣膜性房颤的上游疾病如冠心病、高血压、糖尿病、老龄化等使房颤患者处于较高的炎症、氧化应激及AGEs水平。脑卒中高危的非瓣膜性房颤患者血浆中的氧化应激、炎症指标及AGEs远远高于中低危患者。越来越多的研究表明,炎症、氧化应激及AGEs参与了非瓣膜性房颤高凝状态的形成。上述研究分别提供了各自独立的证据,却无法将这些导致高凝状态的各种机制作为一个整体进行系统性研究,使治疗缺乏特异性。因此,寻找一种能够同时承载上述启动血小板活化的各种机制的载体,以此为工具深入研究房颤时血栓前状态的发生机制,将为探索房颤时高凝状态综合防治的新的有效靶点提供依据。
Microvesicles (MVs)能够作为介导氧化应激、炎症或聚积的AGEs活化血小板的重要载体。炎症、氧化应激及聚积的AGEs是房颤发生、心房纤维化及血栓前状态形成的重要机制已被众多学者肯定。我们有理由推测,房颤患者循环中的microvesicles是氧化应激、炎症及AGEs综合作用的产物,同时携带血小板活化的各种信息,是深入研究房颤时血栓前状态的发生机制的重要载体。我们在论文Ⅱ中证实,在氧化应激、炎症或AGEs条件下,MKK4/JNK2信号转导途径介导了PMVs与血小板膜CD36结合后激活血小板的过程,但是这条通路是否介导了房颤条件下microvesicles激活血小板的过程未见报道。
据此,我们提出如下假说：非瓣膜性房颤状态下,microvesicles是综合诸多基础病因(氧化应激、炎症及聚集的AGEs等)的重要载体,通过与血小板膜CD36结合,启动了MV-CD36结合体介导MKK4/JNK2信号转导途径活化血小板,随之分泌更多Annexin-V阳性的MVs,进一步与更多的血小板CD36结合,通过正反馈机制,大量活化血小板,启动房颤的血栓前状态或高凝状态,进而导致血栓形成及血栓栓塞事件。
研究目的1.非瓣膜性房颤环境中microvesicles对血小板活化的作用及信号转导机制；
2. MV-CD36结合体及其介导的信号通路作为血栓前状态综合防治的新的有效靶点的可能性。
方法
1.正常人血小板的分离：健康献血志愿者未服用过任何抗血小板药物,隔夜禁食12～14小时,次日清晨在轻扎压脉带或不扎压脉带的情况下,抽取空腹肘静脉血20mL于0.109mol/L枸橼酸钠抗凝(1：9)的塑料采血管中。常温下,抗凝血静置10min后,经过120g离心10分钟后获得富含血小板血浆(PRP),后者加入100nmol/LPG-E1后经800g离心后得到血小板沉淀。血小板沉淀经改良台式液(137mmol/L NaCl,2.7mmol/L KC1,12mmol/L NaHCO3,0.4mmol/L NaH2PO4,5mmol/L HEPES,0.1%Glucose,0.35%BSA,100nmol/L PG-E1, pH7.2)洗涤、重悬。血小板悬液的浓度经细胞计数仪调整为1×106/mL,且立即应用于各项实验。
2.流式细胞术检测非瓣膜性房颤病人乏血小板血浆(PFP)中MVs的含量及性质：取100μL脑卒中高危房颤患者(CHADS2评分≥2)的PFP与10μL小鼠抗人PE-cyTM5-CD41a,10μL FITC-Annexin V,100μL缓冲液(10mM Hepes/NaOH,140mM NaCl,2.5mM CaCl2, PH7.4)于BD流式管内轻轻混匀,室温下避光孵育15min后,即刻上机分析。
3.高速梯度离心法分离房颤患者血浆中的MVs：根据CHADS2评分选取非瓣膜性房颤脑卒中高危患者及年龄、性别相匹配的中低危患者,抽取3.8%枸橼酸钠抗凝空腹静脉血,经3,000g离心20分钟后,得PFP。PFP中加入PMSF后,于4℃条件下15,000g高速离心60min,将获得的MVs沉淀经过两次充分洗涤后-80℃保存备用。
4.双色流式细胞术检测血小板膜蛋白CD36的表达：在BD流式管内加入血小板悬液(2.5μL)与5μL小鼠抗人PEcy5-CD41a,5μL小鼠抗人PE-CD36或其同型对照小鼠抗人PE-IgM,轻轻混匀,在室温下避光孵育15min后,用1mL PBS重悬细胞,即刻上机分析。
5.流式细胞术检测血小板膜糖蛋白(GP)Ⅱb/Ⅲa:洗涤血小板悬液(1×106/mL)与MVs (30μg/mL)在常温下孵育30min。于BD流式管内加入处理后的血小板悬液(2.5μL)与5gL小鼠抗人PEcy5-CD41a,5μL小鼠抗人FITC-PAC-1(能够识别与结合因血小板激活而异构的GPⅡb/Ⅲa)轻轻混匀后,室温下避光孵育15min后,用1mL PBS重悬细胞,即刻上机分析。
6.双色流式细胞术检测血小板膜CD40L的表达：洗涤血小板悬液(1x106/mL)与MVs(30μg/mL)在常温下孵育30min。于BD流式管内加入处理后的血小板悬液(2.5μL)与5μL小鼠抗人PEcy5-CD41a,5μL小鼠抗人PE-CD40L,轻轻混匀后,室温下避光孵育15min后,用1mL PBS重悬细胞,即刻上机分析。
7.酶联免疫法测定细胞上清中可溶性P-selectin含量：将MVs (30μg/mL)与洗涤血小板悬液(1×106/mL)于常温下孵育刺激30min后,MV-血小板混合液3,000g离心10min后取上清,后者再次12,000g离心3min避免血小板的干扰。ELISA法检测最终细胞上清液中可溶性P-selectin含量,操作方法及步骤严格按照说明书的要求进行。
8. Western blot检测：裂解经MVs刺激的血小板悬液(1×106/mL),提取蛋白,western blot检测CD36阳性或CD36阴性的血小板内磷酸化及非磷酸化的MKK4和JNK的表达量。具体实验步骤同论文Ⅱ。
9.丹参酮ⅡA对MVs活化血小板的干预试验：在加入MVs(30μg/mL)刺激前,给予洗涤血小板(1×106/mL)不同浓度的丹参酮ⅡA (5μg/mL、20μg/mL、50μg/mL、100μg/mL)预处理15min,然后采用流式细胞术检测处理后的血小板表面GPⅡb/Ⅲa及CD36的表达；采用western blot的方法检测MKK4/JNK信号分子的磷酸化水平。
研究结果
(1)房颤病人来源的MVs对血小板活性的影响
我们采用高速离心的方法从非瓣膜性房颤脑卒中高危患者乏血小板血浆中分离出MVs(AF-MVs),同时从年龄、性别均与高危患者匹配的非瓣膜性房颤脑卒中低危患者乏血小板血浆中分离出MVs作为对照(C-MVs)。流式细胞术分析结果发现,AF-MVs大多为CD41a阳性、Annexin V阳性,即大部分MVs来源于血小板,且具有很强的促凝活性。
与对照组及C-MVs刺激组相比,AF-PMVs刺激组血小板PAC-1的平均荧光强度(MFI)及阳性百分率均显著增加(P<0.05)；此外,AF-MVs能够明显促进血小板表面CD40L的表达(P<0.05)；与对照组相比,C-MVs作用后的血小板PAC-1的MFI及百分率,血小板CD40L表达一定程度上有所增加,但强度较弱。
(2) AF-MVs诱导血小板的活化呈CD36、PS依赖性
AF-MVs(30μg/mL)和C-MVs(30μg/mL)分别与CD36阴性血小板(1×106/mL)在常温下相互作用30min后,流式细胞术结果显示,血小板PAC-1的MFI及阳性百分率都未见明显变化；此外,血小板表面CD40L的表达未见明显增强。
AF-MVs能够显著增加CD36阳性血小板GP Ⅱ b/Ⅲa的表达及可溶性P-selectin的分泌(P<0.05)；阻断血小板膜CD36后,血小板活化指标均明显降低(P<0.05)；采用Annexin V阻断PMVs表面的PS同样能够抑制AF-MVs诱导的血小板活化(P<0.05)。
(3) MKK4/JNK2信号通路对AF-MVs诱导的血小板活化的影响
阻断JNK信号通路能够显著抑制AF-MVs对血小板的激活作用,包括GPⅡ b/Ⅲa的表达及可溶性P-selectin的分泌(P<0.05)。
Western blot结果显示,AF-MVs (30μg/mL)能够显著上调CD36阳性血小板JNK2及其上游信号分子MKK4的磷酸化水平(P<0.05)；在AF-MVs (30μ/mL)作用Omin、5min、15min、30min,JNK2磷酸化水平呈时间依赖性提高,15min达到峰值水平；分别用不同浓度(Oμg/mL、10μg/mL、30μg/mL、50μg/mL) IL-6-PMVs作用CD36阳性血小板30min,发现随着AF-MVs浓度的升高,JNK2磷酸化水平呈剂量依赖性提高。但是对于CD36阴性的血小板,AF-MVs刺激对MKK4/JNK2的磷酸化水平基本无影响。
(4)丹参酮ⅡA干预对AF-MVs诱导的血小板活化的影响
为了验证血小板MV-CD36结合体及其介导的信号通路能否成为房颤时高凝状态综合防治的有效靶点,我们进一步探讨其被药物干预的可能性。我们分别用不同浓度的丹参酮ⅡA (5μg/mL、10μg/mL、20μg/mL、50μ/mL.100μg/mL)预处理血小板15min后加入AF-MVs (30μg/mL)刺激。结果发现,丹参酮ⅡA在体外能够呈剂量依赖性的抑制AF-MVs诱导的血小板活化：10M.g/mL丹参酮ⅡA使血小板PAC-1百分率降低,但效果不显著；10μg/mL丹参酮ⅡA对血小板活化指标的抑制作用达到统计学意义(P<0.05)；随着丹参酮ⅡA浓度的增加,其对血小板活化的抑制作用也逐渐增强。同时,我们检测了丹参酮ⅡA对血小板膜CD36表达的影响。结果显示：丹参酮ⅡA在体外能够呈剂量依赖性的抑制血小板CD36的表达；当丹参酮ⅡA浓度达到20μg/mL时,其抑制作用达到统计学意义(P＜0.05)；随着丹参酮ⅡA浓度的增加,其对血小板CD36表达的抑制作用也逐渐增强。Western blot结果显示,丹参酮ⅡA还能够抑制AF-MVs诱导的JNK2及其的上游信号分子MKK4的磷酸化水平。
结论
(1)非瓣膜性房颤病人循环中的MVs大部分来源于血小板,且具有很强的促凝活性；
(2) AF-MVs能够促进CD36阳性的血小板活化,但对CD36阴性的血小板无效；
(3) AF-MVs对血小板的活化作用呈CD36、PS依赖性；
(4) MKK4/JNK2信号转导通路参与AF-MVs对血小板的激活；
Background
Nonvalvular atrial fibrillation (NVAF) is a major cause of stroke and thromboembolism. Although anticoagulant therapy is recommended for patients with NVAF at high risk for stroke,33%-38%of patients receiving anticoagulant therapy still experience stroke. The mechanisms underlying thromboembolism in NVAF are still incompletely understood. The activation of platelets may initiate thrombosis. Abundant thrombin is generated as a result of a cascade of coagulation factors activated on the phospholipid surface of activated platelets, thereby leading to thrombosis.
However, antiplatelet therapy in NVAF patients has been challenged. Compared with placebo, with aspirin treatment, the risk of thromboembolism in patients with AF decreased by only21%, which was less effective than warfarin. Insufficiency of the present antiplatelet therapy may be associated with the diversity of platelet activation pathways. The molecular mechanisms that underlie platelet activation in NVAF are poorly defined but may involve the etiology of NVAF.
The potential effects of inflammation, oxidative stress and metabolic disorders in the NVAF-related prothrombotic state have aroused much attention. However, these risk factors have not been considered together in targeting prevention of NVAF-related platelet activation. To determine a vector exhibiting all the mechanisms in NVAF, we can study the potential mechanisms of platelet activation and explore new, effective targets for preventing thrombosis in NVAF. Recently, studies of microvesicles (MVs) have enlightened us.
MVs are submicrometer vesicles formed by activated cells. MVs released into the circulation can act as messengers delivering a variety of cargos, including cell surface receptors, proinflammatory cytokines, signature proteomes, and even mRNA, to target cells. Thus, MVs may be the vectors combining multiple pathogenic mechanisms of platelet activation. Platelet-derived microvesicles (PMVs), with surface exposure of phosphatidylserine (PS, with strong affinity for Annexin V), are the main culprit in the development of thrombosis. CD36, a class B scavenger receptor expressed on platelets, monocytes and several other cells. Its role in platelet function has remained obscure. Platelet CD36could recognize and capture PS on the surface of MVs, thus generating an MV-CD36complex. Accordingly, we speculated that the PMVs, generated in response to pathogenesis or accompanying disorders of NVAF, could be endogenous CD36ligands that transmit an activating signal to platelets to induce thrombosis. We investigated healthy controls and the NVAF patients with "low to moderate risk" or "high risk" for stroke to determine the triggers for the release of platelet-derived microvesicles and Annexin V-positive microvesicles and to explore the potential relationship among microvesicles, platelet CD36and platelet activation.
Objectives
1. To screen for the risk factors which trigger the release of the microvesicles and platelet-derived microvesicles in patients with NVAF.
2. To determine the plasma levels of platelet-derived microvesicles and Annexin V-positive microvesicles in NVAF patients, as well as their relationship to stroke risk.
3. To determine the expression of platelet CD36and its relationship to stroke risk in NVAF patients.
4. To explore the relationship among platelet-derived microvesicles, Annexin V-positive microvesicles, platelet CD36and platelet activation indexes in patients with NVAF.
Subjects and methods
We included210patients with persistent or paroxysmal NVAF (mean age62.23±11.47years;127men) and35healthy controls (mean age55.71±7.43;17men). All subjects completed a questionnaire on age, gender, present condition, family and medical histories of cardiovascular risk factors and complications. Height, weight, body mass index (BMI), waist circumference, hip circumference and blood pressure (BP) were measured. Most subjects underwent2D transthoracic echocardiography and carotid ultrasonography evaluation. Furthermore, fasting blood sample was collected from each subject after12-14hours fast to determine the fasting blood glucose (FBG), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), creatinine (Cr) and uric acid (UA); Measurement of8-iso-prostaglandinF2α, interleukin6(IL-6), advanced glycosylation end products (AGEs) and soluble P-selectin involved ELISA commercially; The quantification of platelet-derived microvesicles and Annexin V-positive microvesicles in platelet free plasma (PFP) involved use of a homemade ELISA; The expression of platelet GPⅡ b/Ⅲa and CD36were detected by flow cytometry.
Results
1. Characteristics in NVAF patients:relationship to stroke risk
Patients were classified as being at "low to moderate risk" or "high risk" for stroke according to the CHADS2stroke risk scheme:1point each for presence of congestive heart failure, hypertension, age older than75or diabetes mellitus; and2points for history of stroke or transient ischemic attack. Patients with CHADS2score0or1were considered at "low to moderate risk" and those with score≥2at "high risk". Of the210NVAF patients,113(53.81%) were at high risk of stroke (mean age:66.68±10.46years,62males and51females),97(46.19%) were at low to moderate risk of stroke (mean age:57.05±10.41years,65males and32females).
2. Clinical characteristics of the healthy controls and NVAF patients at "low to moderate risk" or "high risk" for stroke
(1) There were no significant differences in sex, body mass index (BMI), diastolic blood pressure (DBP), cholesterol, triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c) and platelet counts among healthy controls, NVAF patients at "low to moderate risk" and NVAF patients at "high risk" for stroke (P>0.05).
(2) There were no significant differences in age, sex, BMI, DBP, fasting blood glucose, cholesterol, cholesterol, TG, LDL-c, HDL-c, UA, creatinine and platelet counts between healthy controls and NVAF patients at low to moderate risk for stroke (P>0.05). Systolic blood pressure (SBP) and waist-to-hip ratio (WHR) were significantly higher in the patients at low to moderate risk compared with healthy controls (P<0.01).
(3) By definition, the high-risk NVAF patients were older and more likely to have congestive heart failure, hypertension, diabetes mellitus, or history of stroke than those at low to moderate risk (P<0.001for all); Furthermore, SBP, fasting blood glucose, creatinine, white blood cell counts were significantly higher in the patients at low to moderate risk compared with healthy controls and the low to moderate-risk NVAF patients (P<0.01); There was no significant differences in WHR between the high-risk NVAF patients and the low to moderate-risk NVAF patients (P>0.05). WHR was higher in the high-risk NVAF patients compared with healthy controls (P <0.001).
3. Clinical medication of the healthy controls and NVAF patients at "low to moderate risk" or "high risk" for stroke
Compared with the NVAF patients at low to moderate risk for stroke, the application proportion of warfarin, calcium channel blockers, beta-receptor blockers and statins had no significant differences in the high-risk NVAF patients (P>0.05); However, the patients in the high-risk group received more anti-platelet therapy and ACEI/ARB compared with those with low to moderate risk (P<0.01); The healthy controls were free of any medication.
4. Comparison of ultrasonic parameters among the healthy controls and NVAF patients at "low to moderate risk" or "high risk" for stroke
(1) The transthoracic echocardiography results:There were no significant differences in thickness of inter-ventricular septum (IVST), thickness of posterior wall of left ventricle (LVPWT) and left ventricular ejection fraction (LVEF) between NVAF patients at low to moderate risk for stroke and healthy controls (P>0.05), but the diameter of left atrium (LAD), the diameter of left atrium (RAD), ratio of early transmitral flow velocity to early mitral annular diastolic velocity (E/E') and the pulmonary capillary wedge pressure (PCWP) were higher in patients at low to moderate risk (P<0.01); The high-risk NVAF patients showed significantly decreases in LVEF (P<0.01), with significantly increases in LAD, RAD, IVST, LVPWT, E/E'and PCWP (P<0.01) compared with healthy controls; Compared with the NVAF patients at low to moderate risk for stroke, the high-risk patients showed significantly decreases in LVEF (P<0.05), with increases in E/E'(P<0.05) and no differences in PCWP (P>0.05).
(2) The carotid ultrasonography results:The mean intima-media thickness (IMT) and plaque score increased significantly in the NVAF patients at low to moderate risk for stroke compared with healthy controls (P<0.01); Furthermore, the high-risk NVAF patients showed significantly increases in the mean IMT and plaque score than healthy controls (P<0.001); Compared with patients at low to moderate risk, the high-risk NVAF patients showed significantly increases in plaque score (P<0.05), with no differences in IMT (P>0.05).
5. Plasma markers of the healthy controls and NVAF patients at "low to moderate risk" or "high risk" for stroke
(1) Compared with healthy controls, oxLDL and8-iso-PGF2a (indexes of oxidative stress) were significantly higher in the NVAF patients at low to moderate risk for stroke (P<0.01); Meanwhile, oxLDL and8-iso-PGF2a were significantly higher in the high-risk NVAF patients compared with those at low to moderate risk and healthy controls (P<0.05).
(2) Compared with healthy controls, IL-6was significantly higher in the NVAF patients at low to moderate risk for stroke (P<0.05); Furthermore, IL-6was significantly higher in the high-risk NVAF patients compared with those at low to moderate risk and healthy controls (P<0.001).
(3) Compared with healthy controls, the levels of AGEs (markers of glucose metabolic disorders) was significantly higher in the NVAF patients at low to moderate risk for stroke (P<0.001); Furthermore, AGEs was significantly higher in the high-risk NVAF patients compared with those at low to moderate risk and healthy controls (P<0.01).
6. Microvesicles of the healthy controls and NVAF patients at "low to moderate risk" or "high risk" for stroke
Compared with healthy controls, PMVs and Annexin V-positive PMVs were significantly higher in the NVAF patients at low to moderate risk for stroke (P<0.01); PMVs and Annexin V-positive PMVs were significantly higher in the high-risk NVAF patients compared with healthy controls (P<0.001); Furthermore, PMVs and Annexin V-positive PMVs were higher in the high-risk NVAF patients compared with those at low to moderate risk (P<0.01).
7. Platelet markers of the healthy controls and NVAF patients at "low to moderate risk" or "high risk" for stroke
(1) The expression of platelet CD36was significantly increased in the NVAF patients at low to moderate risk for stroke compared with healthy controls (P<0.001); Furthermore, mean fluorescent intensity (MFI) of platelet CD36was significantly increased in the high-risk NVAF patients compared with those at low to moderate risk and healthy controls (P<0.01).
(2) Platelet activation in patients was assessed by surface detection of GPⅡ b/Ⅲa (PAC-1binding) and degranulation of P-selectin, both of which enhanced significantly in the high-risk group compared with those in the low to moderate-risk group (P<0.05) and the healthy controls (P<0.001) although the patients in the high-risk group received more anti-platelet therapy. Furthermore, platelet GP Ⅱb/Ⅲa and plasma soluble P-selectin enhanced significantly in the NVAF patients at low to moderate risk compared with the healthy controls (P<0.001).
8. Correlations of the CHADS2scores and the platelet activation markers
The CHADS2scores was correlated with platelet GP Ⅱb/Ⅲa significantly (r=0.264, P<0.001); Meanwhile, the CHADS2scores was correlated with plasma soluble P-selectin significantly (r=0.448, P<0.001).
9. Correlations of the CHADS2scores and plasma levels of PMVs
The CHADS2scores was correlated with plasma levels of PMVs (r=0.213, P<0.001); Meanwhile, the CHADS2scores was correlated with plasma levels of Annexin V-positive PMVs (r=0.449, P<0.001).
10. Correlations of the CHADS2scores and plasma markers
The CHADS2scores was correlated with plasma levels of8-iso-PGF2a significantly (r=0.353, P<0.001); the CHADS2scores was correlated with plasma levels of ox-LDL significantly (r=0.338, P<0.001); the CHADS2scores was correlated with IL-6significantly (r=0.410, P<0.001); the CHADS2scores was correlated with AGEs significantly (r=0.511, P<0.001).
11. Correlations of PMVs and the plasma markers
PMVs was associated with8-iso-PGF2a significantly (r=0.320, P<0.001); PMVs was associated with IL-6significantly (r=0.150, P=0.027); PMVs was associated with AGEs significantly (r=0.226, P=0.001).
12. Correlations of Annexin V-positive PMVs and the plasma markers
Annexin V-positive PMVs was associated with8-iso-PGF2a significantly (r=0.228, P=0.004); Annexin V-positive PMVs was associated with oxLDL significantly (r=0.321, P<0.001); Annexin V-positive PMVs was associated with IL-6significantly (r=0.176, P=0.008); Annexin V-positive PMVs was associated with AGEs significantly (r=0.228, P=0.001).
13. Correlations of plasma soluble P-selectin and microvesicles
PMVs was correlated with plasma soluble P-selectin (index of platelet activation) significantly (r=0.184, P=0.007); Annexin V-positive PMVs was also correlated with plasma soluble P-selectin significantly (r=0.173, P=0.009). Those results indicated that PMVs and Annexin V-positive PMVs may be involved in the process of platelet activation in NVAF patients.14. Associations of platelet CD36and platelet activation markers
MFI of platelet CD36was correlated with platelet GPⅡb/Ⅲa significantly (r=0.296, P<0.001); Meanwhile, platelet CD36was correlated with plasma soluble P-selectin significantly (r=0.248> P<0.001). Moreover, multivariate linear regression showed that the CD36contributed to the activation of platelet activation (β=0.314, P=0.011for GP Ⅱb/Ⅲa and β=0.114, P=0.045for soluble P-selectin).
Conclusions
(1) Compared with controls and NVAF patients at low to moderate risk for stroke, the high-risk NVAF patients had higher levels of oxidative stress, inflammation and AGEs;
(2) Compared with controls and NVAF patients at low to moderate risk for stroke, the plasma levels of PMVs and Annexin V-positive PMVs were significantly enhanced in the high-risk NVAF patients;
(3) Compared with controls and NVAF patients at low to moderate risk for stroke, the high-risk NVAF patients had higher levels of platelet CD36and platelet activation;
(4) PMVs and Annexin V-positive PMVs were associated with markers of oxidative stress, inflammation and AGEs significantly, indicating that oxidative stress, inflammation and AGEs might be the important stimuli of production of PMVs in NVAF;
(5) PMVs and Annexin V-positive PMVs were associated with markers of platelet activation significantly, indicating that PMVs might be the important mediators of platelet activation mechanisms in NVAF.
(6) Platelet CD36was correlated with markers of platelet activation significantly, indicating that CD36might involved in the signal pathway of platelet activation in NVAF.
Background
Nonvalvular atrial fibrillation (NVAF) is a. major cause of stroke and thromboembolism. It is still controversial whether the prothrombotic state and thrombosis in NVAF due to the atrial arrhythmia alone or the coexisting pathological factors. The results of AFFIRM and RACE trials did not demonstrate any superiority of a rhythm control versus a rate control strategy on occurrence of ischemic stroke. So, the thrombosis is independent of AF itself. The underlying pathological factors, such as oxidative stress and inflammation are speculated be the principal culprit of platelet activation in NVAF.
The upstream diseases of NVAF, including coronary heart diseases, hypertension, diabetes mellitus, could enhance the oxidative stress and inflammatory state. The process of non-enzymatic glycation of proteins and lipids is markedly accelerated in the setting of inflammation and oxidative stress. The resulting new products are defined as advanced glycation endproducts (AGEs), which mediate the atrial fibrosis and contribute to the pathogenesis of NVAF. We have demonstrated that, the plasma levels of oxidative stress, inflammation and AGEs enhanced significantly in NVAF patients at high risk for stroke compared with those at low to moderate risk. So, oxidative stress, inflammation and accumulated AGEs might not only result in occurrence of NVAF, but also be linked to thrombogenesis.
Platelets contribute to thrombosis by assembling into aggregates and by stimulating blood coagulation. Activated platelets play a well established role in thrombosis of NVAF. How the fundamental disorders of NVAF, such as oxidative stress, inflammation and accumulated AGEs, drive the prothrombotic state in atrial fibrillation? Recently, studies of microvesicles (MVs) have enlightened us.
MVs are membranous submicrometer vesicles formed by activated or apoptotic cells. They carry abundant signaling proteins and participate in multiple physiopathologic processes. The most MVs in circulation are platelet-derived microvesicles (PMVs), which are the main culprit in the development of thrombosis. The plasma levels of PMVs and Annexin V-positive PMVs enhanced significantly in NVAF patients at high risk for stroke compared with those at low to moderate risk. Thus, MVs may be the vectors bearing multiple pathogenic mechanisms of platelet activation. To find the source of platelet activation in NVAF, we can study the potential mechanism of the prothrombotic state and explore new, effective targets for preventing thromboembolism.
CD36, a multifunctional membrane receptor expressed on platelets, monocytes and several other cells, takes part in multiple physiopathological processes by engaging with multiple ligands. Platelet CD36could recognize and capture PS on the surface of PMVs, thus generating a PMV-CD36complex. The PMV-CD36complex was speculated be the key part of platelet activation. CD36can be a signaling molecule, but the detailed signal pathway of the PMV-CD36complex is unknown. Chen et al. showed that platelet CD36mediates mitogen-activated protein kinase (MAPK) activation induced by oxLDL. The modified lipids on oxLDL mediate its combination with CD36, and the surface of microvesicles contains multiple modified lipids (such as PS). Therefore, we speculated that the PMV-CD36complex could activate MAPKs, thereby activating platelets.
However, whether the PMVs mediate platelet activation induced by accompanying disorders, such as oxidative stress, inflammation and accumulated AGEs, in the context of NVAF is unclear. Thus, we speculated that the PMVs, generated in response to pathogenesis or accompanying disorders of NVAF, could be endogenous CD36ligands that transmit an activating signal to platelets to induce thrombosis.
Objectives
1. To determine the effects of oxidative stress, inflammation and accumulated AGEs on the production of PMVs.
2. To determine the effects of PMVs bearing signals of oxidative stress, inflammation and AGEs on the platelet activation and the involved signaling transduction.
3. To determine whether the signal pathway mediated by PMV-CD36complex could be a new effective target for preventing the prothrombotic state of NVAF.
Methods
1. Platelet isolation.
All the healthy volunteers had not taken any medication for2weeks. Fasting venous blood was collected in0.109mol/L sodium citrate (ratio1:9) under minimal tourniquet pressure using a sterile22-gauge needle. Platelet-rich plasma (PRP) was prepared by centrifugation at120g for10min at room temperature. Platelets were isolated from PRP after centrifugation at800g for10min in the presence of in the presence of100nmol/L prostaglandin E1. Then platelets were washed and resuspended in modified Tyrode's buffer (137mmol/L NaCl,2.7mmol/L KC1,12mmol/LNaHCO3,0.4mmol/L NaH2PO4,5mmol/LHEPES,0.1%glucose and0.35%bovine serum albumin, pH7.2) in the presence of100nmol/L PG-E1. Platelet concentration was adjusted to1×106/mL by use of a Z2particle counter, then used at once in all experiments.
2. The effects of oxidative stress, inflammation and AGEs on production of PMVs.
Resting platelets (1×106/mL) were incubated with oxidized LDL (oxLDL,50μg/mL), IL-6(1μg/mL) or AGEs (200μg/mL) for proper times at37℃respectively. The blank control (treated with buffer), isotype control and positive control (treated with10μmol/L adenosine diphosphate, ADP) were set for each group at the same time. Then the production of PMVs was detected by flow cytometry and a homemade ELISA.
3. Identification of PMVs by flow cytometry.
Immunofluorescent staining for PMVs:After gently blending,2.5μL platelet suspension (1×106/mL) was incubated with PEcy5-conjugated anti-CD41a antibody (5μL) in the dark for15min. Then the platelets were resuspended by1mL PBS and detected at once. Only cells and particles labeled with CD41a were gated. The PMV region was defined by FSC-SSC dot plot. The lower limit of the platelet gate was set at the left-hand border for resting platelets to distinguish between platelets and microvesicles. For each sample,10,000positive events in the platelet gate were acquired by use of FACS Calibur cytometer.
4. ELISA quantification of PMVs and Annexin V-positive PMVs.
After incubation with oxLDL, IL-6, AGEs or their control reagents, the platelet suspension was centrifuged at3,000g for10min. Then the supernatant was centrifuged again at13,000g for2min to avoid platelet contamination. Quantification of total PMVs and Annexin V-positive PMVs in the last supernatant was involved use of a homemade ELISA.
5. Collection of PMVs derived from platelets stimulated by oxLDL, IL-6or AGEs.
Platelets (1×106/mL) treated with oxLDL, IL-6or AGEs were sedimented at3000g for10min. An amount of2.0mmol/L phenylmethylsulfonyl fluoride was added to the PMV-enriched supernatants, which were then centrifuged at15,000g for1h at4℃. Then PMV pellets were washed twice (to avoid contamination of oxLDL, IL-6or AGEs) and resuspended in Modified Tyrode buffer and stored at-80℃. Freezing had no ad-verse effect on microvesicles. The protein content was measured by the Bradford Protein Assay Kit.
6. Double color flow cytometry of CD36expression.
For CD36quantification and CD36deficiency screening, the platelet suspension was incubated with5μL PEcy5-conjugated anti-CD41a antibody and5μL PE-conjugated anti-CD36antibody or isotype-matched control IgM in the dark for15 min. Then the platelets were resuspended by1mL PBS and detected by flow cytometry at once.
7. Double color flow cytometry of platelet GPⅡb/Ⅲa.
The platelet suspension (2.5μL) treated with PMVs (30μg/mL) was incubated with5μL PEcy5-conjugated anti-CD41a antibody and5μL FITC-conjugated PAC-1antibody (for activated platelet GPⅡb/Ⅲa) at room temperature in the dark for15min. Then the platelets were resuspended by1mL PBS and detected by flow cytometry at once.
8. Platelet aggregation studies.
PRP was obtained by centrifuging fasting venous blood at120g for10min at22℃, and platelet-poor plasma (PPP) was obtained by centrifuging PRP at3000g for10min. The platelet concentration of PRP was adjusted to2.5×108/mL by the addition of PPP. Aggregation was assessed by turbidimetry with a dual channel aggregometer (Chrono-log Corp., Havertown, PA, USA), with2μmol/L ADP used as an agonist. An amount of100%aggregation was defined as the light transmission of PPP, and0%was defined as the light transmission of PRP before the addition of agonists. Then the PRP treated with PMVs (30μg/mL) was stimulated with ADP (2μmol/L), and the change in light transmission was recorded.
9. Immunoassay for soluble P-selectin.
After the treatment described above, the platelet-PMV mixtures were centrifuged at3,000g for10min. Then the supernatant was centrifuged again at13,000g for2min to avoid platelet contamination. The last supernatant was used for detection of soluble P-selectin with use of commercially available immunoassay kits. The lower limit of sensitivity of the assay was0.5ng/mL.10. Western blot analysis.
Platelets treated with PMVs or controls were lysed in2mmol/L Tris-HCl (pH7.5),150mmol/L NaCl,1mmol/L EGTA,1mmol/L EDTA,1%Triton X-100,2.5mmol/L sodium pyrophos-phate,1mmol/L Na3VO4,1mmol/L phenylmethylsulfonyl fluoride and1μg/mL leupeptin, and protein concentrations were measured with use of a Bradford protein assay kit (Beyotime). Lysate protein (40μg) was separated on10% polyacrylamide gel and transferred onto polyvinylidene fluoride membranes. After a blocking for1h at room temperature in5%nonfat milk, the membranes were incubated with mouse anti-phospho-JNK1/2or rabbit anti-phospho-MKK4(1:1000dilution) overnight at4℃, then with HRP-conjugated secondary anti-mouse IgG or anti-rabbit IgG (1:4000dilution). The blots were developed with use of ECL detection reagent, then stripped and re-blotted with antibodies to native proteins for normalization.
11. Tanshinone IIA (TS IIA) treatment
TS IIA was obtained commercially (Xi'an Honson Biotechnology, China). Platelets were incubated with various concentrations of TS IIA (5μg/mL、10μg/mL20μg/mL、50μg/mL、100μg/mL) for15min before being stimulated with various PMVs(30μg/mL). Then the expression of platelet CD36and GPⅡ b/Ⅲa were detected by flow cytometry and the phosphorylation of MKK4/JNK was detected by western blot analysis.
Results
1. Platelet function studies with PMVs derived from oxLDL-treated platelets (oxLDL-PMVs)
(1) OxLDL treatment induced the release of PMVs and Annexin V-positive PMVs
Flow cytometry detected the formation of microvesicles by labeling with PEcy5-conjugated anti-CD41a antibody (M gates). The release of microvesicles increased after stimulation with oxLDL (50μg/mL) or ADP (10μmol/L), as quantified by CD41a-positive microparticles (P<0.05). For further confirmation, the supernatant of platelets was tested by ELISA. The amount of total PMVs and Annexin V-positive PMVs increased significantly after treatment with oxLDL or ADP (P<0.05), but not native LDL.
(2) OxLDLr-PMVs induced platelet activation
Next, we tested whether the PMVs collected from oxLDL-treated platelets could enhance platelet activation. We incubated resting platelets (1×106/mL) with oxLDL-PMVs (30μg/mL) for30min at22℃. Platelet activation is characterized by a conformation change in glycoprotein Ⅱ b/Ⅲa (GP Ⅱ b/Ⅲa). As expected, the MFI and percentage of PAC-1(recognizing the activated platelet GP Ⅱ b/Ⅲa) were enhanced significantly compared with the control (P<0.05). OxLDL-PMVs had almost the same effect as ADP. Furthermore, oxLDL-PMVs increased platelet aggregation in response to ADP.
(3) OxLDL-PMVs were ineffective in CD36-deficient platelets
We screened2CD36-deficient male donors in our laboratory. The CD36-deficient platelets were unable to bind PE-conjugated anti-CD36antibody. We incubated the CD36-deficient platelets (1×106/mL) with oxLDL-PMVs (30μg/mL) for30min at22℃. The PMV-enhanced platelet GP Ⅱ b/Ⅲa expression and platelet aggregation were absent.
(4) OxLDL-PMVs activated platelets in a CD36-and PS-dependent manner
To define the influence of CD36and PS on PMV-platelet interaction, we determined the expression of platelet GP Ⅱ b/Ⅲa and the secretion of P-selection. oxLDL-PMVs substantially increased the expression of GP Ⅱ b/Ⅲa and secretion of P-selectin (P<0.05). In all cases, the blocking of CD36(by a CD36neutralizing antibody) or PS (by Annexin V) could diminish the enhancement by oxLDL-PMVs (P<0.05), but IgG, an isotype-matched control of the CD36neutralizing antibody, had no effect. As well, the CD36neutralizing antibody decreased the binding of PE-conjugated anti-CD36antibody to platelets, with the expression of CD36for other groups not changed significantly.
(5) Platelet activation induced by oxLDL-PMVs was mediated by JNK signals
To elucidate the potential effect of JNK signaling in PMV-induced platelet activation, platelets were treated with the JNK inhibitor SP600125before incubation with PMVs. Pharmacological inhibition of JNK reduced platelet activation by PMVs, including expression of GP Ⅱ b/Ⅲa and secretion of P-selectin (P<0.05). Thus, JNK signaling may contribute to PMV-induced platelet activation.
(6) Phosphorylation of platelet JNK2and its upstream activator MKK4 induced by oxLDL-PMVs.
To confirm the role of the JNK pathway in PMV-induced activation of platelets, we compared the phosphorylation of JNK2in CD36-deficient and-positive platelets by immunoblotting. CD36-positive but not CD36-deficient platelets exposed to oxLDL-PMVs for30min showed a significant increase in phosphorylation of JNK2and its upstream activator MKK4(P<0.05). The JNK2phosphorylation was time and dose dependent, peaking at15min to approximately1.3-fold that of baseline with30μg/mL oxLDL-PMVs and with increased concentrations of oxLDL-PMVs inducing stronger phosphorylation of JNK2.
2. Platelet function studies with PMVs derived from IL-6-treated platelets (IL-6-PMVs)
(1) IL-6treatment promoted the formation of PMVs and Annexin Ⅴ-positive PMVs
The formation of PMVs was identified with labeling with PEcy5-conjugated anti-CD41a antibody (M gates). We observed an increase in total PMVs and Annexin V-positive PMVs released from IL-6treated compared to untreated platelets (P<0.05)
(2) IL-6-PMVs induced platelet activation
The MFI and percentage of PAC-1of the CD36-positive platelets were increased significantly stimulated by IL-6-PMVs, with similar effect as ADP (10μmol/L)(P <0.05). Furthermore, IL-6-PMVs increased platelet aggregation in response to ADP.
(3) IL-6-PMVs induced platelet activation in a CD36-and PS-dependent way
The PMV-enhanced platelet GPⅡ b/Ⅲa expression and platelet aggregation were absent when the CD36-deficient platelets (1×106/mL) were incubated with IL-6-PMVs (30μg/mL).
IL-6-PMVs substantially increased the expression of GP Ⅱb/Ⅲa and secretion of P-selectin (P<0.05). In all cases, the blocking of CD36(by a CD36neutralizing antibody) or PS (by Annexin V) could diminish the enhancement by IL-6-PMVs (P <0.05).
(4) Activation of platelets by IL-6-PMVs depends on MKK4/JNK pathway
Inhibition of JNK blocked the effect of IL-6-PMVs on platelet activation, including the expression of integrin αHbβ3and secretion of P-selectin (P<0.05).
CD36-positive but not-deficient platelets exposed to IL-6-PMVs for30min showed a significant increase in phosphorylation of JNK2and its upstream activator MKK4(P<0.05). The JNK2phosphorylation was time and dose dependent.
(5) Tanshinone ⅡA (TS Ⅱ A) treatment inhibits platelet activation induced by IL-6-PMVs To test whether the signal pathway mediated by IL-6-PMV/CD36complex could be a potential target for preventing the prothrombotic state of NVAF, we treated resting platelets with doses of TS ⅡA (5μg/mL、10μg/mL.20μg/mL、50μg/mL、100μg/mL) for15min before incubation with IL-6-PMVs. We found that TSⅡA dose-dependently inhibited the activation of platelets in vitro. At the same time, we detected the platelet CD36expression and found that TS ⅡA dose-dependently decreased the expression of platelet CD36. Furthermore, TS ⅡA could downregulate the phosphorylation of MKK4/JNK2activated by IL-6-PMVs.
3. Platelet function studies with PMVs derived from AGE-treated platelets (AGE-PMVs)
(1) AGEs treatment promoted the formation of PMVs and Annexin V-positive PMVs
We observed an increase in total PMVs and Annexin V-positive PMVs released from AGE (200μg/mL)-treated compared to bovine serum albumin (BSA,200μg/mL)-treated platelets or untreated platelets (P<0.05).
(2) Effect of AGE-PMVs on platelet activation
The MFI and percentage of platelet PAC-1were enhanced significantly after incubation with AGE-PMVs (P<0.05), which had similar effect as ADP. Furthermore, Surface exposure of CD40L (an index of platelet activation and assumed to originate from the platelet cytosol) increased after incubati
非瓣膜性心房颤动(房颤)是脑卒中及血栓栓塞事件强烈的独立危险因素。抗凝治疗是预防房颤卒中最为有效的手段,但仍然有33%-38%的卒中不能被防治,提示非瓣膜性房颤患者血栓形成的机制复杂,可能与房颤的基础病因相关。晚近,人们认为,血小板活化是高凝状态及血栓形成的始动因素。一系列凝血因子借助活化血小板的磷脂表面为反应平台,通过级联放大反应产生了数量庞大的凝血酶,最终形成血栓。
但是以抗血小板作为首选的治疗方案同样受到以往研究的挑战。与安慰剂相比,阿司匹林可使房颤患者血栓栓塞的相对危险降低21%,其效果显著低于华法林。其中最重要的原因之一就是与血小板激活途径的多样性有关。事实上非瓣膜性房颤中血小板被激活的机制尤其是被房颤基础病因激活的机制复杂,至今仍未完全阐明。
近期的研究强调了炎症、氧化应激以及代谢紊乱在非瓣膜性房颤血栓前状态形成中的潜在作用。上述研究分别提供了各自独立的证据,却无法将这些导致高凝状态的各种机制作为一个整体进行系统性研究,使治疗缺乏特异性。因此,寻找一种能够同时承载上述启动血小板活化的各种机制的载体,以此为工具深入研究房颤时血栓前状态的发生机制,将为探索房颤时血栓前状态综合防治的新的有效靶点提供依据。晚近有关microvesicles(微囊泡)的研究为我们提供了线索。
Microvesicles是由激活或凋亡的多种细胞通过分泌或脱落的形式释放到细胞外的小囊泡体。其中,与高凝状态和血栓形成关系最为密切的是血小板源的microvesicles,其在促凝的同时携带了多种血小板活化的信息。由此,我们推测,血小板来源的]microvesicles是房颤时传递血小板活化信息的重要载体,是启动凝血过程的关键环节。已有研究证实,microvesicles的强促凝活性主要依赖于其表达的PS(与Annexin V有很强的亲和力),而血小板膜糖蛋白CD36是PS的主要受体。CD36属于B类清道夫受体,是一种细胞膜表面单链糖蛋白,广泛存在于单核细胞、血小板、内皮细胞及脂肪细胞中。CD36可与多种配体结合,通过与不同配体的结合,在多种病理生理过程中发挥重要作用。因此,我们推测,在非瓣膜性房颤各组基础病因环境下,microvesicles是通过与血小板膜CD36结合途径活化血小板,并以级联放大的方式启动血栓前状态或高凝状态。迄今为止,国内外尚缺乏这方面的研究。因此,本课题拟采用病例对照研究,以正常对照组、非瓣膜性房颤脑卒中中低危组及非瓣膜性房颤脑卒中高危组的临床患者为研究对象,筛选出调控microvesicles特别是血小板源的microvesicles的各种危险因素；研究血小板源microvesicles和Annexin V阳性的microvesicles的表达及其与血小板活化指标的相关性；探讨血小板膜CD36的表达情况及其与microvesicles、血小板活化指标的相关性。
研究目的
1.非瓣膜性房颤状态下,筛选出调控血小板源microvesicles释放的各种危险因素；
2.非瓣膜性房颤患者血小板源microvesicles和Annexin V阳性的microvesicles的表达及其与房颤脑卒中风险的关系；
3.非瓣膜性房颤患者血小板膜糖蛋白CD36的表达水平及其与房颤脑卒中风险的关系；
4.探讨在非瓣膜性房颤状态下,血小板来源的microvesicles和Annexin V阳性的microvesicles的水平,血小板膜糖蛋白CD36的表达,血小板活化指标等各因素之间的相关性及内在联系。
研究对象与方法
选择阵发性或持续性非瓣膜性房颤患者210例进行病例—对照研究,平均年龄62.23±11.47岁,男性127例,女性83例。正常对照组35例,平均年龄55.71±7.43岁,男性17例,女性18例,均来自健康人群志愿者。对所有参与者进行体格检查,记录年龄、性别,测量身高、体重、腰围、臀围和血压等,并询问现病史、既往史、烟酒史、用药史和家族史等。部分参与者进行了超声心动图和颈动脉超声检查。所有参与者均经隔夜禁食12～14小时,次日清晨抽取空腹肘静脉血,测定血常规、葡萄糖(GLU)、总胆固醇(cholesterol, Cho)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-c)、低密度脂蛋白胆固醇(LDL-c)、尿酸(UA)等指标；采用ELISA法检测血浆中8-表氧-前列腺素F2a (8-iso-prostaglandinF2a)、氧化型低密度脂蛋白(ox-LDL)、白介素6(IL-6)、晚期糖基化终术产物(AGEs)及可溶性P选择素(P-selectin)的水平；固相免疫吸附法检测血小板源的microvesicles及Annexin V阳性的microvesicles;采用流式细胞术测定血小板血小板膜CD36和血小板糖蛋白(GP)Ⅱb/Ⅲa的表达。
结果
1.非瓣膜性房颤患者卒中风险分层：
所有入选的非瓣膜性房颤患者根据CHADS2卒中危险评分标准分为脑卒中高危组和中低危组。CHADS2评分参数包括：充血性心力衰竭、高血压、年龄>75岁、糖尿病、既往卒中或TIA史,前四项危险因素各1分,最后一项危险因素为2分。CHADS2评分总分为6分,0-1分为中低危组,2-6分为高危组。所有房颤患者中,113例(53.81%)属于卒中高危组(平均年龄：66.68±10.46岁,男性62例,女性51例),97例(46.19%)属于卒中中低危组(平均年龄：57.05±10.41岁,男性65例,女性32例)。
2.正常对照组、非瓣膜性房颤卒中中低危组及高危组临床特征的比较：
(1)正常对照组、非瓣膜性房颤脑卒中中低危组及高危组间性别、体重指数、舒张压、胆固醇、甘油三酯、LDL-C、HDL-C和血小板计数均无显著性差异(P>0.05)。
(2)正常对照组和非瓣膜性房颤脑卒中中低危组之间年龄、性别、体重指数、舒张压、空腹血糖、胆固醇、甘油三酯、LDL-C、HDL-C、尿酸、血肌酐和血小板计数差别均无统计学意义(P>0.05)；与对照相比,中低危组患者收缩压、腰臀比显著增高(P<0.01)。
(3)按照CHADS2卒中危险评分标准,非瓣膜性房颤卒中高危组除年龄较大外,其心衰、高血压、糖尿病及卒中史的发生率也远高于正常对照组和非瓣膜性房颤脑卒中中低危组(P<0.001)；此外,高危组患者的收缩压、空腹血糖、血肌酐、白细胞计数等指标较对照组和中低危组显著增高(P<0.01)；高危组患者腰臀比较对照组显著增高(P<0.001),较中低危组无显著性差异(P>0.05)。
3.正常对照组、非瓣膜性房颤卒中中低危组及高危组临床用药情况的比较：
与中低危组相比,非瓣膜性房颤卒中高危组华法林、钙通道阻滞剂、p受体阻断剂和它汀类药物的应用比例无显著差异(P>0.05),但是阿司匹林、ACEI/ARB的应用比例显著增高(P<0.01)。正常对照组未用任何上述心血管疾病治疗相关药物。
4.正常对照组、非瓣膜性房颤卒中中低危组及高危组超声指标的比较：
(1)超声心动图结果显示：非瓣膜性房颤卒中中低危组患者与正常对照组之间IVST、LVPWT及LVEF差别无统计学意义,但是中低危组患者LAD、RAD、E/E’及PCWP均有明显升高(P<0.01)；非瓣膜性房颤卒中高危组患者与正常对照组相比LVEF显著降低(P<0.01), LAD、RAD、IVST、LVPWT、E/E'及PCWP显著升高(P<0.01)；与中低危组相比,高危组LVEF降低(P<0.05)、E/E’升高(P<0.05), LAD、RAD、IVST、LVPWT及PCWP均无显著差异。
(2)颈动脉超声发现,非瓣膜性房颤卒中中低危组患者IMT及斑块指数较正常对照明显增高(P<0.01)；与正常对照相比,高危组患者IMT及斑块指数显著增高(P<0.001)；与中低危组相比,高危组患者IMT无显著差异,但是斑块指数显著增高(P<0.05)。
5.正常对照组、非瓣膜性房颤卒中中低危组及高危组氧化应激、炎症等指标的比较：
(1)非瓣膜性房颤卒中中低危组患者血浆中氧化应激指标oxLDL、8-iso-PGF2α较正常对照组显著升高(P<0.01)；房颤卒中高危组患者血浆oxLDL、8-iso-PGF2α水平较正常对照及中低危组房颤患者均明显升高(P<0.05)。
(2)房颤卒中中低危组患者血浆中IL-6水平较正常对照组明显升高(P<0.05)；高危组患者血浆IL-6水平较正常对照及中低危组均显著升高(P<0.001)。
(3)AGEs是糖代谢紊乱的产物及指标。房颤卒中中低危组患者血浆中AGEs水平较正常对照组明显升高(P<0.001)；高危组患者血浆AGEs水平较正常对照组及中低危组均显著升高(P<0.01)。
6.正常对照组、非瓣膜性房颤卒中中低危组及高危组microvesicles水平的比较：
与正常对照组相比,非瓣膜性房颤脑卒中中低危组患者血小板来源的microvesicles (PMVs)及Annexin V阳性的PMVs含量均增高(P<0.01)；与对照组相比,房颤卒中高危组患者PMVs及Annexin V阳性的PMVs含量显著增高(P<0.001)；此外,高危组患者PMVs及Annexin V阳性的PMVs含量较中低危组有所增高(P<0.01)。
7.正常对照组、非瓣膜性房颤卒中中低危组及高危组血小板相关指标的比较：
(1)与正常对照组相比,非瓣膜性房颤房颤卒中中低危组患者血小板膜蛋白CD36的表达显著增强(P<0.001)；房颤卒中高危组患者血小板膜蛋白CD36的表达较正常对照及中低危组明显增强(P<0.01)。
(2)血小板活化状态的检测包括流式细胞术测定血小板GPⅡb/Ⅲa的表达及ELISA测定血浆中可溶性P-selectin的水平。与对照组相比,中低危组患者血小板GPⅡb/Ⅲa的表达及可溶性P-selectin水平均显著增高(P<0.001)；高危组患者血小板GPⅡb/Ⅲa的表达及可溶性P-selectin水平较正常对照组(P<0.001)或中低危组患者均显著增高(P<0.05)。
8. CHADS2评分与血小板活化指标的相关分析
CHADS2评分与血小板活化指标GPⅡb/Ⅲa相关(r=0.264,P<0.001)；CHADS2评分与血小板可溶性P-selectin的分泌亦密切相关(r=0.448,P<0.001),提示非瓣膜性房颤患者脑卒中高风险与血小板激活密切相关。
9. CHADS2评分与microvesicles的相关分析
CHADS2评分与血浆中PMVs水平相关(r=0.213, P<0.001); CHADS2评分与Annexin V阳性的PMVs亦密切相关(r=0.449,P<0.001),提示血浆中PMVs,尤其是Annexin V阳性的PMVs有可能参与了房颤患者血栓前状态的发生。
10. CHADS2评分与氧化应激、炎症、糖代谢紊乱等指标的相关性分析
CHADS2评分与8-iso-PGF2α、ox-LDL(反映氧化应激指标)相关(r=0.353,P<0.001; r=0.338, P<0.001); CHADS2评分与炎症指标IL-6密切相关(r=0.410,P<0.001); CHADS2评分与AGEs(糖代谢紊乱相关指标)显著相关(r=0.511,P<0.001),提示氧化应激、炎症及糖代谢紊乱有可能是非瓣膜性房颤患者血栓前状态发生的危险因素。
11. PMVs与氧化应激、炎症、糖代谢紊乱等指标的相关性分析：
PMVs与8-iso-PGF2a显著相关(r=0.320, P<0.001); PMVs与IL-6显著相关(r=0.150,P=0.027); PMVs与AGEs相关(r=0.226,P=0.001)。
12. Annexin V阳性的PMVs与氧化应激、炎症、糖代谢紊乱等指标的相关性分析：
Annexin V阳性的PMVs与8-iso-PGF2a相关(r=0.228, P=0.004); Annexin V阳性的PMVs与ox-LDL显著相关(r=0.321, P<0.001); Annexin V阳性的PMVs与IL-6相关(r=0.176,P=0.008); Annexin V阳性的PMVs与AGEs相关(r=0.228,P=0.001)。
13.血浆中可溶性P-selectin与microvesicles的相关分析：
PMVs与可溶性P-selectin(反映血小板活化指标)显著相关(r=0.184, P=0.007); Annexin V阳性的PMVs与可溶性P-selectin相关(r=0.173,P=0.009),提示PMVs及Annexin V阳性的PMVs可能参与了非瓣膜性房颤状态下血小板的活化。
14.血小板CD36与血小板活化指标的相关分析：
血小板CD36的表达与血小板活化指标GPⅡ b/Ⅲa显著相关(r=0.296,P<0.001)；血小板CD36与可溶性P-selectin亦密切相关(r=0.248,P<0.001)。以年龄、收缩压、BMI、WHR、空腹血糖、胆固醇、血小板计数、血小板CD36表达为自变量,以血小板GPⅡb/Ⅲa为因变量,进行逐步回归多元线性回归分析,血小板CD36(β=0.314,P=0.011)和BMI (β=0.474,P<0.001)进入方程；以年龄、收缩压、BMI、WHR、空腹血糖、胆固醇、血小板计数、血小板CD36表达为自变量,以血浆可溶性P-selectin为因变量,进行多元线性回归分析,血小板CD36(β=0.114,P=0.045)、年龄(β=0.360,P=0.004)和WHR(β=0.501,P<0.001)进入方程。
结论：
(1)与正常对照组及脑卒中中低危组患者相比,非瓣膜性房颤卒中高危组具有较高的氧化应激、炎症及AGEs水平；
(2)与正常对照组及脑卒中中低危组患者相比,非瓣膜性房颤卒中高危组具有较高的PMVs及Annexin V阳性的PMVs水平；
(3)与正常对照组及脑卒中中低危组患者相比,非瓣膜性房颤卒中高危组具有较高的血小板膜CD36水平及血小板活化水平；
(4) PMVs及Annexin V阳性的PMVs与氧化应激、炎症、AGEs等指标密切相关,提示炎症、氧化应激及AGEs有可能是调控非瓣膜性房颤状态下PMVs释放的重要因素；
(5) PMVs及Annexin V阳性的PMVs分别与血小板活化指标密切相关,提示PMVs可能是传递血小板活化信息的重要载体；
(6)血小板膜CD36与血小板活化指标密切相关,提示血小板CD36作为信号分子有可能介导非瓣膜性房颤状态下血小板激活。
研究背景
非瓣膜性心房颤动(房颤)是脑卒中及血栓栓塞事件最强烈的独立危险因素。房颤时血栓前状态及血栓的形成是由房颤本身引起的还是由房颤伴随的基础病因引起的,目前仍存在争议。近年来相继揭晓的有关房颤治疗的临床研究(AFFIRM、RACE及AF/CHF)证实,“节律”控制治疗并不优于“室率”控制治疗,2种方法的卒中发生率无显著性差异。此外,孤立性房颤的卒中发生率不比正常人高。这提示我们,房颤时血栓前状态及血栓的形成并不完全依赖于房颤本身,推测可能是与房颤伴随的基础病因有关。
非瓣膜性房颤的上游疾病如冠心病、高血压、糖尿病、老龄化等使房颤患者处于较高的炎症、氧化应激水平。高水平的炎症、氧化应激状态可显著加速蛋白质、核酸或脂质等大分子物质的糖基化反应,得到的产物即晚期糖基化终产物(advanced glycation end products, AGEs)。AGEs的形成参与了房颤的发病机制,如动脉粥样硬化、心房纤维化等。我们在论文Ⅰ中证实,脑卒中高危的非瓣膜性房颤患者血浆中的氧化应激、炎症指标及AGEs水平远远高于中低危患者。综上,氧化应激、炎症及聚积的AGEs不仅参与了房颤的发生机制,而且很可能与房颤时血栓前状态及血栓的形成密切相关。
血小板粘集堆的形成是血栓形成的第一步,活化血小板的磷脂表面为凝血瀑布的激活及纤维蛋白的形成提供了一个反应平台。可见,血小板在非瓣膜性房颤血栓前状态及血栓形成中发挥着重要作用。房颤发生发展的基础病因,如氧化应激、炎症及聚积的AGEs是如何激活血小板导致血栓前状态的?至今仍未完全阐明。晚近有关microvesicles(微囊泡)的研究为我们提供了线索。
Microvesicles是细胞在活化或凋亡时从胞膜表面以生芽方式脱落的一些磷脂膜包裹的膜性小囊泡结构,其表面携带大量的信息蛋白等,参与多种病理生理过程。血小板源microvesicles (PMVs)是循环中微囊泡的主要来源,在血栓形成和止血的过程中发挥重要作用。脑卒中高危的非瓣膜性房颤患者血浆中的PMVs的总量及具有促凝活性的Annexin V阳性的PMVs的数量远远高于中低危患者。这提示我们,PMVs有可能是承载上述启动血小板活化的各种机制的载体,将有利于我们研究房颤时血栓前状态的发生机制,从而为探索房颤时高凝状态综合防治的新的有效靶点提供依据。
PMVs依靠何种途径传递血小板活化信息?PMVs的强促凝活性主要依赖于其表达的PS(与Annexin V有很强的亲和力),而血小板膜糖蛋白CD36是PS的主要候选受体之一。因此,PMV-CD36结合体可能是活化血小板的关键环节。CD36是一种多功能膜受体,广泛存在于血小板、单核细胞、内皮细胞及脂肪细胞中。它能够与多种配体结合,参与多种病理生理过程。PMV-CD36结合体如何启动血小板的活化过程?Chen等研究发现,MKK4/JNK2信号转导通路介导了ox-LDL与血小板表面CD36结合后激活血小板的过程。深入研究发现,CD36与oxLDL的结合是由与oxLDL上的脂质成分介导的。蛋白质组学证实microvesicles表面含有大量脂质成分。因此,PMV-CD36结合体活化血小板的信号转导途径与ox-LDL激活血小板的过程可能完全相同。由此我们推断,MKK4/JNK2信号转导通路同样介导了PMV-CD36结合体激活血小板的过程。
在非瓣膜性房颤状态下,PMVs能否介导房颤基础病因诱导的血小板激活,从而启动高凝状态尚不清楚。因此,我们提出如下假说：非瓣膜性房颤的基础病因,如氧化应激、炎症或聚集的AGEs,使体内PMVs总量增多,通过与血小板膜CD36结合,启动了PMV-CD36结合体介导的MKK4/JNK2信号转导途径活化血小板,从而参与房颤的血栓前状态或高凝状态的形成。
研究目的
1.氧化应激、炎症或聚积的AGEs对PMVs产生的影响；
2.携带氧化应激、炎症或AGEs刺激信息的PMVs对血小板活化的作用及信号转导机制；
3. PMV-CD36结合体及其介导的信号通路作为血栓前状态综合防治的新的有效靶点的可能性。
方法
1.正常人血小板的分离：健康献血志愿者均来自本实验室,近两周未服用任何抗血小板药物。志愿者隔夜禁食12～14小时,次日清晨在轻扎压脉带或不扎压脉带的情况下,抽取空腹肘静脉血20mL于0.109mol/L枸橼酸钠抗凝(1：9)的塑料采血管中。常温下,抗凝血静置10min后,经过120g离心10分钟后获得富含血小板血浆(PRP),后者加入100nmol/L PG-E1后经800g离心后得到血小板沉淀。血小板沉淀经改良台式液(137mmol/L NaCl,2.7mmol/L KCl,12mmol/L NaHCO3,0.4mmol/L NaH2PO4,5mmol/L HEPES,0.1%Glucose,0.35%BSA,100nmol/L PG-E1, pH7.2)洗涤、重悬。血小板悬液的浓度经细胞计数仪调整为1×106/mL,且立即应用于各项实验。
2.氧化应激、炎症及AGEs刺激对PMVs生成的影响：于37℃条件下,分别采用oxLDL (oxidized LDL,50μg/mL)、IL-6(1μg/mL)或AGEs (200μg/mL)孵育刺激洗涤血小板悬液(1×106/mL),同时各刺激组均设空白对照(等量缓冲液)、同型对照或阳性对照——二磷酸腺苷刺激(ADP,10μmol/L)。分别采用流式细胞术和固相免疫吸附法检测microvesicles的生成。
3.流式细胞术观察PMVs的释放：
PMVs免疫荧光标记：于BD流式管中先加入5μL PE-cyTM5标记抗人CD41a抗体,再加入2.5μL刺激后或未刺激的血小板悬液(1×106/mL),轻轻混匀后,于常温下避光孵育15min后,用1mL PBS悬浮细胞立即上机检测。
流式细胞仪测定及检测原理：开机校准流式细胞仪后,在Cell Quest软件环境下,将流式细胞仪的前向角散射(FSC),侧向角散射(SSC)及荧光(FL1-FL3)检测信号均设置为对数放大,以PE-cyTM5-CD41a阳性为获取条件,在CD41a vs. SSC点图中设门找出血小板群圈门,以血小板的位置定位设门找出PMVs群圈门,计数门内10,000个血小板,每管均在同一检测条件及程序设定下检测,数据分析采用Cell Quest软件。
4.固相免疫吸附法检测PMVs的生成
OxLDL、IL-6、AGEs及各组对照试剂孵育刺激后的血小板悬液,经过3,000g离心10min后,上清液再经12,000g离心3min以去除血小板的干扰,采用固相免疫吸附法检测最终上清液的PMVs,总量及Annexin V阳性的PMVs含量。具体步骤同论文Ⅰ。
5.氧化应激、炎症及AGEs刺激条件下PMVs的制备：分别将经过oxLDL (50μg/mL)、IL-6(1μg/mL)或AGEs (200μg/mL)孵育刺激后的血小板悬液(1×106/mL)3,000g离心10min后,取上清。上清中加入2.0mmol/L苯甲基黄酰化氟(PMSF)后,于4℃条件下15,000g高速离心60min,此时获得的microvesicles沉淀经过两次充分洗涤以去除oxLDL、IL-6或者AGEs的污染,采用BCA法检测提取的PMVs的蛋白浓度,-80℃保存备用。
6.双色流式细胞术检测血小板膜蛋白CD36的表达：在BD流式管内加入血小板悬液(2.5μL)与5μL小鼠抗人PEcy5-CD41a,5μL小鼠抗人PE-CD36轻轻混匀；同型对照管内加入等量样本及小鼠抗人PEcy5-CD41a,5μL小鼠抗人PE-IgM。样本与抗体在室温下避光孵育15min后,用1mL PBS重悬细胞,即刻上机分析。
7.双色流式细胞术检测血小板膜糖蛋白(GP)Ⅱb/Ⅲa:分别将步骤5中提取的各种来源的PMVs (30μg/mL)与洗涤血小板悬液(1×106/mL)于常温下孵育刺激30min。于BD流式管内加入处理后的血小板悬液(2.5μL)与5μL小鼠抗人PEcy5-CD41a,5μL FITC结合PAC-1抗体(能够识别与结合因血小板激活而异构的GPⅡb/Ⅲa),轻轻混匀后,室温下避光孵育15min后,用1mL PBS重悬细胞,即刻上机分析。
8.比浊法测定血小板聚集：将正常人空腹静脉血以120g离心10min,小心取出上层血浆为PRP,将取出PRP后的标本以3,000g离心10min,其上层较为透明的液体即为PFP。分别计数PRP与PFP中的血小板数,用PFP将PRP中的血小板数调节至25万/mm3。分别将步骤5中提取的各种来源的PMVs(30μg/mL)与PRP常温下孵育30min。取225μL的PFP于比浊管中作为空白对照,取等量的经PMVs刺激后的PRP于比浊管内,放入一磁棒,在37℃温育3min后,放入测定仪。用对照管内的PFP调零后,加入25μL ADP的同时开始记录,测定时间为5min,根据悬液透光度变化的程度及速度即可确定血小板聚集的程度与速度,并同时记录下血小板聚集曲线。
9.酶联免疫法测定细胞上清中可溶性P-selectin含量：分别将步骤5中提取的各种来源的PMVs (30μg/mL)与洗涤血小板悬液(1×106/mL)于常温下孵育刺激30min后,将PMV-血小板混合液3,000g离心10min后取上清,后者再次12,000g离心3min以去除血小板的污染。ELISA法检测最终细胞上清液中可溶性P-selectin含量,操作方法及步骤严格按照说明书的要求进行。
10. Western blot检测：裂解经各种来源PMVs刺激的血小板悬液(1×106/mL),提取蛋白,western blot检测CD36阳性或CD36阴性的血小板内磷酸化及非磷酸化的MKK4和JNK的表达量。
11.丹参酮ⅡA对PMVs活化血小板的干预试验：在加入各种刺激来源的PMVs (30μg/mL)前,给予洗涤血小板(1×106/mL)不同浓度的丹参酮ⅡA(5μg/mL、10μg/mL、20μg/mL、50μg/mL、100μg/mL)预处理15min,然后采用流式细胞术检测处理后的血小板表面GPⅡb/Ⅲa及CD36的表达；采用western blot的方法检测MKK4/JNK信号分子的磷酸化水平。
研究结果
1. PMVs介导氧化应激对血小板功能影响的研究
(1) OxLDL能够促进PMVs及Annexin V阳性的PMVs的释放
OxLDL (50μg/mL)及经典的血小板激活剂ADP(10μmol/L)在与洗涤血小板(1×106/mL)作用15min后,均能显著促进PMVs的释放,表现为M门内PE-cyTM5-CD41a阳性的微粒数显著增多(P<0.05),但是,天然的未经氧化的低密度脂蛋白(nLDL)却对PMVs的生成基本无影响。ELISA结果显示,与对照组及nLDL(?)相比,oxLDL能够显著促进PMVs总量及Annexin V阳性的PMVs的释放,其作用可与ADP相比(P<0.05)。
(2) OxLDL依赖的PMVs对血小板活性的影响
为了验证oxLDL刺激产生的PMVs (oxLDL-PMVs)对血小板活性的影响,我们将洗涤血小板(1×106/mL)与oxLDL-PMVs(30μg/mL)于常温下孵育30min。血小板激活以血小板膜糖蛋白Ⅱb/Ⅲa (GPⅡb/Ⅲa)的空间构象改变为特点,从而显露出纤维蛋白原等粘附分子的识别与结合部位,由此导致血小板聚集。抗体PAC-1能够识别并结合血小板因激活而异构的GPⅡb/Ⅲa,是检测血小板活化最常用的指标之一。本实验发现,与对照组相比,oxLDL-PMVs能够显著促进血小板PAC-1的平均荧光强度(MFI)增强(P<0.05), PAC-1阳性的血小板百分率增加(P<0.05)；此外,oxLDL-PMVs能够明显促进ADP诱导下的血小板的聚集。
(3) OxLDL-PMVs对CD36阴性的血小板活性的影响
OxLDL-PMVs (30μg/mL)与CD36阴性的血小板(1×106/mL)在常温下相互作用30min,血小板PAC-1的MFI及阳性百分率都未见明显变化；血小板聚集率未见明显增强。
(4) OxLDL-PMVs诱导的血小板活化呈CD36、磷脂酰丝氨酸(PS)依赖性为了进一步验证PMVs表面的PS及血小板表面的CD36对PMV-血小板相互作用的影响,在oxLDL-PMVs与血小板相互作用之前,我们分别采用CD36中和抗体预处理洗涤血小板和Annexin V预处理oxLDL-PMVs,并检测了血小板活化指标——GP Ⅱb/Ⅲ a的表达及可溶性P-selectin的分泌。结果发现,oxLDL-PMVs (30μg/mL)能够显著增加CD36阳性血小板GPⅡb/Ⅲa的表达及可溶性P-selectin的分泌(P<0.05)；阻断血小板膜CD36后,血小板活化指标均明显降低(P<0.05),但是相同条件下经CD36中和抗体的同型对照IgG预处理却不能抑制oxLDL-PMVs对血小板的活化作用；采用Annexin V阻断PMVs表面的PS同样能够抑制oxLDL-PMVs诱导的血小板活化(P<0.05)。同时,我们检测了经过上述各种处理后血小板表面CD36的表达情况,发现中和CD36后血小板膜CD36表达有所降低,其它各组并无显著变化。
(5) OxLDL-PMVs诱导血小板的活化呈JNK信号通路依赖性为了明确JNK信号传导通路对oxLDL-PMVs诱导血小板的活化的潜在作用,我们用JNK抑制剂SP600125预处理血小板后加入oxLDL-PMVs刺激。结果显示,阻断JNK信号通路能够显著抑制oxLDL-PMVs对血小板的激活作用,包括GPⅡb/Ⅲa的表达及可溶性P-selectin的分泌(P<0.05)；经SP600125预处理后,血小板表面的CD36表达并无显著变化。
(6) OxLDL-PMVs能够上调血小板JNK2及其上游MKK4的磷酸化水平对于CD36阳性血小板,oxLDL-PMVs刺激能够显著促进JNK2及其上游信号分子MKK4的磷酸化水平升高(P<0.05)；当oxLDL-PMVs (30μg/mL)分别作用Omin、5min、15min、30min时,JNK2磷酸化水平呈时间依赖性提高；分别用不同浓度(0.μg/mL、10μg/mL、30μg/mL、50μg/mL) oxLDL-PMVs作用于CD36阳性血小板30min,发现随着oxLDL-PMVs浓度的升高,JNK2磷酸化水平呈剂量依赖性提高。但是对于CD36阴性的血小板,oxLDL-PMVs刺激对MKK4/JNK2的磷酸化水平基本无影响。
2. PMVs介导炎症对血小板功能影响的研究
(1)IL-6刺激能够促进PMVs及Annexin V阳性的PMVs的释放
与对照组相比,IL-6能够显著促进PMVs总量及Annexin V阳性的PMVs的生成,其作用可与血小板激活剂ADP相比(P<0.05)。
(2)IL-6依赖的PMVs(IL-6-PMVs)对血小板活性的影响
与对照组相比,IL-6-PMVs刺激组血小板PAC-1的MFI及阳性百分率均显著增加(P<0.05)；此外,IL-6-PMVs能够明显促进ADP诱导下的血小板的聚集。
(3) IL-6-PMVs诱导血小板的活化呈CD36、PS依赖性
IL-6-PMVs (30μg/mL)与CD36阴性血小板(1×106/mL)在常温下相互作用30min后,血小板PAC-1的MFI及阳性百分率都未见明显变化；ADP诱导的.血小板聚集率未见明显增强。
IL-6-PMVs能够显著增加CD36阳性血小板GPⅡ b/Ⅲa的表达及可溶性P-selectin的分泌(P<0.05)；阻断血小板膜CD36后,血小板活化指标均明显降低(P<0.05)；采用Annexin V阻断PMVs表面的PS同样能够抑制IL-6-PMVs诱导的血小板活化(P<0.05)。
(4) MKK4/JNK2信号通路对IL-6-PMVs诱导血小板活化的影响
阻断JNK信号通路能够显著抑制IL-6-PMVs对血小板的激活作用,包括GP Ⅱb/Ⅲa的表达及可溶性P-selectin的分泌(P<0.05)。
Western blot结果显示,对于CD36阳性血小板,IL-6-PMVs (30μg/mL)能够显著上调JNK2及其上游信号分子MKK4的磷酸化水平(P<0.05)；当IL-6-PMVs (30μg/mL)作用0min、5min、15min、30min, JNK2磷酸化水平呈时间依赖性提高；分别用不同浓度(Oμg/mL、10μg/mL、30μg/mL、50μg/mL) IL-6-PMVs作用CD36阳性血小板30min,发现随着IL-6-PMVs浓度的升高,JNK2磷酸化水平呈剂量依赖性提高。但是对于CD36阴性的血小板,IL-6-PMVs刺激对MKK4/JNK2的磷酸化水平基本无影响。
(5)丹参酮ⅡA能够抑制IL-6-PMVs诱导的血小板活化
为了验证血小板PMV-CD36结合体及其介导的信号通路能否成为高水平炎症状态下血小板过度激活的干预靶点,我们进一步探讨其被药物干预的可能性。在加入IL-6-PMVs(30μg/mL)刺激前给予血小板不同浓度的丹参酮ⅡA(5μg/mL、10μg/mL、20μg/mL、50μg/mL、100μg/mL)预处理15min。结果发现,丹参酮ⅡA在体外能够呈剂量依赖性的抑制IL-6-PMVs诱导的血小板活化：5μg/mL丹参酮ⅡA使血小板PAC-1百分率降低,但效果不显著；10μg/mL丹参酮ⅡA对血小板活化指标的抑制作用达到统计学意义(P<0.05)；随着丹参酮ⅡA浓度的增加,其对血小板活化的抑制作用也逐渐增强。同时,我们检测了丹参酮ⅡA对血小板膜CD36表达的影响。结果显示：丹参酮ⅡA在体外能够呈剂量依赖性的抑制血小板CD36的表达；当丹参酮ⅡA浓度达到20μg/mL时,其抑制作用达到统计学意义(P<0.05)；随着丹参酮ⅡA浓度的增加,其对血小板CD36表达的抑制作用也逐渐增强。Western blot结果显示,丹参酮ⅡA能够有效下调IL-6-PMVs诱导的JNK2及其上游信号分子MKK4磷酸化水平(P<0.05)。
3. PMVs介导AGEs对血小板功能影响的研究
(1) AGEs刺激能够促进PMVs及Annexin V阳性的PMVs的释放
AGEs (200μg/mL)及ADP (10μmol/mL)刺激组M门内的PMVs数量显著增多,BSA (200μg/mL)刺激组较对照组未见明显变化。定量分析流式细胞术和ELISA的结果显示：与对照组及BSA处理组相比,AGEs能够显著促进PMVs总量及Annexin V阳性的PMVs的生成(P<0.05),其作用可与血小板激活剂ADP相比。
(2) AGEs依赖的PMVs (AGE-PMVs)对血小板活性的影响
与对照组相比,AGE-PMVs刺激组及ADP刺激组的血小板被明显活化,表现为GPⅡb/Ⅲa的MFI及阳性百分率均明显升高(P<0.05)；此外,AGE-PMVs能够显著增强ADP诱导的血小板的聚集。
(3) AGE-PMVs诱导血小板的活化呈CD36、PS依赖性
AGE-PMVs (30μg/mL与CD36阴性血小板(1×106/mL)在常温下相互作用30min后,流式细胞术结果显示,血小板PAC-1的MFI及阳性百分率都未见明显变化；ADP诱导的血小板聚集率未见明显增强。
AGE-PMVs能够显著增加CD36阳性的血小板GPⅡb/Ⅲa的表达及可溶性P-selectin的分泌(P<0.05)；阻断血小板膜CD36后,血小板活化指标均明显降低(P<0.05)；采用Annexin V阻断PMVs表面的PS同样能够抑制AGE-PMVs诱导的血小板活化(P<0.05)。
(4) MKK4/JNK2信号通路对AGE-PMVs诱导的血小板活化的影响
阻断JNK信号通路能够显著抑制AGE-PMVs对血小板的激活作用,包括GP Ⅱ b/Ⅲa的表达及可溶性P-selectin的分泌(P<0.05)。
Western blot结果显示,对于CD36阳性血小板,AGE-PMVs (30μg/mL)能够显著上调JNK2及其上游信号分子MKK4的磷酸化水平(P<0.05)；当AGE-PMVs (30μg/mL)分别作用0min、5min、15min、30min时,JNK2磷酸化水平呈时间依赖性提高,15min达到峰值水平；分别用不同浓度(Oμg/mL、10μng/mL)、30μg/mL、50μg/mL) AGE-PMVs作用CD36阳性血小板30min,发现随着AGE-PMVs浓度的升高,JNK2磷酸化水平呈剂量依赖性提高。但是对于CD36阴性的血小板,AGE-PMVs刺激对MKK4/JNK2的磷酸化水平基本无影响。
(5)丹参酮ⅡA能够抑制AGE-PMVs诱导的血小板活化
在加入AGE-PMVs (30μg/mL)刺激前,用不同浓度的丹参酮ⅡA (5μg/mL、10μg/mL、20μg/mL、50μg/mL)预处理洗涤血小板15min,然后检测血小板活化指标GPⅡb/Ⅲa。流式细胞术结果显示,丹参酮ⅡA在体外能够呈剂量依赖性的抑制AGE-PMVs诱导的血小板活化；当丹参酮ⅡA浓度达到10μg/mL时,其对血小板活化的抑制作用达到统计学意义(P<0.05)；随着丹参酮ⅡA浓度的增加,其对血小板活化的抑制作用也逐渐增强。同时,我们检测了丹参酮ⅡA对血小板膜CD36表达的影响。流式细胞术结果显示：丹参酮ⅡA在体外能够呈剂量依赖性的抑制血小板CD36的表达；当丹参酮ⅡA浓度达到20μg/mL时,其抑制作用达到统计学意义(P<0.05)；随着丹参酮ⅡA浓度的增加,其对血小板CD36表达的抑制作用也逐渐增强。Western blot结果显示,丹参酮ⅡA和SP600125(JNK抑制剂,阳性对照)均能够显著下调AGE-PMVs诱导的JNK2磷酸化水平升高(P<0.05)；丹参酮ⅡA还能够有效抑制AGE-PMVs诱导的MKK4的磷酸化水平(P<0.05)。
结论
(1)OxLDL、IL-6及AGEs刺激均能够促进PMVs的释放；
(2)各种刺激来源的PMVs均能够促进CD36阳性的血小板活化,但对CD36阴性的血小板几乎无影响；
(3)各种刺激来源的PMVs对血小板的活化作用呈CD36、PS依赖性；
(4) MKK4/JNK2信号转导通路参与各种刺激来源的PMVs对血小板的激活；
(5)丹参酮ⅡA能够抑制各种刺激来源的PMVs对血小板的激活；
(6)丹参酮ⅡA对PMVs作用的抑制是通过对CD36/MKK4/JNK2信号通路的抑制而发挥作用的,提示PMV-CD36及其介导的信号通路可以作为高氧化应激、高炎症及AGEs刺激条件下防治血小板过度激活的有效靶点。
研究背景
非瓣膜性心房颤动(房颤)是最常见的快速性心律失常,导致房颤患者致死、致残的最主要原因是血栓栓塞所致的脑卒中。血小板活化在血栓形成中起着非常重要的作用。脑卒中高危的非瓣膜性房颤患者血小板活化水平远远高于中低危患者。但是,非瓣膜性房颤中血小板被激活的机制复杂,至今仍未完全阐明。
药物维持窦性心律和控制心室率的循证医学(AFFIRM、RACE及AF/CHF)结果表明,“节律”控制治疗并不优于“心率”控制治疗,2种方法在卒中发生率方面并无显著性差异。这提示我们,房颤时血栓前状态及血栓的形成并不完全依赖于房颤本身,推测可能是与房颤伴随的基础病因有关。非瓣膜性房颤的上游疾病如冠心病、高血压、糖尿病、老龄化等使房颤患者处于较高的炎症、氧化应激及AGEs水平。脑卒中高危的非瓣膜性房颤患者血浆中的氧化应激、炎症指标及AGEs远远高于中低危患者。越来越多的研究表明,炎症、氧化应激及AGEs参与了非瓣膜性房颤高凝状态的形成。上述研究分别提供了各自独立的证据,却无法将这些导致高凝状态的各种机制作为一个整体进行系统性研究,使治疗缺乏特异性。因此,寻找一种能够同时承载上述启动血小板活化的各种机制的载体,以此为工具深入研究房颤时血栓前状态的发生机制,将为探索房颤时高凝状态综合防治的新的有效靶点提供依据。
Microvesicles (MVs)能够作为介导氧化应激、炎症或聚积的AGEs活化血小板的重要载体。炎症、氧化应激及聚积的AGEs是房颤发生、心房纤维化及血栓前状态形成的重要机制已被众多学者肯定。我们有理由推测,房颤患者循环中的microvesicles是氧化应激、炎症及AGEs综合作用的产物,同时携带血小板活化的各种信息,是深入研究房颤时血栓前状态的发生机制的重要载体。我们在论文Ⅱ中证实,在氧化应激、炎症或AGEs条件下,MKK4/JNK2信号转导途径介导了PMVs与血小板膜CD36结合后激活血小板的过程,但是这条通路是否介导了房颤条件下microvesicles激活血小板的过程未见报道。
据此,我们提出如下假说：非瓣膜性房颤状态下,microvesicles是综合诸多基础病因(氧化应激、炎症及聚集的AGEs等)的重要载体,通过与血小板膜CD36结合,启动了MV-CD36结合体介导MKK4/JNK2信号转导途径活化血小板,随之分泌更多Annexin-V阳性的MVs,进一步与更多的血小板CD36结合,通过正反馈机制,大量活化血小板,启动房颤的血栓前状态或高凝状态,进而导致血栓形成及血栓栓塞事件。
研究目的1.非瓣膜性房颤环境中microvesicles对血小板活化的作用及信号转导机制；
2. MV-CD36结合体及其介导的信号通路作为血栓前状态综合防治的新的有效靶点的可能性。
方法
1.正常人血小板的分离：健康献血志愿者未服用过任何抗血小板药物,隔夜禁食12～14小时,次日清晨在轻扎压脉带或不扎压脉带的情况下,抽取空腹肘静脉血20mL于0.109mol/L枸橼酸钠抗凝(1：9)的塑料采血管中。常温下,抗凝血静置10min后,经过120g离心10分钟后获得富含血小板血浆(PRP),后者加入100nmol/LPG-E1后经800g离心后得到血小板沉淀。血小板沉淀经改良台式液(137mmol/L NaCl,2.7mmol/L KC1,12mmol/L NaHCO3,0.4mmol/L NaH2PO4,5mmol/L HEPES,0.1%Glucose,0.35%BSA,100nmol/L PG-E1, pH7.2)洗涤、重悬。血小板悬液的浓度经细胞计数仪调整为1×106/mL,且立即应用于各项实验。
2.流式细胞术检测非瓣膜性房颤病人乏血小板血浆(PFP)中MVs的含量及性质：取100μL脑卒中高危房颤患者(CHADS2评分≥2)的PFP与10μL小鼠抗人PE-cyTM5-CD41a,10μL FITC-Annexin V,100μL缓冲液(10mM Hepes/NaOH,140mM NaCl,2.5mM CaCl2, PH7.4)于BD流式管内轻轻混匀,室温下避光孵育15min后,即刻上机分析。
3.高速梯度离心法分离房颤患者血浆中的MVs：根据CHADS2评分选取非瓣膜性房颤脑卒中高危患者及年龄、性别相匹配的中低危患者,抽取3.8%枸橼酸钠抗凝空腹静脉血,经3,000g离心20分钟后,得PFP。PFP中加入PMSF后,于4℃条件下15,000g高速离心60min,将获得的MVs沉淀经过两次充分洗涤后-80℃保存备用。
4.双色流式细胞术检测血小板膜蛋白CD36的表达：在BD流式管内加入血小板悬液(2.5μL)与5μL小鼠抗人PEcy5-CD41a,5μL小鼠抗人PE-CD36或其同型对照小鼠抗人PE-IgM,轻轻混匀,在室温下避光孵育15min后,用1mL PBS重悬细胞,即刻上机分析。
5.流式细胞术检测血小板膜糖蛋白(GP)Ⅱb/Ⅲa:洗涤血小板悬液(1×106/mL)与MVs (30μg/mL)在常温下孵育30min。于BD流式管内加入处理后的血小板悬液(2.5μL)与5gL小鼠抗人PEcy5-CD41a,5μL小鼠抗人FITC-PAC-1(能够识别与结合因血小板激活而异构的GPⅡb/Ⅲa)轻轻混匀后,室温下避光孵育15min后,用1mL PBS重悬细胞,即刻上机分析。
6.双色流式细胞术检测血小板膜CD40L的表达：洗涤血小板悬液(1x106/mL)与MVs(30μg/mL)在常温下孵育30min。于BD流式管内加入处理后的血小板悬液(2.5μL)与5μL小鼠抗人PEcy5-CD41a,5μL小鼠抗人PE-CD40L,轻轻混匀后,室温下避光孵育15min后,用1mL PBS重悬细胞,即刻上机分析。
7.酶联免疫法测定细胞上清中可溶性P-selectin含量：将MVs (30μg/mL)与洗涤血小板悬液(1×106/mL)于常温下孵育刺激30min后,MV-血小板混合液3,000g离心10min后取上清,后者再次12,000g离心3min避免血小板的干扰。ELISA法检测最终细胞上清液中可溶性P-selectin含量,操作方法及步骤严格按照说明书的要求进行。
8. Western blot检测：裂解经MVs刺激的血小板悬液(1×106/mL),提取蛋白,western blot检测CD36阳性或CD36阴性的血小板内磷酸化及非磷酸化的MKK4和JNK的表达量。具体实验步骤同论文Ⅱ。
9.丹参酮ⅡA对MVs活化血小板的干预试验：在加入MVs(30μg/mL)刺激前,给予洗涤血小板(1×106/mL)不同浓度的丹参酮ⅡA (5μg/mL、20μg/mL、50μg/mL、100μg/mL)预处理15min,然后采用流式细胞术检测处理后的血小板表面GPⅡb/Ⅲa及CD36的表达；采用western blot的方法检测MKK4/JNK信号分子的磷酸化水平。
研究结果
(1)房颤病人来源的MVs对血小板活性的影响
我们采用高速离心的方法从非瓣膜性房颤脑卒中高危患者乏血小板血浆中分离出MVs(AF-MVs),同时从年龄、性别均与高危患者匹配的非瓣膜性房颤脑卒中低危患者乏血小板血浆中分离出MVs作为对照(C-MVs)。流式细胞术分析结果发现,AF-MVs大多为CD41a阳性、Annexin V阳性,即大部分MVs来源于血小板,且具有很强的促凝活性。
与对照组及C-MVs刺激组相比,AF-PMVs刺激组血小板PAC-1的平均荧光强度(MFI)及阳性百分率均显著增加(P<0.05)；此外,AF-MVs能够明显促进血小板表面CD40L的表达(P<0.05)；与对照组相比,C-MVs作用后的血小板PAC-1的MFI及百分率,血小板CD40L表达一定程度上有所增加,但强度较弱。
(2) AF-MVs诱导血小板的活化呈CD36、PS依赖性
AF-MVs(30μg/mL)和C-MVs(30μg/mL)分别与CD36阴性血小板(1×106/mL)在常温下相互作用30min后,流式细胞术结果显示,血小板PAC-1的MFI及阳性百分率都未见明显变化；此外,血小板表面CD40L的表达未见明显增强。
AF-MVs能够显著增加CD36阳性血小板GP Ⅱ b/Ⅲa的表达及可溶性P-selectin的分泌(P<0.05)；阻断血小板膜CD36后,血小板活化指标均明显降低(P<0.05)；采用Annexin V阻断PMVs表面的PS同样能够抑制AF-MVs诱导的血小板活化(P<0.05)。
(3) MKK4/JNK2信号通路对AF-MVs诱导的血小板活化的影响
阻断JNK信号通路能够显著抑制AF-MVs对血小板的激活作用,包括GPⅡ b/Ⅲa的表达及可溶性P-selectin的分泌(P<0.05)。
Western blot结果显示,AF-MVs (30μg/mL)能够显著上调CD36阳性血小板JNK2及其上游信号分子MKK4的磷酸化水平(P<0.05)；在AF-MVs (30μ/mL)作用Omin、5min、15min、30min,JNK2磷酸化水平呈时间依赖性提高,15min达到峰值水平；分别用不同浓度(Oμg/mL、10μg/mL、30μg/mL、50μg/mL) IL-6-PMVs作用CD36阳性血小板30min,发现随着AF-MVs浓度的升高,JNK2磷酸化水平呈剂量依赖性提高。但是对于CD36阴性的血小板,AF-MVs刺激对MKK4/JNK2的磷酸化水平基本无影响。
(4)丹参酮ⅡA干预对AF-MVs诱导的血小板活化的影响
为了验证血小板MV-CD36结合体及其介导的信号通路能否成为房颤时高凝状态综合防治的有效靶点,我们进一步探讨其被药物干预的可能性。我们分别用不同浓度的丹参酮ⅡA (5μg/mL、10μg/mL、20μg/mL、50μ/mL.100μg/mL)预处理血小板15min后加入AF-MVs (30μg/mL)刺激。结果发现,丹参酮ⅡA在体外能够呈剂量依赖性的抑制AF-MVs诱导的血小板活化：10M.g/mL丹参酮ⅡA使血小板PAC-1百分率降低,但效果不显著；10μg/mL丹参酮ⅡA对血小板活化指标的抑制作用达到统计学意义(P<0.05)；随着丹参酮ⅡA浓度的增加,其对血小板活化的抑制作用也逐渐增强。同时,我们检测了丹参酮ⅡA对血小板膜CD36表达的影响。结果显示：丹参酮ⅡA在体外能够呈剂量依赖性的抑制血小板CD36的表达；当丹参酮ⅡA浓度达到20μg/mL时,其抑制作用达到统计学意义(P＜0.05)；随着丹参酮ⅡA浓度的增加,其对血小板CD36表达的抑制作用也逐渐增强。Western blot结果显示,丹参酮ⅡA还能够抑制AF-MVs诱导的JNK2及其的上游信号分子MKK4的磷酸化水平。
结论
(1)非瓣膜性房颤病人循环中的MVs大部分来源于血小板,且具有很强的促凝活性；
(2) AF-MVs能够促进CD36阳性的血小板活化,但对CD36阴性的血小板无效；
(3) AF-MVs对血小板的活化作用呈CD36、PS依赖性；
(4) MKK4/JNK2信号转导通路参与AF-MVs对血小板的激活；
Background
Nonvalvular atrial fibrillation (NVAF) is a major cause of stroke and thromboembolism. Although anticoagulant therapy is recommended for patients with NVAF at high risk for stroke,33%-38%of patients receiving anticoagulant therapy still experience stroke. The mechanisms underlying thromboembolism in NVAF are still incompletely understood. The activation of platelets may initiate thrombosis. Abundant thrombin is generated as a result of a cascade of coagulation factors activated on the phospholipid surface of activated platelets, thereby leading to thrombosis.
However, antiplatelet therapy in NVAF patients has been challenged. Compared with placebo, with aspirin treatment, the risk of thromboembolism in patients with AF decreased by only21%, which was less effective than warfarin. Insufficiency of the present antiplatelet therapy may be associated with the diversity of platelet activation pathways. The molecular mechanisms that underlie platelet activation in NVAF are poorly defined but may involve the etiology of NVAF.
The potential effects of inflammation, oxidative stress and metabolic disorders in the NVAF-related prothrombotic state have aroused much attention. However, these risk factors have not been considered together in targeting prevention of NVAF-related platelet activation. To determine a vector exhibiting all the mechanisms in NVAF, we can study the potential mechanisms of platelet activation and explore new, effective targets for preventing thrombosis in NVAF. Recently, studies of microvesicles (MVs) have enlightened us.
MVs are submicrometer vesicles formed by activated cells. MVs released into the circulation can act as messengers delivering a variety of cargos, including cell surface receptors, proinflammatory cytokines, signature proteomes, and even mRNA, to target cells. Thus, MVs may be the vectors combining multiple pathogenic mechanisms of platelet activation. Platelet-derived microvesicles (PMVs), with surface exposure of phosphatidylserine (PS, with strong affinity for Annexin V), are the main culprit in the development of thrombosis. CD36, a class B scavenger receptor expressed on platelets, monocytes and several other cells. Its role in platelet function has remained obscure. Platelet CD36could recognize and capture PS on the surface of MVs, thus generating an MV-CD36complex. Accordingly, we speculated that the PMVs, generated in response to pathogenesis or accompanying disorders of NVAF, could be endogenous CD36ligands that transmit an activating signal to platelets to induce thrombosis. We investigated healthy controls and the NVAF patients with "low to moderate risk" or "high risk" for stroke to determine the triggers for the release of platelet-derived microvesicles and Annexin V-positive microvesicles and to explore the potential relationship among microvesicles, platelet CD36and platelet activation.
Objectives
1. To screen for the risk factors which trigger the release of the microvesicles and platelet-derived microvesicles in patients with NVAF.
2. To determine the plasma levels of platelet-derived microvesicles and Annexin V-positive microvesicles in NVAF patients, as well as their relationship to stroke risk.
3. To determine the expression of platelet CD36and its relationship to stroke risk in NVAF patients.
4. To explore the relationship among platelet-derived microvesicles, Annexin V-positive microvesicles, platelet CD36and platelet activation indexes in patients with NVAF.
Subjects and methods
We included210patients with persistent or paroxysmal NVAF (mean age62.23±11.47years;127men) and35healthy controls (mean age55.71±7.43;17men). All subjects completed a questionnaire on age, gender, present condition, family and medical histories of cardiovascular risk factors and complications. Height, weight, body mass index (BMI), waist circumference, hip circumference and blood pressure (BP) were measured. Most subjects underwent2D transthoracic echocardiography and carotid ultrasonography evaluation. Furthermore, fasting blood sample was collected from each subject after12-14hours fast to determine the fasting blood glucose (FBG), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), creatinine (Cr) and uric acid (UA); Measurement of8-iso-prostaglandinF2α, interleukin6(IL-6), advanced glycosylation end products (AGEs) and soluble P-selectin involved ELISA commercially; The quantification of platelet-derived microvesicles and Annexin V-positive microvesicles in platelet free plasma (PFP) involved use of a homemade ELISA; The expression of platelet GPⅡ b/Ⅲa and CD36were detected by flow cytometry.
Results
1. Characteristics in NVAF patients:relationship to stroke risk
Patients were classified as being at "low to moderate risk" or "high risk" for stroke according to the CHADS2stroke risk scheme:1point each for presence of congestive heart failure, hypertension, age older than75or diabetes mellitus; and2points for history of stroke or transient ischemic attack. Patients with CHADS2score0or1were considered at "low to moderate risk" and those with score≥2at "high risk". Of the210NVAF patients,113(53.81%) were at high risk of stroke (mean age:66.68±10.46years,62males and51females),97(46.19%) were at low to moderate risk of stroke (mean age:57.05±10.41years,65males and32females).
2. Clinical characteristics of the healthy controls and NVAF patients at "low to moderate risk" or "high risk" for stroke
(1) There were no significant differences in sex, body mass index (BMI), diastolic blood pressure (DBP), cholesterol, triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c) and platelet counts among healthy controls, NVAF patients at "low to moderate risk" and NVAF patients at "high risk" for stroke (P>0.05).
(2) There were no significant differences in age, sex, BMI, DBP, fasting blood glucose, cholesterol, cholesterol, TG, LDL-c, HDL-c, UA, creatinine and platelet counts between healthy controls and NVAF patients at low to moderate risk for stroke (P>0.05). Systolic blood pressure (SBP) and waist-to-hip ratio (WHR) were significantly higher in the patients at low to moderate risk compared with healthy controls (P<0.01).
(3) By definition, the high-risk NVAF patients were older and more likely to have congestive heart failure, hypertension, diabetes mellitus, or history of stroke than those at low to moderate risk (P<0.001for all); Furthermore, SBP, fasting blood glucose, creatinine, white blood cell counts were significantly higher in the patients at low to moderate risk compared with healthy controls and the low to moderate-risk NVAF patients (P<0.01); There was no significant differences in WHR between the high-risk NVAF patients and the low to moderate-risk NVAF patients (P>0.05). WHR was higher in the high-risk NVAF patients compared with healthy controls (P <0.001).
3. Clinical medication of the healthy controls and NVAF patients at "low to moderate risk" or "high risk" for stroke
Compared with the NVAF patients at low to moderate risk for stroke, the application proportion of warfarin, calcium channel blockers, beta-receptor blockers and statins had no significant differences in the high-risk NVAF patients (P>0.05); However, the patients in the high-risk group received more anti-platelet therapy and ACEI/ARB compared with those with low to moderate risk (P<0.01); The healthy controls were free of any medication.
4. Comparison of ultrasonic parameters among the healthy controls and NVAF patients at "low to moderate risk" or "high risk" for stroke
(1) The transthoracic echocardiography results:There were no significant differences in thickness of inter-ventricular septum (IVST), thickness of posterior wall of left ventricle (LVPWT) and left ventricular ejection fraction (LVEF) between NVAF patients at low to moderate risk for stroke and healthy controls (P>0.05), but the diameter of left atrium (LAD), the diameter of left atrium (RAD), ratio of early transmitral flow velocity to early mitral annular diastolic velocity (E/E') and the pulmonary capillary wedge pressure (PCWP) were higher in patients at low to moderate risk (P<0.01); The high-risk NVAF patients showed significantly decreases in LVEF (P<0.01), with significantly increases in LAD, RAD, IVST, LVPWT, E/E'and PCWP (P<0.01) compared with healthy controls; Compared with the NVAF patients at low to moderate risk for stroke, the high-risk patients showed significantly decreases in LVEF (P<0.05), with increases in E/E'(P<0.05) and no differences in PCWP (P>0.05).
(2) The carotid ultrasonography results:The mean intima-media thickness (IMT) and plaque score increased significantly in the NVAF patients at low to moderate risk for stroke compared with healthy controls (P<0.01); Furthermore, the high-risk NVAF patients showed significantly increases in the mean IMT and plaque score than healthy controls (P<0.001); Compared with patients at low to moderate risk, the high-risk NVAF patients showed significantly increases in plaque score (P<0.05), with no differences in IMT (P>0.05).
5. Plasma markers of the healthy controls and NVAF patients at "low to moderate risk" or "high risk" for stroke
(1) Compared with healthy controls, oxLDL and8-iso-PGF2a (indexes of oxidative stress) were significantly higher in the NVAF patients at low to moderate risk for stroke (P<0.01); Meanwhile, oxLDL and8-iso-PGF2a were significantly higher in the high-risk NVAF patients compared with those at low to moderate risk and healthy controls (P<0.05).
(2) Compared with healthy controls, IL-6was significantly higher in the NVAF patients at low to moderate risk for stroke (P<0.05); Furthermore, IL-6was significantly higher in the high-risk NVAF patients compared with those at low to moderate risk and healthy controls (P<0.001).
(3) Compared with healthy controls, the levels of AGEs (markers of glucose metabolic disorders) was significantly higher in the NVAF patients at low to moderate risk for stroke (P<0.001); Furthermore, AGEs was significantly higher in the high-risk NVAF patients compared with those at low to moderate risk and healthy controls (P<0.01).
6. Microvesicles of the healthy controls and NVAF patients at "low to moderate risk" or "high risk" for stroke
Compared with healthy controls, PMVs and Annexin V-positive PMVs were significantly higher in the NVAF patients at low to moderate risk for stroke (P<0.01); PMVs and Annexin V-positive PMVs were significantly higher in the high-risk NVAF patients compared with healthy controls (P<0.001); Furthermore, PMVs and Annexin V-positive PMVs were higher in the high-risk NVAF patients compared with those at low to moderate risk (P<0.01).
7. Platelet markers of the healthy controls and NVAF patients at "low to moderate risk" or "high risk" for stroke
(1) The expression of platelet CD36was significantly increased in the NVAF patients at low to moderate risk for stroke compared with healthy controls (P<0.001); Furthermore, mean fluorescent intensity (MFI) of platelet CD36was significantly increased in the high-risk NVAF patients compared with those at low to moderate risk and healthy controls (P<0.01).
(2) Platelet activation in patients was assessed by surface detection of GPⅡ b/Ⅲa (PAC-1binding) and degranulation of P-selectin, both of which enhanced significantly in the high-risk group compared with those in the low to moderate-risk group (P<0.05) and the healthy controls (P<0.001) although the patients in the high-risk group received more anti-platelet therapy. Furthermore, platelet GP Ⅱb/Ⅲa and plasma soluble P-selectin enhanced significantly in the NVAF patients at low to moderate risk compared with the healthy controls (P<0.001).
8. Correlations of the CHADS2scores and the platelet activation markers
The CHADS2scores was correlated with platelet GP Ⅱb/Ⅲa significantly (r=0.264, P<0.001); Meanwhile, the CHADS2scores was correlated with plasma soluble P-selectin significantly (r=0.448, P<0.001).
9. Correlations of the CHADS2scores and plasma levels of PMVs
The CHADS2scores was correlated with plasma levels of PMVs (r=0.213, P<0.001); Meanwhile, the CHADS2scores was correlated with plasma levels of Annexin V-positive PMVs (r=0.449, P<0.001).
10. Correlations of the CHADS2scores and plasma markers
The CHADS2scores was correlated with plasma levels of8-iso-PGF2a significantly (r=0.353, P<0.001); the CHADS2scores was correlated with plasma levels of ox-LDL significantly (r=0.338, P<0.001); the CHADS2scores was correlated with IL-6significantly (r=0.410, P<0.001); the CHADS2scores was correlated with AGEs significantly (r=0.511, P<0.001).
11. Correlations of PMVs and the plasma markers
PMVs was associated with8-iso-PGF2a significantly (r=0.320, P<0.001); PMVs was associated with IL-6significantly (r=0.150, P=0.027); PMVs was associated with AGEs significantly (r=0.226, P=0.001).
12. Correlations of Annexin V-positive PMVs and the plasma markers
Annexin V-positive PMVs was associated with8-iso-PGF2a significantly (r=0.228, P=0.004); Annexin V-positive PMVs was associated with oxLDL significantly (r=0.321, P<0.001); Annexin V-positive PMVs was associated with IL-6significantly (r=0.176, P=0.008); Annexin V-positive PMVs was associated with AGEs significantly (r=0.228, P=0.001).
13. Correlations of plasma soluble P-selectin and microvesicles
PMVs was correlated with plasma soluble P-selectin (index of platelet activation) significantly (r=0.184, P=0.007); Annexin V-positive PMVs was also correlated with plasma soluble P-selectin significantly (r=0.173, P=0.009). Those results indicated that PMVs and Annexin V-positive PMVs may be involved in the process of platelet activation in NVAF patients.14. Associations of platelet CD36and platelet activation markers
MFI of platelet CD36was correlated with platelet GPⅡb/Ⅲa significantly (r=0.296, P<0.001); Meanwhile, platelet CD36was correlated with plasma soluble P-selectin significantly (r=0.248> P<0.001). Moreover, multivariate linear regression showed that the CD36contributed to the activation of platelet activation (β=0.314, P=0.011for GP Ⅱb/Ⅲa and β=0.114, P=0.045for soluble P-selectin).
Conclusions
(1) Compared with controls and NVAF patients at low to moderate risk for stroke, the high-risk NVAF patients had higher levels of oxidative stress, inflammation and AGEs;
(2) Compared with controls and NVAF patients at low to moderate risk for stroke, the plasma levels of PMVs and Annexin V-positive PMVs were significantly enhanced in the high-risk NVAF patients;
(3) Compared with controls and NVAF patients at low to moderate risk for stroke, the high-risk NVAF patients had higher levels of platelet CD36and platelet activation;
(4) PMVs and Annexin V-positive PMVs were associated with markers of oxidative stress, inflammation and AGEs significantly, indicating that oxidative stress, inflammation and AGEs might be the important stimuli of production of PMVs in NVAF;
(5) PMVs and Annexin V-positive PMVs were associated with markers of platelet activation significantly, indicating that PMVs might be the important mediators of platelet activation mechanisms in NVAF.
(6) Platelet CD36was correlated with markers of platelet activation significantly, indicating that CD36might involved in the signal pathway of platelet activation in NVAF.
Background
Nonvalvular atrial fibrillation (NVAF) is a. major cause of stroke and thromboembolism. It is still controversial whether the prothrombotic state and thrombosis in NVAF due to the atrial arrhythmia alone or the coexisting pathological factors. The results of AFFIRM and RACE trials did not demonstrate any superiority of a rhythm control versus a rate control strategy on occurrence of ischemic stroke. So, the thrombosis is independent of AF itself. The underlying pathological factors, such as oxidative stress and inflammation are speculated be the principal culprit of platelet activation in NVAF.
The upstream diseases of NVAF, including coronary heart diseases, hypertension, diabetes mellitus, could enhance the oxidative stress and inflammatory state. The process of non-enzymatic glycation of proteins and lipids is markedly accelerated in the setting of inflammation and oxidative stress. The resulting new products are defined as advanced glycation endproducts (AGEs), which mediate the atrial fibrosis and contribute to the pathogenesis of NVAF. We have demonstrated that, the plasma levels of oxidative stress, inflammation and AGEs enhanced significantly in NVAF patients at high risk for stroke compared with those at low to moderate risk. So, oxidative stress, inflammation and accumulated AGEs might not only result in occurrence of NVAF, but also be linked to thrombogenesis.
Platelets contribute to thrombosis by assembling into aggregates and by stimulating blood coagulation. Activated platelets play a well established role in thrombosis of NVAF. How the fundamental disorders of NVAF, such as oxidative stress, inflammation and accumulated AGEs, drive the prothrombotic state in atrial fibrillation? Recently, studies of microvesicles (MVs) have enlightened us.
MVs are membranous submicrometer vesicles formed by activated or apoptotic cells. They carry abundant signaling proteins and participate in multiple physiopathologic processes. The most MVs in circulation are platelet-derived microvesicles (PMVs), which are the main culprit in the development of thrombosis. The plasma levels of PMVs and Annexin V-positive PMVs enhanced significantly in NVAF patients at high risk for stroke compared with those at low to moderate risk. Thus, MVs may be the vectors bearing multiple pathogenic mechanisms of platelet activation. To find the source of platelet activation in NVAF, we can study the potential mechanism of the prothrombotic state and explore new, effective targets for preventing thromboembolism.
CD36, a multifunctional membrane receptor expressed on platelets, monocytes and several other cells, takes part in multiple physiopathological processes by engaging with multiple ligands. Platelet CD36could recognize and capture PS on the surface of PMVs, thus generating a PMV-CD36complex. The PMV-CD36complex was speculated be the key part of platelet activation. CD36can be a signaling molecule, but the detailed signal pathway of the PMV-CD36complex is unknown. Chen et al. showed that platelet CD36mediates mitogen-activated protein kinase (MAPK) activation induced by oxLDL. The modified lipids on oxLDL mediate its combination with CD36, and the surface of microvesicles contains multiple modified lipids (such as PS). Therefore, we speculated that the PMV-CD36complex could activate MAPKs, thereby activating platelets.
However, whether the PMVs mediate platelet activation induced by accompanying disorders, such as oxidative stress, inflammation and accumulated AGEs, in the context of NVAF is unclear. Thus, we speculated that the PMVs, generated in response to pathogenesis or accompanying disorders of NVAF, could be endogenous CD36ligands that transmit an activating signal to platelets to induce thrombosis.
Objectives
1. To determine the effects of oxidative stress, inflammation and accumulated AGEs on the production of PMVs.
2. To determine the effects of PMVs bearing signals of oxidative stress, inflammation and AGEs on the platelet activation and the involved signaling transduction.
3. To determine whether the signal pathway mediated by PMV-CD36complex could be a new effective target for preventing the prothrombotic state of NVAF.
Methods
1. Platelet isolation.
All the healthy volunteers had not taken any medication for2weeks. Fasting venous blood was collected in0.109mol/L sodium citrate (ratio1:9) under minimal tourniquet pressure using a sterile22-gauge needle. Platelet-rich plasma (PRP) was prepared by centrifugation at120g for10min at room temperature. Platelets were isolated from PRP after centrifugation at800g for10min in the presence of in the presence of100nmol/L prostaglandin E1. Then platelets were washed and resuspended in modified Tyrode's buffer (137mmol/L NaCl,2.7mmol/L KC1,12mmol/LNaHCO3,0.4mmol/L NaH2PO4,5mmol/LHEPES,0.1%glucose and0.35%bovine serum albumin, pH7.2) in the presence of100nmol/L PG-E1. Platelet concentration was adjusted to1×106/mL by use of a Z2particle counter, then used at once in all experiments.
2. The effects of oxidative stress, inflammation and AGEs on production of PMVs.
Resting platelets (1×106/mL) were incubated with oxidized LDL (oxLDL,50μg/mL), IL-6(1μg/mL) or AGEs (200μg/mL) for proper times at37℃respectively. The blank control (treated with buffer), isotype control and positive control (treated with10μmol/L adenosine diphosphate, ADP) were set for each group at the same time. Then the production of PMVs was detected by flow cytometry and a homemade ELISA.
3. Identification of PMVs by flow cytometry.
Immunofluorescent staining for PMVs:After gently blending,2.5μL platelet suspension (1×106/mL) was incubated with PEcy5-conjugated anti-CD41a antibody (5μL) in the dark for15min. Then the platelets were resuspended by1mL PBS and detected at once. Only cells and particles labeled with CD41a were gated. The PMV region was defined by FSC-SSC dot plot. The lower limit of the platelet gate was set at the left-hand border for resting platelets to distinguish between platelets and microvesicles. For each sample,10,000positive events in the platelet gate were acquired by use of FACS Calibur cytometer.
4. ELISA quantification of PMVs and Annexin V-positive PMVs.
After incubation with oxLDL, IL-6, AGEs or their control reagents, the platelet suspension was centrifuged at3,000g for10min. Then the supernatant was centrifuged again at13,000g for2min to avoid platelet contamination. Quantification of total PMVs and Annexin V-positive PMVs in the last supernatant was involved use of a homemade ELISA.
5. Collection of PMVs derived from platelets stimulated by oxLDL, IL-6or AGEs.
Platelets (1×106/mL) treated with oxLDL, IL-6or AGEs were sedimented at3000g for10min. An amount of2.0mmol/L phenylmethylsulfonyl fluoride was added to the PMV-enriched supernatants, which were then centrifuged at15,000g for1h at4℃. Then PMV pellets were washed twice (to avoid contamination of oxLDL, IL-6or AGEs) and resuspended in Modified Tyrode buffer and stored at-80℃. Freezing had no ad-verse effect on microvesicles. The protein content was measured by the Bradford Protein Assay Kit.
6. Double color flow cytometry of CD36expression.
For CD36quantification and CD36deficiency screening, the platelet suspension was incubated with5μL PEcy5-conjugated anti-CD41a antibody and5μL PE-conjugated anti-CD36antibody or isotype-matched control IgM in the dark for15 min. Then the platelets were resuspended by1mL PBS and detected by flow cytometry at once.
7. Double color flow cytometry of platelet GPⅡb/Ⅲa.
The platelet suspension (2.5μL) treated with PMVs (30μg/mL) was incubated with5μL PEcy5-conjugated anti-CD41a antibody and5μL FITC-conjugated PAC-1antibody (for activated platelet GPⅡb/Ⅲa) at room temperature in the dark for15min. Then the platelets were resuspended by1mL PBS and detected by flow cytometry at once.
8. Platelet aggregation studies.
PRP was obtained by centrifuging fasting venous blood at120g for10min at22℃, and platelet-poor plasma (PPP) was obtained by centrifuging PRP at3000g for10min. The platelet concentration of PRP was adjusted to2.5×108/mL by the addition of PPP. Aggregation was assessed by turbidimetry with a dual channel aggregometer (Chrono-log Corp., Havertown, PA, USA), with2μmol/L ADP used as an agonist. An amount of100%aggregation was defined as the light transmission of PPP, and0%was defined as the light transmission of PRP before the addition of agonists. Then the PRP treated with PMVs (30μg/mL) was stimulated with ADP (2μmol/L), and the change in light transmission was recorded.
9. Immunoassay for soluble P-selectin.
After the treatment described above, the platelet-PMV mixtures were centrifuged at3,000g for10min. Then the supernatant was centrifuged again at13,000g for2min to avoid platelet contamination. The last supernatant was used for detection of soluble P-selectin with use of commercially available immunoassay kits. The lower limit of sensitivity of the assay was0.5ng/mL.10. Western blot analysis.
Platelets treated with PMVs or controls were lysed in2mmol/L Tris-HCl (pH7.5),150mmol/L NaCl,1mmol/L EGTA,1mmol/L EDTA,1%Triton X-100,2.5mmol/L sodium pyrophos-phate,1mmol/L Na3VO4,1mmol/L phenylmethylsulfonyl fluoride and1μg/mL leupeptin, and protein concentrations were measured with use of a Bradford protein assay kit (Beyotime). Lysate protein (40μg) was separated on10% polyacrylamide gel and transferred onto polyvinylidene fluoride membranes. After a blocking for1h at room temperature in5%nonfat milk, the membranes were incubated with mouse anti-phospho-JNK1/2or rabbit anti-phospho-MKK4(1:1000dilution) overnight at4℃, then with HRP-conjugated secondary anti-mouse IgG or anti-rabbit IgG (1:4000dilution). The blots were developed with use of ECL detection reagent, then stripped and re-blotted with antibodies to native proteins for normalization.
11. Tanshinone IIA (TS IIA) treatment
TS IIA was obtained commercially (Xi'an Honson Biotechnology, China). Platelets were incubated with various concentrations of TS IIA (5μg/mL、10μg/mL20μg/mL、50μg/mL、100μg/mL) for15min before being stimulated with various PMVs(30μg/mL). Then the expression of platelet CD36and GPⅡ b/Ⅲa were detected by flow cytometry and the phosphorylation of MKK4/JNK was detected by western blot analysis.
Results
1. Platelet function studies with PMVs derived from oxLDL-treated platelets (oxLDL-PMVs)
(1) OxLDL treatment induced the release of PMVs and Annexin V-positive PMVs
Flow cytometry detected the formation of microvesicles by labeling with PEcy5-conjugated anti-CD41a antibody (M gates). The release of microvesicles increased after stimulation with oxLDL (50μg/mL) or ADP (10μmol/L), as quantified by CD41a-positive microparticles (P<0.05). For further confirmation, the supernatant of platelets was tested by ELISA. The amount of total PMVs and Annexin V-positive PMVs increased significantly after treatment with oxLDL or ADP (P<0.05), but not native LDL.
(2) OxLDLr-PMVs induced platelet activation
Next, we tested whether the PMVs collected from oxLDL-treated platelets could enhance platelet activation. We incubated resting platelets (1×106/mL) with oxLDL-PMVs (30μg/mL) for30min at22℃. Platelet activation is characterized by a conformation change in glycoprotein Ⅱ b/Ⅲa (GP Ⅱ b/Ⅲa). As expected, the MFI and percentage of PAC-1(recognizing the activated platelet GP Ⅱ b/Ⅲa) were enhanced significantly compared with the control (P<0.05). OxLDL-PMVs had almost the same effect as ADP. Furthermore, oxLDL-PMVs increased platelet aggregation in response to ADP.
(3) OxLDL-PMVs were ineffective in CD36-deficient platelets
We screened2CD36-deficient male donors in our laboratory. The CD36-deficient platelets were unable to bind PE-conjugated anti-CD36antibody. We incubated the CD36-deficient platelets (1×106/mL) with oxLDL-PMVs (30μg/mL) for30min at22℃. The PMV-enhanced platelet GP Ⅱ b/Ⅲa expression and platelet aggregation were absent.
(4) OxLDL-PMVs activated platelets in a CD36-and PS-dependent manner
To define the influence of CD36and PS on PMV-platelet interaction, we determined the expression of platelet GP Ⅱ b/Ⅲa and the secretion of P-selection. oxLDL-PMVs substantially increased the expression of GP Ⅱ b/Ⅲa and secretion of P-selectin (P<0.05). In all cases, the blocking of CD36(by a CD36neutralizing antibody) or PS (by Annexin V) could diminish the enhancement by oxLDL-PMVs (P<0.05), but IgG, an isotype-matched control of the CD36neutralizing antibody, had no effect. As well, the CD36neutralizing antibody decreased the binding of PE-conjugated anti-CD36antibody to platelets, with the expression of CD36for other groups not changed significantly.
(5) Platelet activation induced by oxLDL-PMVs was mediated by JNK signals
To elucidate the potential effect of JNK signaling in PMV-induced platelet activation, platelets were treated with the JNK inhibitor SP600125before incubation with PMVs. Pharmacological inhibition of JNK reduced platelet activation by PMVs, including expression of GP Ⅱ b/Ⅲa and secretion of P-selectin (P<0.05). Thus, JNK signaling may contribute to PMV-induced platelet activation.
(6) Phosphorylation of platelet JNK2and its upstream activator MKK4 induced by oxLDL-PMVs.
To confirm the role of the JNK pathway in PMV-induced activation of platelets, we compared the phosphorylation of JNK2in CD36-deficient and-positive platelets by immunoblotting. CD36-positive but not CD36-deficient platelets exposed to oxLDL-PMVs for30min showed a significant increase in phosphorylation of JNK2and its upstream activator MKK4(P<0.05). The JNK2phosphorylation was time and dose dependent, peaking at15min to approximately1.3-fold that of baseline with30μg/mL oxLDL-PMVs and with increased concentrations of oxLDL-PMVs inducing stronger phosphorylation of JNK2.
2. Platelet function studies with PMVs derived from IL-6-treated platelets (IL-6-PMVs)
(1) IL-6treatment promoted the formation of PMVs and Annexin Ⅴ-positive PMVs
The formation of PMVs was identified with labeling with PEcy5-conjugated anti-CD41a antibody (M gates). We observed an increase in total PMVs and Annexin V-positive PMVs released from IL-6treated compared to untreated platelets (P<0.05)
(2) IL-6-PMVs induced platelet activation
The MFI and percentage of PAC-1of the CD36-positive platelets were increased significantly stimulated by IL-6-PMVs, with similar effect as ADP (10μmol/L)(P <0.05). Furthermore, IL-6-PMVs increased platelet aggregation in response to ADP.
(3) IL-6-PMVs induced platelet activation in a CD36-and PS-dependent way
The PMV-enhanced platelet GPⅡ b/Ⅲa expression and platelet aggregation were absent when the CD36-deficient platelets (1×106/mL) were incubated with IL-6-PMVs (30μg/mL).
IL-6-PMVs substantially increased the expression of GP Ⅱb/Ⅲa and secretion of P-selectin (P<0.05). In all cases, the blocking of CD36(by a CD36neutralizing antibody) or PS (by Annexin V) could diminish the enhancement by IL-6-PMVs (P <0.05).
(4) Activation of platelets by IL-6-PMVs depends on MKK4/JNK pathway
Inhibition of JNK blocked the effect of IL-6-PMVs on platelet activation, including the expression of integrin αHbβ3and secretion of P-selectin (P<0.05).
CD36-positive but not-deficient platelets exposed to IL-6-PMVs for30min showed a significant increase in phosphorylation of JNK2and its upstream activator MKK4(P<0.05). The JNK2phosphorylation was time and dose dependent.
(5) Tanshinone ⅡA (TS Ⅱ A) treatment inhibits platelet activation induced by IL-6-PMVs To test whether the signal pathway mediated by IL-6-PMV/CD36complex could be a potential target for preventing the prothrombotic state of NVAF, we treated resting platelets with doses of TS ⅡA (5μg/mL、10μg/mL.20μg/mL、50μg/mL、100μg/mL) for15min before incubation with IL-6-PMVs. We found that TSⅡA dose-dependently inhibited the activation of platelets in vitro. At the same time, we detected the platelet CD36expression and found that TS ⅡA dose-dependently decreased the expression of platelet CD36. Furthermore, TS ⅡA could downregulate the phosphorylation of MKK4/JNK2activated by IL-6-PMVs.
3. Platelet function studies with PMVs derived from AGE-treated platelets (AGE-PMVs)
(1) AGEs treatment promoted the formation of PMVs and Annexin V-positive PMVs
We observed an increase in total PMVs and Annexin V-positive PMVs released from AGE (200μg/mL)-treated compared to bovine serum albumin (BSA,200μg/mL)-treated platelets or untreated platelets (P<0.05).
(2) Effect of AGE-PMVs on platelet activation
The MFI and percentage of platelet PAC-1were enhanced significantly after incubation with AGE-PMVs (P<0.05), which had similar effect as ADP. Furthermore, Surface exposure of CD40L (an index of platelet activation and assumed to originate from the platelet cytosol) increased after incubati
引文
1. Singer DE, Albers GW, Dalen JE, Go AS, Halperin JL, Manning WJ. Antithrombotic therapy in atrial fibrillation:the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest.2004; 126(3 Suppl):429S-456S.
2. Watson T, Shantsila E, Lip GY. Mechanisms of thrombogenesis in atrial fibrillation:Virchow's triad revisited. Lancet 2009;373(9658):155-166.
3. Pongratz G, Brandt-Pohlmann M, Henneke KH, Pohle C, Zink D, Gehling Q Bachmann K. Platelet activation in embolic and preembolic status of patients with nonrheumatic atrial fibrillation. Chest.1997;111(4):929-933.
4. Katritsis DG. Is atrial fibrillation an inflammatory disorder? Eur Heart J. 2006;27(7):886.
5. Cocucci E, Racchetti G, Meldolesi J. Shedding microvesicles:artefacts no more. Trends Cell Biol.2009;19(2):43-51.
6. Del Conde I, Shrimpton CN, Thiagarajan P, Lopez JA. Tissue-factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. Blood 2005;106(5):1604-1611.
7. Bastida E, Ordinas A, Escolar G, Jamieson GA. Tissue factor in microvesicles shed from U87MG human glioblastoma cells induces coagulation, platelet aggregation, and thrombogenesis. Blood.1984;64(1):177-184.
8. Ratajczak J, Wysoczynski M, Hayek F, Janowska-Wieczorek A, Ratajczak MZ. Membrane-derived microvesicles:important and underappreciated mediators of cell-to-cell communication. Leukemia.2006;20(9):1487-1495.
9. Sinauridze EI, Kireev DA, Popenko NY, Pichugin AV, Panteleev MA, Krymskaya OV, Ataullakhanov FI. Platelet microparticle membranes have 50-to 100-fold higher specific procoagulant activity than activated platelets. Thrombo Haemostasis.2007;97(3):425-434.
10. Shantsila E, Kamphuisen PW, Lip GY. Circulating microparticles in cardiovascular disease:implications for atherogenesis and atherothrombosis. J Thromb Haemost.2010; 8(11):2358-2368.
11. Pignoli P, Tremoli E, Poli A, Oreste P, Paoletti R. Intimal plus medial thickness of the arterial wall:a direct measurement with ultrasound imaging. Circulation.1986;74(6):1399-1406.
12. Sutton-Tyrrell K, Alcorn HG, Wolfson SK, Jr., Kelsey SF, Kuller LH. Predictors of carotid stenosis in older adults with and without isolated systolic hypertension. Stroke.1993;24(3):355-361.
13. Deng JT, Parsons PG. Solid phase immunoassay for high molecular weight alkaline phosphatase in human sera using a specific monoclonal antibody. Clin chimacta.1988;176(3):291-301.
14. Wang H, Yan HM, Tang MX, Wang ZH, Zhong M, Zhang Y, Deng JT, Zhang W. Increased serum levels of microvesicles in nonvalvular atrial fibrillation determinated by ELISA using a specific monoclonal antibody AD-1. Clin chim acta.2010;411(21-22):1700-1704.
15. Wattigney WA, Mensah GA, Croft JB. Increasing trends in hospitalization for atrial fibrillation in the United States,1985 through 1999:implications for primary prevention. Circulation.2003;108(6):711-716.
16. Bornstein N, Corea F, Galllai V, Parnetti L. Heart-brain relationship:atrial fibrillation and stroke. Clin Exp Hypertens.2002;24(7-8):493-499.
17. Wolf PA, Abbott RD, Kannel WB. Atrial fibrillation:a major contributor to stroke in the elderly. The Framingham Study. Arch Intern Med. 1987;147(9):1561-1564.
18. Tayal AH, Tian M, Kelly KM, Jones SC, Wright DG, Singh D, Jarouse J, Brillman J, Murali S, Gupta R. Atrial fibrillation detected by mobile cardiac outpatient telemetry in cryptogenic TIA or stroke. Neurology. 2008;71(21):1696-1701.
19. Das RR, Seshadri S, Beiser AS, Kelly-Hayes M, Au R, Himali JJ, Kase CS, Benjamin EJ, Polak JF, O'Donnell CJ, Yoshita M, D'Agostino RB, Sr., DeCarli C, Wolf PA. Prevalence and correlates of silent cerebral infarcts in the Framingham offspring study. Stroke.2008;39(11):2929-2935.
20. Pink J, Lane S, Pirmohamed M, Hughes DA. Dabigatran etexilate versus warfarin in management of non-valvular atrial fibrillation in UK context: quantitative benefit-harm and economic analyses. BMJ.2011; 343:d6333.
21. Singer DE, Chang Y, Fang MC, Borowsky LH, Pomernacki NK, Udaltsova N, Go AS. The net clinical benefit of warfarin anticoagulation in atrial fibrillation. Ann Intern Med.2009;151(5):297-305.
22. Gage BF, van Walraven C, Pearce L, Hart RG, Koudstaal PJ, Boode BS, Petersen P. Selecting patients with atrial fibrillation for anticoagulation:stroke risk stratification in patients taking aspirin. Circulation. 2004; 110(16):2287-2292.
23.张学义,张薇,张运.非瓣膜性心房颤动缺血性脑卒中危险因素的超声研究.中华超声影像学杂志,2005；14(2)：96-98.
24. Vinogradova O, elyvis A, Velyviene A, Hu B, Haas T, Plow E, Qin J. A structural mechanism of integrin alpha(IIb)beta(3) "inside-out" activation as regulated by its cytoplasmic face. Cell.2002;110(5):587-597.
25. Michelson AD, Barnard MR, Krueger LA, Frelinger AL,3rd, Furman MI. Evaluation of platelet function by flow cytometry. Methods. 2000;21(3):259-270.
26. Minamino T, Kitakaze M, Sanada S, Asanuama H, Kurotobi T, Koretsune Y, Fukunami M, Kuzuya T, Hoki N, Hori M. Increased expression of P-selectin on platelets is a risk factor for silent cerebral infarction in patients with atrial fibrillation:role of nitric oxide. Circulation.1998;98(17):1721-1727.
27. Kamath S, Blann AD, Caine GJ, Gurney D, Chin BS, Lip GY. Platelet P-selectin levels in relation to plasma soluble P-selectin and beta-thromboglobulin levels in atrial fibrillation. Strok.2002;33(5):1237-1242.
28. Li-Saw-Hee FL, Blann AD, Gurney D, Lip GY. Plasma von Willebrand factor, fibrinogen and soluble P-selectin levels in paroxysmal, persistent and permanent atrial fibrillation. Effects of cardioversion and return of left atrial function. Eur Heart J.2001;22(18):1741-1747.
29. Li-Saw-Hee FL, Blann AD, Lip GY. A cross-sectional and diurnal study of thrombogenesis among patients with chronic atrial fibrillation. J Am Coll Cardiol. 2000;35(7):1926-1931.
30. Ord D, Ord T. Mouse NIPK interacts with ATF4 and affects its transcriptional activity. Exp Cell Res.2003;286(2):308-320.
31. Li R, Duncan BB, Metcalf PA, Crouse JR,3rd, Sharrett AR, Tyroler HA, Barnes R, Heiss G. B-mode-detected carotid artery plaque in a general population. Atherosclerosis Risk in Communities (ARIC) Study Investigators. Stroke.1994;25(12):2377-2383.
32. Burke GL, Evans GW, Riley WA, Sharrett AR, Howard G, Barnes RW, Rosamond W, Crow RS, Rautaharju PM, Heiss G. Arterial wall thickness is associated with prevalent cardiovascular disease in middle-aged adults. The Atherosclerosis Risk in Communities (ARIC) Study. Stroke. 1995;26(3):386-391.
33. Watanabe T, Takeishi Y, Hirono O, Itoh M, Matsui M, Nakamura K, Tamada Y, Kubota I. C-reactive protein elevation predicts the occurrence of atrial structural remodeling in patients with paroxysmal atrial fibrillation. Heart Vessels.2005;20(2):45-49.
34. Watanabe E, Arakawa T, Uchiyama T, Kodama I, Hishida H. High-sensitivity C-reactive protein is predictive of successful cardioversion for atrial fibrillation and maintenance of sinus rhythm after conversion. Intern J Cardiol. 2006;108(3):346-353.
35. Dernellis J, Panaretou M. Effects of C-reactive protein and the third and fourth components of complement (C3 and C4) on incidence of atrial fibrillation. Am J Cardiol.2006;97(2):245-248.
36. Kim YH, Lim DS, Lee JH, Shim WJ, Ro YM, Park GH, Becker KG, Cho-Chung YS, Kim MK. Gene expression profiling of oxidative stress on atrial fibrillation in humans. Exp Mol Med.2003;35(5):336-349.
37. Carnes CA, Chung MK, Nakayama T, Nakayama H, Baliga RS, Piao S, Kanderian A, Pavia S, Hamlin RL, McCarthy PM, Bauer JA, Van Wagoner DR. Ascorbate attenuates atrial pacing-induced peroxynitrite formation and electrical remodeling and decreases the incidence of postoperative atrial fibrillation. Circ Res.2001;89(6):E32-38.
38. Kim YM, Guzik TJ, Zhang YH, Zhang MH, Kattach H, Ratnatunga C, Pillai R, Channon KM, Casadei B. A myocardial Nox2 containing NAD(P)H oxidase contributes to oxidative stress in human atrial fibrillation. Circ Res. 2005;97(7):629-636.
39. Inal JM, Ansa-Addo EA, Stratton D, Kholia S, Antwi-Baffour SS, Jorfi S, Lange S. Microvesicles in health and disease. Arch Immunol Ther Ex. 2012;60(2):107-121.
40. Simpson RJ, Lim JW, Moritz RL, Mathivanan S. Exosomes:proteomic insights and diagnostic potential. Expert Rev Proteomic.2009;6(3):267-283.
41. Scanu A, Molnarfi N, Brandt KJ, Gruaz L, Dayer JM, Burger D. Stimulated T cells generate microparticles, which mimic cellular contact activation of human monocytes:differential regulation of pro-and anti-inflammatory cytokine production by high-density lipoproteins. J Leukocyte Biol. 2008;83(4):921-927.
42. Nolan S, Dixon R, Norman K, Hellewell P, Ridger V. Nitric oxide regulates neutrophil migration through microparticle formation. Am J Pathol. 2008;172(1):265-273.
43. Kim JW, Wieckowski E, Taylor DD, Reichert TE, Watkins S, Whiteside TL. Fas ligand-positive membranous vesicles isolated from sera of patients with oral cancer induce apoptosis of activated T lymphocytes. Clin Cancer Res. 2005;11(3):1010-1020.
44. Siljander PR. Platelet-derived microparticles-an updated perspective. Thrombo Res.2011;127 Suppl 2:S30-33.
45. Hunter MP, Ismail N, Zhang X, Aguda BD, Lee EJ, Yu L, Xiao T, Schafer J, Lee ML, Schmittgen TD, Nana-Sinkam SP, Jarjoura D, Marsh CB. Detection of microRNA expression in human peripheral blood microvesicles. PloS one. 2008;3(11):e3694.
46. Aatonen M, Gronholm M, Siljander PR. Platelet-derived microvesicles: multitalented participants in intercellular communication. Semin Thromb Hemost.2012;38(1):102-113.
47. Ardoin SP, Shanahan JC, Pisetsky DS. The role of microparticles in inflammation and thrombosis. Scand J Immunol.2007;66(2-3):159-165.
48. Deng JT, Hoylaerts MF, Van Hoof VO, De Broe ME. Differential release of human intestinal alkaline phosphatase in duodenal fluid and serum. Clin Chem. 1992;38(12):2532-2538.
49. Van Hoof VO, Deng JT, De Broe ME. How do plasma membranes reach the circulation? Clin Chim Acta.1997;266(1):23-31.
50. Choudhury A, Chung I, Blann AD, Lip GY. Elevated platelet microparticle levels in nonvalvular atrial fibrillation:relationship to p-selectin and antithrombotic therapy. Chest.2007;131(3):809-815.
51. Ederhy S, Di Angelantonio E, Mallat Z, Hugel B, Janower S, Meuleman C, Boccara F, Freyssinet JM, Tedgui A, Cohen A. Levels of circulating procoagulant microparticles in nonvalvular atrial fibrillation. Am J Cardiol. 2007;100(6):989-994.
52. Ghosh A, Li W, Febbraio M, Espinola RG, McCrae KR, Cockrell E, Silverstein RL. Platelet CD36 mediates interactions with endothelial cell-derived microparticles and contributes to thrombosis in mice. J Clin Invest. May 2008;118(5):1934-1943.
1. Caldeira D, David C, Sampaio C. Rate versus rhythm control in atrial fibrillation and clinical outcomes:updated systematic review and meta-analysis of randomized controlled trials. Arch Cardiovasc Dis. 2012;105(4):226-238.
2. Katritsis DG. Is atrial fibrillation an inflammatory disorder? Eur Heart J. 2006;27(7):886.
3. Watson T, Shantsila E, Lip GY. Mechanisms of thrombogenesis in atrial fibrillation:Virchow's triad revisited. Lancet.2009;373(9658):155-166.
4. Oleksowicz L, Mrowiec Z, Isaacs R, Dutcher JP, Puszkin E. Morphologic and ultrastructural evidence of interleukin-6 induced platelet activation. Am J hematol.1995;48(2):92-99.
5. Raposeiras-Roubin S, Rodino-Janeiro BK, Grigorian-Shamagian L, Seoane-Blanco A, Moure-Gonzalez M, Varela-Roman A, Alvarez E, Gonzalez-Juanatey JR. Evidence for a role of advanced glycation end products in atrial fibrillation.Int J Cardiol.2012;157(3):397-402.
6. Del Conde I, Shrimpton CN, Thiagarajan P, Lopez JA. Tissue-factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. Blood.2005;106(5):1604-1611.
7. Cocucci E, Racchetti G, Meldolesi J. Shedding microvesicles:artefacts no more. Trends Cell Biol.2009;19(2):43-51.
8. Bastida E, Ordinas A, Escolar G, Jamieson GA. Tissue factor in microvesicles shed from U87MG human glioblastoma cells induces coagulation, platelet aggregation, and thrombogenesis. Blood.1984;64(1):177-184.
9. Aatonen M, Gronholm M, Siljander PR. Platelet-derived microvesicles: multitalented participants in intercellular communication. Semin Thromb Hemost.2012;38(1):102-113.
10. Ratajczak J, Wysoczynski M, Hayek F, Janowska-Wieczorek A, Ratajczak MZ. Membrane-derived microvesicles:important and underappreciated mediators of cell-to-cell communication. Leukemia.2006;20(9):1487-1495.
11. Mackman N. On the trail of microparticles. Circ Res.2009; 104(8):925-927.
12. Chen K, Febbraio M, Li W, Silverstein RL. A specific CD36-dependent signaling pathway is required for platelet activation by oxidized low-density lipoprotein. Circ Res.2008;102(12):1512-1519.
13. Abe H, Matsubara T, Iehara N, Nagai K, Takahashi T, Arai H, Kita T, Doi T. Type IV collagen is transcriptionally regulated by Smadl under advanced glycation end product (AGE) stimulation. J Biol Chem. 2004;279(14):14201-14206.
14. Wattigney WA, Mensah GA, Croft JB. Increasing trends in hospitalization for atrial fibrillation in the United States,1985 through 1999:implications for primary prevention. Circulation.2003;108(6):711-716.
15. Chien KL, Su TC, Hsu HC, Chang WT, Chen PC, Chen MF, Lee YT. Atrial fibrillation prevalence, incidence and risk of stroke and all-cause death among Chinese. Int J Cardiol.2010;139(2):173-180.
16. Lloyd-Jones DM, Wang TJ, Leip EP, Larson MG, Levy D, Vasan RS, D'Agostino RB, Massaro JM, Beiser A, Wolf PA, Benjamin EJ. Lifetime risk for development of atrial fibrillation:the Framingham Heart Study. Circulation.2004; 110(9):1042-1046.
17. Bornstein N, Corea F, Galllai V, Parnetti L. Heart-brain relationship:atrial fibrillation and stroke. Clin Exp Hypertens.2002;24(7-8):493-499.
18. Wolf PA, Abbott RD, Kannel WB. Atrial fibrillation:a major contributor to stroke in the elderly. The Framingham Study. Arch Internal Med. 1987;147(9):1561-1564.
19. Lin HJ, Wolf PA, Kelly-Hayes M, Beiser AS, Kase CS, Benjamin EJ, D'Agostino RB. Stroke severity in atrial fibrillation. The Framingham Study. Stroke.1996;27(10):1760-1764.
20. Pink J, Lane S, Pirmohamed M, Hughes DA. Dabigatran etexilate versus warfarin in management of non-valvular atrial fibrillation in UK context: quantitative benefit-harm and economic analyses.BMJ.2011.343:d6333.
21. Singer DE, Albers GW, Dalen JE, Go AS, Halperin JL, Manning WJ. Antithrombotic therapy in atrial fibrillation:the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest.2004; 126(3 Suppl):429S-456S.
22. The AFFIRM Investigators. A comparison of rate control and rhythm control in patients with atrial fibrillation. NEngl J Med.2002; 347:1825-1833
23. Steinberg JS, Sandaniantz A, Kron J, Krahn A, Denny DM, Daubert J, Campbell WB, Havranek E, Murray K, Olshansky B, O' Neill G, Sami M, Schmidt S, Storm R, Zabalgoitia M, Miller J, Chandler M, Nasco EM, Greene HL. Analysis of cause-specific mortality in the Atrial Fibrillation Follow-up Investigation of Rhythm Management AFFIRM) study. Circulation.2004; 109(16):1973-1980.
24. Corley SD, Epstein AE, DiMarco JP, Domanski MJ, Geller N, Greene HL, Josephson RA, Kellen JC, Klein RC, Krahn AD, Mickel M, Mitchell LB, Nelson JD, Rosenberg Y, Schron E, Shemanski L, Waldo AL, Wyse DG; AFFIRM Investigators. Relationships between sinus rhythm, treatment, and survival in the Atrial Fibrillation Follow-up Investigation of Rhythm Management(AFFIRM) study. Circulation.2004; 109(12):1509-1513.
25. Cleland JG, Coletta AP, Abdellah AT, Cullington D, Clark AL, Rigby AS. Clinical trials update from the American Heart Association 2007:CORONA, RethinQ, MASCOT, AF-CHF, HART, MASTER, POISE and stem cell therapy. Eur J Heart Fail.2008; 10(1):102-108.
26. Van Gelder IC, Hagens VE, Bosker HA, Kingma JH, Kamp O, Kingma T, Said SA, Darmanata JI, Timmermans AJ, Tijssen JG, Crijns HJ; Rate Control versus Electrical Cardioversion for Persistent Atrial Fibrillation Study Group. A comparison of rate control and rhythm control in patients with recurrent persistent atrial fibrillation. NEnglJMed.2002; 347(23):1834-1840.
27. Marin F, Corral J, Roldan Ⅴ, Gonzalez-Conejero R, del Rey ML, Sogorb F, Lip GY, Vicente V. Factor ⅩⅢ Val34Leu polymorphism modulates the prothrombotic and inflammatory state associated with atrial fibrillation. J Mol Cell Cardiol.2004;37(3):699-704.
28. Cesari M, Penninx BW, Newman AB, Kritchevsky SB, Nicklas BJ, Sutton-Tyrrell K, Rubin SM, Ding J, Simonsick EM, Harris TB, Pahor M. Inflammatory markers and onset of cardiovascular events:results from the Health ABC study. Circulation.2003;108(19):2317-2322.
29. Goldin A, Beckman JA, Schmidt AM, Creager MA. Advanced glycation end products:sparking the development of diabetic vascular injury. Circulation. 2006;114(6):597-605.
30. Friedman EA. Advanced glycosylated end products and hyperglycemia in the pathogenesis of diabetic complications. Diabetes care.1999;22 Suppl 2:B65-71.
31. Smit AJ, Lutgers HL. The clinical relevance of advanced glycation endproducts (AGE) and recent developments in pharmaceutics to reduce AGE accumulation. Curr Med Chem.2004;11(20):2767-2784.
32. Weinrauch LA, Berger AJ, Aronson D, Gleason RE, Lee AT, D' Elia JA. Regression of left ventricular hypertrophy in diabetic nephropathy:loss of parasympathetic function predicts response to treatment. J Clin Hypertens. 2006;8(5):330-335.
33. McNulty M, Mahmud A, Feely J. Advanced glycation end-products and arterial stiffness in hypertension. Am J Hypertension.2007;20(3):242-247.
34. Meerwaldt R, Lutgers HL, Links TP, Graaff R, Baynes JW, Gans RO, Smit AJ. Skin autofluorescence is a strong predictor of cardiac mortality in diabetes. Diabetes care.2007;30(1):107-112.
35. Aronson D. Pharmacological prevention of cardiovascular aging--targeting the Maillard reaction. Brit J Pharmacol.2004;142(7):1055-1058.
36. Reddy S, Bichler J, Wells-Knecht KJ, Thorpe SR, Baynes JW. N epsilon-(carboxymethyl)lysine is a dominant advanced glycation end product (AGE) antigen in tissue proteins. Biochemistry.1995;34(34):10872-10878.
37. Verbeke P, Clark BF, Rattan SI. Modulating cellular aging in vitro:hormetic effects of repeated mild heat stress on protein oxidation and glycation. Exp Gerontol.2000;35(6-7):787-794.
38. Schmidt AM, Yan SD, Stern DM. The dark side of glucose. Nat Med. 1995;1(10):1002-1004.
39. Inal JM, Ansa-Addo EA, Stratton D, Kholia S, Antwi-Baffour SS, Jorfi S, Lange S. Microvesicles in health and disease. Arch Immunol Ther Ex. 2012;60(2):107-121.
40. Lima LG, Chammas R, Monteiro RQ, Moreira ME, Barcinski MA. Tumor-derived microvesicles modulate the establishment of metastatic melanoma in a phosphatidylserine-dependent manner. Cancer lett. 2009;283(2):168-175.
41. Hugel B, Martinez MC, Kunzelmann C, Freyssinet JM. Membrane microparticles:two sides of the coin. Physiology.2005;20:22-27.
42. Muralidharan-Chari V, Clancy JW, Sedgwick A, D'Souza-Schorey C. Microvesicles:mediators of extracellular communication during cancer progression. J Cell Sci.2010;123(Pt 10):1603-1611.
43. Siljander PR. Platelet-derived microparticles-an updated perspective. Thromb Res.2011; 127 Suppl 2:S30-33.
44. Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, Geuze HJ. B lymphocytes secrete antigen-presenting vesicles. J Exp Med. 1996;183(3):1161-1172.
45. Febbraio M, Hajjar DP, Silverstein RL. CD36:a class B scavenger receptor involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism. J Clin Invest.2001; 108(6):785-791.
46. Greenberg ME, Sun M, Zhang R, Febbraio M, Silverstein R, Hazen SL. Oxidized phosphatidylserine-CD36 interactions play an essential role in macrophage-dependent phagocytosis of apoptotic cells. J Exp Med. 2006;203(12):2613-2625.
47. Nicholson AC, Frieda S, Pearce A, Silverstein RL. Oxidized LDL binds to CD36 on human monocyte-derived macrophages and transfected cell lines. Evidence implicating the lipid moiety of the lipoprotein as the binding site. Arterioscl Throm Vas.1995;15(2):269-275.
48. Boullier A, Bird DA, Chang MK, Dennis EA, Friedman P, Gillotre-Taylor K, Horkko S, Palinski W, Quehenberger O, Shaw P, Steinberg D, Terpstra V, Witztum JL. Scavenger receptors, oxidized LDL, and atherosclerosis. Ann NY Acad Sci.2001;947:214-222.
49. Nicholson AC, Hajjar DP. CD36, oxidized LDL and PPAR gamma: pathological interactions in macrophages and atherosclerosis. Vasc Pharm. 2004;41(4-5):139-146.
50. Rahaman SO, Lennon DJ, Febbraio M, Podrez EA, Hazen SL, Silverstein RL. A CD36-dependent signaling cascade is necessary for macrophage foam cell formation. Cell metah.2006;4(3):211-221.
51. Ge Y, Elghetany M. CD36:a multiligand molecule. Lab Hematol.2005; 11(1): 31-37.
52. Lin M, Shieh SH, Yang TF. Frequency of platelet-specific antigens among Chinese in Taiwan. Transfusion.1993;33(2):155-157.
53. Hirano K, Kuwasako T, Nakagawa-Toyama Y, Janabi M, Yamashita S, Matsuzawa Y Pathophysiology of human genetic CD36 deficiency. Trends Cardiovasc Med.2003; 13(4):136-141.
54. Ikeda H, Mitani T, Ohnuma M, Haga H, Ohtzuka S, Kato T, Nakase T, Sekiguchi S. A new platelet-specific antigen, Naka involved in the refractories of HLA-matched platelet transfusion. Vox Sang.1989; 57(3):213-217.
55. Yanai H, Chiba H, Fujiwara H, Morimoto M, Abe K, Yoshida S, Takahashi Y, Fuda H, Hui SP, Akita H, Kobayashi K, Matsuno K. Phenotype-genotype correlation in CD36 deficiency types Ⅰ and Ⅱ. Thromb Haemost.2000; 84(3):436-441.
56. Curtis BR, Aster RH. Incidence of the Nak(a)-negative platelet phenotype in African Americans is similar to that of Asians. Transfusion.1996; 36(4): 331-334.
57. Nozaki S, Kashiwagi H, Yamashita S, Nakagawa T, Kostner B, Tomiyama Y, Nakata A, Ishigami M, Miyagawa J, Kameda-Takemura K, et al. Reduced uptake of oxidized low density lipoproteins in monocyte-derived macrophages from CD36-deficient subjects. J Clin Invest.1995;96(4):1859-1865.
58. Febbraio M, Abumrad NA, Hajjar DP, Sharma K, Cheng W, Pearce SF, Silverstein RL. A null mutation in murine CD36 reveals an important role in fatty acid and lipoprotein metabolism. J Bio lChem. 1999;274(27):19055-19062.
59. Han J, Zhou X, Yokoyama T, Hajjar DP, Gotto AM, Jr., Nicholson AC. Pitavastatin downregulates expression of the macrophage type B scavenger receptor, CD36. Circulation.2004;109(6):790-796.
60. Fuhrman B, Koren L, Volkova N, Keidar S, Hayek T, Aviram M. Atorvastatin therapy in hypercholesterolemic patients suppresses cellular uptake of oxidized-LDL by differentiating monocytes. Atherosclerosis. 2002;164(1):179-185.
61. Ozer NK, Negis Y, Aytan N, Villacorta L, Ricciarelli R, Zingg JM, Azzi A. Vitamin E inhibits CD36 scavenger receptor expression in hypercholesterolemic rabbits. Atherosclerosis.2006; 184(1):15-20.
62. Ghosh A, Li W, Febbraio M, Espinola RG, McCrae KR, Cockrell E, Silverstein RL. Platelet CD36 mediates interactions with endothelial cell-derived microparticles and contributes to thrombosis in mice. J Clin Invest. 2008;118(5):1934-1943.
63. Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell. 2000;103(2):239-252.
64. Wu TW, Zeng LH, Fung KP, Wu J, Pang H, Grey AA, Weisel RD, Wang JY. Effect of sodium tanshinone ⅡA sulfonate in the rabbit myocardium and on human cardiomyocytes and vascular endothelial cells. Biochem Pharmcol. 1993;46(12):2327-2332.
65. Takahashi K, Ouyang X, Komatsu K, Nakamura N, Hattori M, Baba A, Azuma J. Sodium tanshinone ⅡA sulfonate derived from Danshen (Salvia miltiorrhiza) attenuates hypertrophy induced by angiotensin Ⅱ in cultured neonatal rat cardiac cells. Biochem Pharmcol.2002;64(4):745-749.
66. Lin HC, Ding HY, Chang WL. Two new fatty diterpenoids from Salvia miltiorrhiza. JNa t Prod.2001;64(5):648-650.
67. Wang N, Luo HW, Niwa M, Ji J. A new platelet aggregation inhibitor from Salvia miltiorrhiza. Planta Med.1989;55(4):390-391.
1. Wattigney WA, Mensah GA, Croft JB. Increasing trends in hospitalization for atrial fibrillation in the United States,1985 through 1999:implications for primary prevention. Circulation.2003;108(6):711-716.
2. Bornstein N, Corea F, Galllai V, Parnetti L. Heart-brain relationship:atrial fibrillation and stroke. Clin Exp Hypertens.2002;24(7-8):493-499.
3. Wolf PA, Abbott RD, Kannel WB. Atrial fibrillation:a major contributor to stroke in the elderly. The Framingham Study. Arch Intern Med. 1987;147(9):1561-1564.
4.张学义,张薇,张运.非瓣膜性心房颤动缺血性脑卒中危险因素的超声研究.中华超声影像学杂志,2005；14(2)：96-98.
5. Watson T, Shantsila E, Lip GY. Mechanisms of thrombogenesis in atrial fibrillation:Virchow's triad revisited. Lancet.2009;373(9658):155-166.
6. Caldeira D, David C, Sampaio C. Rate versus rhythm control in atrial fibrillation and clinical outcomes:updated systematic review and meta-analysis of randomized controlled trials. Arch Cardiovasc Dis. 2012;105(4):226-238.
7. Raposeiras-Roubin S, Rodino-Janeiro BK, Grigorian-Shamagian L, Seoane-Blanco A, Moure-Gonzalez M, Varela-Roman A, Alvarez E, Gonzalez-Juanatey JR. Evidence for a role of advanced glycation end products in atrial fibrillation. Int J Cardiol.2012;157(3):397-402.
8. Ardoin SP, Shanahan JC, Pisetsky DS. The role of microparticles in inflammation and thrombosis. Scand J Immunol.2007;66(2-3):159-165.
9. Janiszewski M, Do Carmo AO, Pedro MA, Silva E, Knobel E, Laurindo FR. Platelet-derived exosomes of septic individuals possess proapoptotic NAD(P)H oxidase activity:A novel vascular redox pathway. Criti Care Med. 2004;32(3):818-825.
10. Benjamin EJ, Wolf PA, D' Agostino RB, Silbershatz H, Kannel WB, Levy D. Impact of atrial fibrillation on the risk of death:the Framingham Heart Study. Circulation.1998;98(10):946-952.
11. Lip GY, Boos CJ. Antithrombotic treatment in atrial fibrillation. Heart. 2006;92(2):155-161.
12. Lakshminarayan K, Solid CA, Collins AJ, Anderson DC, Herzog CA. Atrial fibrillation and stroke in the general medicare population:a 10-year perspective (1992 to 2002). Stroke.2006;37(8):1969-1974.
13. Pongratz G, Brandt-Pohlmann M, Henneke KH, Pohle C, Zink D, Gehling G, Bachmann K. Platelet activation in embolic and preembolic status of patients with nonrheumatic atrial fibrillation. Chest.1997;111(4):929-933.
14. Lip GY. The prothrombotic state in atrial fibrillation:new insights, more questions, and clear answers needed. Am Heart J.2000;140(3):348-350.
15. Minamino T, Kitakaze M, Sanada S, Asanuama H, Kurotobi T, Koretsune Y, Fukunami M, Kuzuya T, Hoki N, Hori M. Increased expression of P-selectin on platelets is a risk factor for silent cerebral infarction in patients with atrial fibrillation:role of nitric oxide. Circulation.1998;98(17):1721-1727.
16. Gage BF, van Walraven C, Pearce L, Hart RG, Koudstaal PJ, Boode BS, Petersen P. Selecting patients with atrial fibrillation for anticoagulation:stroke risk stratification in patients taking aspirin. Circulation. 2004;110(16):2287-2292.
17. Inal JM, Ansa-Addo EA, Stratton D, Kholia S, Antwi-Baffour SS, Jorfi S, Lange S. Microvesicles in health and disease. Arch Immunol Ther Ex. 2012;60(2):107-121.
18. Heijnen HF, Schiel AE, Fijnheer R, Geuze HJ, Sixma JJ. Activated platelets release two types of membrane vesicles:microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood.1999;94(11):3791-3799.
19. Aatonen M, Gronholm M, Siljander PR. Platelet-derived microvesicles: multitalented participants in intercellular communication. Semin Thromb Hemost.2012;38(1):102-113.
20. Ratajczak J, Wysoczynski M, Hayek F, Janowska-Wieczorek A, Ratajczak MZ. Membrane-derived microvesicles:important and underappreciated mediators of cell-to-cell communication. Leukemia.2006;20(9):1487-1495.
21. Mackman N. On the trail of microparticles. Circ Re.2009;104(8):925-927.
22. Podrez EA, Byzova TV, Febbraio M, Salomon RG, Ma Y, Valiyaveettil M, Poliakov E, Sun M, Finton PJ, Curtis BR, Chen J, Zhang R, Silverstein RL, Hazen SL. Platelet CD36 links hyperlipidemia, oxidant stress and a prothrombotic phenotype. Nat Med. 2007; 13(9):1086-1095.
23. Ghosh A, Li W, Febbraio M, Espinola RG, McCrae KR, Cockrell E, Silverstein RL. Platelet CD36 mediates interactions with endothelial cell-derived microparticles and contributes to thrombosis in mice. J Clin Invest. 2008; 118(5):1934-1943.
24. Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell. 2000;103(2):239-252.
25. Nozaki S, Kashiwagi H, Yamashita S, Nakagawa T, Kostner B, Tomiyama Y, Nakata A, Ishigami M, Miyagawa J, Kameda-Takemura K, et al. Reduced uptake of oxidized low density Hpoproteins in monocyte-derived macrophages from CD36-deficient subjects. JClin Invest.1995;96(4):1859-1865.
26. Wu TW, Zeng LH, Fung KP, Wu J, Pang H, Grey AA, Weisel RD, Wang JY. Effect of sodium tanshinone ⅡA sulfonate in the rabbit myocardium and on human cardiomyocytes and vascular endothelial cells. Biochem pharmacol. 1993;46(12):2327-2332.
27. Takahashi K, Ouyang X, Komatsu K, Nakamura N, Hattori M, Baba A, Azuma J. Sodium tanshinone ⅡA sulfonate derived from Danshen (Salvia miltiorrhiza) attenuates hypertrophy induced by angiotensin Ⅱ in cultured neonatal rat cardiac cells. Biochem pharmacol.2002;64(4):745-749.
28. Lin HC, Ding HY, Chang WL. Two new fatty diterpenoids from Salvia miltiorrhiza. J Nat Prod.2001;64(5):648-650.
29. Wang N, Luo HW, Niwa M, Ji J. A new platelet aggregation inhibitor from Salvia miltiorrhiza. Planta Med.1989;55(4):390-391.
2. Watson T, Shantsila E, Lip GY. Mechanisms of thrombogenesis in atrial fibrillation:Virchow's triad revisited. Lancet 2009;373(9658):155-166.
3. Pongratz G, Brandt-Pohlmann M, Henneke KH, Pohle C, Zink D, Gehling Q Bachmann K. Platelet activation in embolic and preembolic status of patients with nonrheumatic atrial fibrillation. Chest.1997;111(4):929-933.
4. Katritsis DG. Is atrial fibrillation an inflammatory disorder? Eur Heart J. 2006;27(7):886.
5. Cocucci E, Racchetti G, Meldolesi J. Shedding microvesicles:artefacts no more. Trends Cell Biol.2009;19(2):43-51.
6. Del Conde I, Shrimpton CN, Thiagarajan P, Lopez JA. Tissue-factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. Blood 2005;106(5):1604-1611.
7. Bastida E, Ordinas A, Escolar G, Jamieson GA. Tissue factor in microvesicles shed from U87MG human glioblastoma cells induces coagulation, platelet aggregation, and thrombogenesis. Blood.1984;64(1):177-184.
8. Ratajczak J, Wysoczynski M, Hayek F, Janowska-Wieczorek A, Ratajczak MZ. Membrane-derived microvesicles:important and underappreciated mediators of cell-to-cell communication. Leukemia.2006;20(9):1487-1495.
9. Sinauridze EI, Kireev DA, Popenko NY, Pichugin AV, Panteleev MA, Krymskaya OV, Ataullakhanov FI. Platelet microparticle membranes have 50-to 100-fold higher specific procoagulant activity than activated platelets. Thrombo Haemostasis.2007;97(3):425-434.
10. Shantsila E, Kamphuisen PW, Lip GY. Circulating microparticles in cardiovascular disease:implications for atherogenesis and atherothrombosis. J Thromb Haemost.2010; 8(11):2358-2368.
11. Pignoli P, Tremoli E, Poli A, Oreste P, Paoletti R. Intimal plus medial thickness of the arterial wall:a direct measurement with ultrasound imaging. Circulation.1986;74(6):1399-1406.
12. Sutton-Tyrrell K, Alcorn HG, Wolfson SK, Jr., Kelsey SF, Kuller LH. Predictors of carotid stenosis in older adults with and without isolated systolic hypertension. Stroke.1993;24(3):355-361.
13. Deng JT, Parsons PG. Solid phase immunoassay for high molecular weight alkaline phosphatase in human sera using a specific monoclonal antibody. Clin chimacta.1988;176(3):291-301.
14. Wang H, Yan HM, Tang MX, Wang ZH, Zhong M, Zhang Y, Deng JT, Zhang W. Increased serum levels of microvesicles in nonvalvular atrial fibrillation determinated by ELISA using a specific monoclonal antibody AD-1. Clin chim acta.2010;411(21-22):1700-1704.
15. Wattigney WA, Mensah GA, Croft JB. Increasing trends in hospitalization for atrial fibrillation in the United States,1985 through 1999:implications for primary prevention. Circulation.2003;108(6):711-716.
16. Bornstein N, Corea F, Galllai V, Parnetti L. Heart-brain relationship:atrial fibrillation and stroke. Clin Exp Hypertens.2002;24(7-8):493-499.
17. Wolf PA, Abbott RD, Kannel WB. Atrial fibrillation:a major contributor to stroke in the elderly. The Framingham Study. Arch Intern Med. 1987;147(9):1561-1564.
18. Tayal AH, Tian M, Kelly KM, Jones SC, Wright DG, Singh D, Jarouse J, Brillman J, Murali S, Gupta R. Atrial fibrillation detected by mobile cardiac outpatient telemetry in cryptogenic TIA or stroke. Neurology. 2008;71(21):1696-1701.
19. Das RR, Seshadri S, Beiser AS, Kelly-Hayes M, Au R, Himali JJ, Kase CS, Benjamin EJ, Polak JF, O'Donnell CJ, Yoshita M, D'Agostino RB, Sr., DeCarli C, Wolf PA. Prevalence and correlates of silent cerebral infarcts in the Framingham offspring study. Stroke.2008;39(11):2929-2935.
20. Pink J, Lane S, Pirmohamed M, Hughes DA. Dabigatran etexilate versus warfarin in management of non-valvular atrial fibrillation in UK context: quantitative benefit-harm and economic analyses. BMJ.2011; 343:d6333.
21. Singer DE, Chang Y, Fang MC, Borowsky LH, Pomernacki NK, Udaltsova N, Go AS. The net clinical benefit of warfarin anticoagulation in atrial fibrillation. Ann Intern Med.2009;151(5):297-305.
22. Gage BF, van Walraven C, Pearce L, Hart RG, Koudstaal PJ, Boode BS, Petersen P. Selecting patients with atrial fibrillation for anticoagulation:stroke risk stratification in patients taking aspirin. Circulation. 2004; 110(16):2287-2292.
23.张学义,张薇,张运.非瓣膜性心房颤动缺血性脑卒中危险因素的超声研究.中华超声影像学杂志,2005；14(2)：96-98.
24. Vinogradova O, elyvis A, Velyviene A, Hu B, Haas T, Plow E, Qin J. A structural mechanism of integrin alpha(IIb)beta(3) "inside-out" activation as regulated by its cytoplasmic face. Cell.2002;110(5):587-597.
25. Michelson AD, Barnard MR, Krueger LA, Frelinger AL,3rd, Furman MI. Evaluation of platelet function by flow cytometry. Methods. 2000;21(3):259-270.
26. Minamino T, Kitakaze M, Sanada S, Asanuama H, Kurotobi T, Koretsune Y, Fukunami M, Kuzuya T, Hoki N, Hori M. Increased expression of P-selectin on platelets is a risk factor for silent cerebral infarction in patients with atrial fibrillation:role of nitric oxide. Circulation.1998;98(17):1721-1727.
27. Kamath S, Blann AD, Caine GJ, Gurney D, Chin BS, Lip GY. Platelet P-selectin levels in relation to plasma soluble P-selectin and beta-thromboglobulin levels in atrial fibrillation. Strok.2002;33(5):1237-1242.
28. Li-Saw-Hee FL, Blann AD, Gurney D, Lip GY. Plasma von Willebrand factor, fibrinogen and soluble P-selectin levels in paroxysmal, persistent and permanent atrial fibrillation. Effects of cardioversion and return of left atrial function. Eur Heart J.2001;22(18):1741-1747.
29. Li-Saw-Hee FL, Blann AD, Lip GY. A cross-sectional and diurnal study of thrombogenesis among patients with chronic atrial fibrillation. J Am Coll Cardiol. 2000;35(7):1926-1931.
30. Ord D, Ord T. Mouse NIPK interacts with ATF4 and affects its transcriptional activity. Exp Cell Res.2003;286(2):308-320.
31. Li R, Duncan BB, Metcalf PA, Crouse JR,3rd, Sharrett AR, Tyroler HA, Barnes R, Heiss G. B-mode-detected carotid artery plaque in a general population. Atherosclerosis Risk in Communities (ARIC) Study Investigators. Stroke.1994;25(12):2377-2383.
32. Burke GL, Evans GW, Riley WA, Sharrett AR, Howard G, Barnes RW, Rosamond W, Crow RS, Rautaharju PM, Heiss G. Arterial wall thickness is associated with prevalent cardiovascular disease in middle-aged adults. The Atherosclerosis Risk in Communities (ARIC) Study. Stroke. 1995;26(3):386-391.
33. Watanabe T, Takeishi Y, Hirono O, Itoh M, Matsui M, Nakamura K, Tamada Y, Kubota I. C-reactive protein elevation predicts the occurrence of atrial structural remodeling in patients with paroxysmal atrial fibrillation. Heart Vessels.2005;20(2):45-49.
34. Watanabe E, Arakawa T, Uchiyama T, Kodama I, Hishida H. High-sensitivity C-reactive protein is predictive of successful cardioversion for atrial fibrillation and maintenance of sinus rhythm after conversion. Intern J Cardiol. 2006;108(3):346-353.
35. Dernellis J, Panaretou M. Effects of C-reactive protein and the third and fourth components of complement (C3 and C4) on incidence of atrial fibrillation. Am J Cardiol.2006;97(2):245-248.
36. Kim YH, Lim DS, Lee JH, Shim WJ, Ro YM, Park GH, Becker KG, Cho-Chung YS, Kim MK. Gene expression profiling of oxidative stress on atrial fibrillation in humans. Exp Mol Med.2003;35(5):336-349.
37. Carnes CA, Chung MK, Nakayama T, Nakayama H, Baliga RS, Piao S, Kanderian A, Pavia S, Hamlin RL, McCarthy PM, Bauer JA, Van Wagoner DR. Ascorbate attenuates atrial pacing-induced peroxynitrite formation and electrical remodeling and decreases the incidence of postoperative atrial fibrillation. Circ Res.2001;89(6):E32-38.
38. Kim YM, Guzik TJ, Zhang YH, Zhang MH, Kattach H, Ratnatunga C, Pillai R, Channon KM, Casadei B. A myocardial Nox2 containing NAD(P)H oxidase contributes to oxidative stress in human atrial fibrillation. Circ Res. 2005;97(7):629-636.
39. Inal JM, Ansa-Addo EA, Stratton D, Kholia S, Antwi-Baffour SS, Jorfi S, Lange S. Microvesicles in health and disease. Arch Immunol Ther Ex. 2012;60(2):107-121.
40. Simpson RJ, Lim JW, Moritz RL, Mathivanan S. Exosomes:proteomic insights and diagnostic potential. Expert Rev Proteomic.2009;6(3):267-283.
41. Scanu A, Molnarfi N, Brandt KJ, Gruaz L, Dayer JM, Burger D. Stimulated T cells generate microparticles, which mimic cellular contact activation of human monocytes:differential regulation of pro-and anti-inflammatory cytokine production by high-density lipoproteins. J Leukocyte Biol. 2008;83(4):921-927.
42. Nolan S, Dixon R, Norman K, Hellewell P, Ridger V. Nitric oxide regulates neutrophil migration through microparticle formation. Am J Pathol. 2008;172(1):265-273.
43. Kim JW, Wieckowski E, Taylor DD, Reichert TE, Watkins S, Whiteside TL. Fas ligand-positive membranous vesicles isolated from sera of patients with oral cancer induce apoptosis of activated T lymphocytes. Clin Cancer Res. 2005;11(3):1010-1020.
44. Siljander PR. Platelet-derived microparticles-an updated perspective. Thrombo Res.2011;127 Suppl 2:S30-33.
45. Hunter MP, Ismail N, Zhang X, Aguda BD, Lee EJ, Yu L, Xiao T, Schafer J, Lee ML, Schmittgen TD, Nana-Sinkam SP, Jarjoura D, Marsh CB. Detection of microRNA expression in human peripheral blood microvesicles. PloS one. 2008;3(11):e3694.
46. Aatonen M, Gronholm M, Siljander PR. Platelet-derived microvesicles: multitalented participants in intercellular communication. Semin Thromb Hemost.2012;38(1):102-113.
47. Ardoin SP, Shanahan JC, Pisetsky DS. The role of microparticles in inflammation and thrombosis. Scand J Immunol.2007;66(2-3):159-165.
48. Deng JT, Hoylaerts MF, Van Hoof VO, De Broe ME. Differential release of human intestinal alkaline phosphatase in duodenal fluid and serum. Clin Chem. 1992;38(12):2532-2538.
49. Van Hoof VO, Deng JT, De Broe ME. How do plasma membranes reach the circulation? Clin Chim Acta.1997;266(1):23-31.
50. Choudhury A, Chung I, Blann AD, Lip GY. Elevated platelet microparticle levels in nonvalvular atrial fibrillation:relationship to p-selectin and antithrombotic therapy. Chest.2007;131(3):809-815.
51. Ederhy S, Di Angelantonio E, Mallat Z, Hugel B, Janower S, Meuleman C, Boccara F, Freyssinet JM, Tedgui A, Cohen A. Levels of circulating procoagulant microparticles in nonvalvular atrial fibrillation. Am J Cardiol. 2007;100(6):989-994.
52. Ghosh A, Li W, Febbraio M, Espinola RG, McCrae KR, Cockrell E, Silverstein RL. Platelet CD36 mediates interactions with endothelial cell-derived microparticles and contributes to thrombosis in mice. J Clin Invest. May 2008;118(5):1934-1943.
1. Caldeira D, David C, Sampaio C. Rate versus rhythm control in atrial fibrillation and clinical outcomes:updated systematic review and meta-analysis of randomized controlled trials. Arch Cardiovasc Dis. 2012;105(4):226-238.
2. Katritsis DG. Is atrial fibrillation an inflammatory disorder? Eur Heart J. 2006;27(7):886.
3. Watson T, Shantsila E, Lip GY. Mechanisms of thrombogenesis in atrial fibrillation:Virchow's triad revisited. Lancet.2009;373(9658):155-166.
4. Oleksowicz L, Mrowiec Z, Isaacs R, Dutcher JP, Puszkin E. Morphologic and ultrastructural evidence of interleukin-6 induced platelet activation. Am J hematol.1995;48(2):92-99.
5. Raposeiras-Roubin S, Rodino-Janeiro BK, Grigorian-Shamagian L, Seoane-Blanco A, Moure-Gonzalez M, Varela-Roman A, Alvarez E, Gonzalez-Juanatey JR. Evidence for a role of advanced glycation end products in atrial fibrillation.Int J Cardiol.2012;157(3):397-402.
6. Del Conde I, Shrimpton CN, Thiagarajan P, Lopez JA. Tissue-factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. Blood.2005;106(5):1604-1611.
7. Cocucci E, Racchetti G, Meldolesi J. Shedding microvesicles:artefacts no more. Trends Cell Biol.2009;19(2):43-51.
8. Bastida E, Ordinas A, Escolar G, Jamieson GA. Tissue factor in microvesicles shed from U87MG human glioblastoma cells induces coagulation, platelet aggregation, and thrombogenesis. Blood.1984;64(1):177-184.
9. Aatonen M, Gronholm M, Siljander PR. Platelet-derived microvesicles: multitalented participants in intercellular communication. Semin Thromb Hemost.2012;38(1):102-113.
10. Ratajczak J, Wysoczynski M, Hayek F, Janowska-Wieczorek A, Ratajczak MZ. Membrane-derived microvesicles:important and underappreciated mediators of cell-to-cell communication. Leukemia.2006;20(9):1487-1495.
11. Mackman N. On the trail of microparticles. Circ Res.2009; 104(8):925-927.
12. Chen K, Febbraio M, Li W, Silverstein RL. A specific CD36-dependent signaling pathway is required for platelet activation by oxidized low-density lipoprotein. Circ Res.2008;102(12):1512-1519.
13. Abe H, Matsubara T, Iehara N, Nagai K, Takahashi T, Arai H, Kita T, Doi T. Type IV collagen is transcriptionally regulated by Smadl under advanced glycation end product (AGE) stimulation. J Biol Chem. 2004;279(14):14201-14206.
14. Wattigney WA, Mensah GA, Croft JB. Increasing trends in hospitalization for atrial fibrillation in the United States,1985 through 1999:implications for primary prevention. Circulation.2003;108(6):711-716.
15. Chien KL, Su TC, Hsu HC, Chang WT, Chen PC, Chen MF, Lee YT. Atrial fibrillation prevalence, incidence and risk of stroke and all-cause death among Chinese. Int J Cardiol.2010;139(2):173-180.
16. Lloyd-Jones DM, Wang TJ, Leip EP, Larson MG, Levy D, Vasan RS, D'Agostino RB, Massaro JM, Beiser A, Wolf PA, Benjamin EJ. Lifetime risk for development of atrial fibrillation:the Framingham Heart Study. Circulation.2004; 110(9):1042-1046.
17. Bornstein N, Corea F, Galllai V, Parnetti L. Heart-brain relationship:atrial fibrillation and stroke. Clin Exp Hypertens.2002;24(7-8):493-499.
18. Wolf PA, Abbott RD, Kannel WB. Atrial fibrillation:a major contributor to stroke in the elderly. The Framingham Study. Arch Internal Med. 1987;147(9):1561-1564.
19. Lin HJ, Wolf PA, Kelly-Hayes M, Beiser AS, Kase CS, Benjamin EJ, D'Agostino RB. Stroke severity in atrial fibrillation. The Framingham Study. Stroke.1996;27(10):1760-1764.
20. Pink J, Lane S, Pirmohamed M, Hughes DA. Dabigatran etexilate versus warfarin in management of non-valvular atrial fibrillation in UK context: quantitative benefit-harm and economic analyses.BMJ.2011.343:d6333.
21. Singer DE, Albers GW, Dalen JE, Go AS, Halperin JL, Manning WJ. Antithrombotic therapy in atrial fibrillation:the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest.2004; 126(3 Suppl):429S-456S.
22. The AFFIRM Investigators. A comparison of rate control and rhythm control in patients with atrial fibrillation. NEngl J Med.2002; 347:1825-1833
23. Steinberg JS, Sandaniantz A, Kron J, Krahn A, Denny DM, Daubert J, Campbell WB, Havranek E, Murray K, Olshansky B, O' Neill G, Sami M, Schmidt S, Storm R, Zabalgoitia M, Miller J, Chandler M, Nasco EM, Greene HL. Analysis of cause-specific mortality in the Atrial Fibrillation Follow-up Investigation of Rhythm Management AFFIRM) study. Circulation.2004; 109(16):1973-1980.
24. Corley SD, Epstein AE, DiMarco JP, Domanski MJ, Geller N, Greene HL, Josephson RA, Kellen JC, Klein RC, Krahn AD, Mickel M, Mitchell LB, Nelson JD, Rosenberg Y, Schron E, Shemanski L, Waldo AL, Wyse DG; AFFIRM Investigators. Relationships between sinus rhythm, treatment, and survival in the Atrial Fibrillation Follow-up Investigation of Rhythm Management(AFFIRM) study. Circulation.2004; 109(12):1509-1513.
25. Cleland JG, Coletta AP, Abdellah AT, Cullington D, Clark AL, Rigby AS. Clinical trials update from the American Heart Association 2007:CORONA, RethinQ, MASCOT, AF-CHF, HART, MASTER, POISE and stem cell therapy. Eur J Heart Fail.2008; 10(1):102-108.
26. Van Gelder IC, Hagens VE, Bosker HA, Kingma JH, Kamp O, Kingma T, Said SA, Darmanata JI, Timmermans AJ, Tijssen JG, Crijns HJ; Rate Control versus Electrical Cardioversion for Persistent Atrial Fibrillation Study Group. A comparison of rate control and rhythm control in patients with recurrent persistent atrial fibrillation. NEnglJMed.2002; 347(23):1834-1840.
27. Marin F, Corral J, Roldan Ⅴ, Gonzalez-Conejero R, del Rey ML, Sogorb F, Lip GY, Vicente V. Factor ⅩⅢ Val34Leu polymorphism modulates the prothrombotic and inflammatory state associated with atrial fibrillation. J Mol Cell Cardiol.2004;37(3):699-704.
28. Cesari M, Penninx BW, Newman AB, Kritchevsky SB, Nicklas BJ, Sutton-Tyrrell K, Rubin SM, Ding J, Simonsick EM, Harris TB, Pahor M. Inflammatory markers and onset of cardiovascular events:results from the Health ABC study. Circulation.2003;108(19):2317-2322.
29. Goldin A, Beckman JA, Schmidt AM, Creager MA. Advanced glycation end products:sparking the development of diabetic vascular injury. Circulation. 2006;114(6):597-605.
30. Friedman EA. Advanced glycosylated end products and hyperglycemia in the pathogenesis of diabetic complications. Diabetes care.1999;22 Suppl 2:B65-71.
31. Smit AJ, Lutgers HL. The clinical relevance of advanced glycation endproducts (AGE) and recent developments in pharmaceutics to reduce AGE accumulation. Curr Med Chem.2004;11(20):2767-2784.
32. Weinrauch LA, Berger AJ, Aronson D, Gleason RE, Lee AT, D' Elia JA. Regression of left ventricular hypertrophy in diabetic nephropathy:loss of parasympathetic function predicts response to treatment. J Clin Hypertens. 2006;8(5):330-335.
33. McNulty M, Mahmud A, Feely J. Advanced glycation end-products and arterial stiffness in hypertension. Am J Hypertension.2007;20(3):242-247.
34. Meerwaldt R, Lutgers HL, Links TP, Graaff R, Baynes JW, Gans RO, Smit AJ. Skin autofluorescence is a strong predictor of cardiac mortality in diabetes. Diabetes care.2007;30(1):107-112.
35. Aronson D. Pharmacological prevention of cardiovascular aging--targeting the Maillard reaction. Brit J Pharmacol.2004;142(7):1055-1058.
36. Reddy S, Bichler J, Wells-Knecht KJ, Thorpe SR, Baynes JW. N epsilon-(carboxymethyl)lysine is a dominant advanced glycation end product (AGE) antigen in tissue proteins. Biochemistry.1995;34(34):10872-10878.
37. Verbeke P, Clark BF, Rattan SI. Modulating cellular aging in vitro:hormetic effects of repeated mild heat stress on protein oxidation and glycation. Exp Gerontol.2000;35(6-7):787-794.
38. Schmidt AM, Yan SD, Stern DM. The dark side of glucose. Nat Med. 1995;1(10):1002-1004.
39. Inal JM, Ansa-Addo EA, Stratton D, Kholia S, Antwi-Baffour SS, Jorfi S, Lange S. Microvesicles in health and disease. Arch Immunol Ther Ex. 2012;60(2):107-121.
40. Lima LG, Chammas R, Monteiro RQ, Moreira ME, Barcinski MA. Tumor-derived microvesicles modulate the establishment of metastatic melanoma in a phosphatidylserine-dependent manner. Cancer lett. 2009;283(2):168-175.
41. Hugel B, Martinez MC, Kunzelmann C, Freyssinet JM. Membrane microparticles:two sides of the coin. Physiology.2005;20:22-27.
42. Muralidharan-Chari V, Clancy JW, Sedgwick A, D'Souza-Schorey C. Microvesicles:mediators of extracellular communication during cancer progression. J Cell Sci.2010;123(Pt 10):1603-1611.
43. Siljander PR. Platelet-derived microparticles-an updated perspective. Thromb Res.2011; 127 Suppl 2:S30-33.
44. Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, Geuze HJ. B lymphocytes secrete antigen-presenting vesicles. J Exp Med. 1996;183(3):1161-1172.
45. Febbraio M, Hajjar DP, Silverstein RL. CD36:a class B scavenger receptor involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism. J Clin Invest.2001; 108(6):785-791.
46. Greenberg ME, Sun M, Zhang R, Febbraio M, Silverstein R, Hazen SL. Oxidized phosphatidylserine-CD36 interactions play an essential role in macrophage-dependent phagocytosis of apoptotic cells. J Exp Med. 2006;203(12):2613-2625.
47. Nicholson AC, Frieda S, Pearce A, Silverstein RL. Oxidized LDL binds to CD36 on human monocyte-derived macrophages and transfected cell lines. Evidence implicating the lipid moiety of the lipoprotein as the binding site. Arterioscl Throm Vas.1995;15(2):269-275.
48. Boullier A, Bird DA, Chang MK, Dennis EA, Friedman P, Gillotre-Taylor K, Horkko S, Palinski W, Quehenberger O, Shaw P, Steinberg D, Terpstra V, Witztum JL. Scavenger receptors, oxidized LDL, and atherosclerosis. Ann NY Acad Sci.2001;947:214-222.
49. Nicholson AC, Hajjar DP. CD36, oxidized LDL and PPAR gamma: pathological interactions in macrophages and atherosclerosis. Vasc Pharm. 2004;41(4-5):139-146.
50. Rahaman SO, Lennon DJ, Febbraio M, Podrez EA, Hazen SL, Silverstein RL. A CD36-dependent signaling cascade is necessary for macrophage foam cell formation. Cell metah.2006;4(3):211-221.
51. Ge Y, Elghetany M. CD36:a multiligand molecule. Lab Hematol.2005; 11(1): 31-37.
52. Lin M, Shieh SH, Yang TF. Frequency of platelet-specific antigens among Chinese in Taiwan. Transfusion.1993;33(2):155-157.
53. Hirano K, Kuwasako T, Nakagawa-Toyama Y, Janabi M, Yamashita S, Matsuzawa Y Pathophysiology of human genetic CD36 deficiency. Trends Cardiovasc Med.2003; 13(4):136-141.
54. Ikeda H, Mitani T, Ohnuma M, Haga H, Ohtzuka S, Kato T, Nakase T, Sekiguchi S. A new platelet-specific antigen, Naka involved in the refractories of HLA-matched platelet transfusion. Vox Sang.1989; 57(3):213-217.
55. Yanai H, Chiba H, Fujiwara H, Morimoto M, Abe K, Yoshida S, Takahashi Y, Fuda H, Hui SP, Akita H, Kobayashi K, Matsuno K. Phenotype-genotype correlation in CD36 deficiency types Ⅰ and Ⅱ. Thromb Haemost.2000; 84(3):436-441.
56. Curtis BR, Aster RH. Incidence of the Nak(a)-negative platelet phenotype in African Americans is similar to that of Asians. Transfusion.1996; 36(4): 331-334.
57. Nozaki S, Kashiwagi H, Yamashita S, Nakagawa T, Kostner B, Tomiyama Y, Nakata A, Ishigami M, Miyagawa J, Kameda-Takemura K, et al. Reduced uptake of oxidized low density lipoproteins in monocyte-derived macrophages from CD36-deficient subjects. J Clin Invest.1995;96(4):1859-1865.
58. Febbraio M, Abumrad NA, Hajjar DP, Sharma K, Cheng W, Pearce SF, Silverstein RL. A null mutation in murine CD36 reveals an important role in fatty acid and lipoprotein metabolism. J Bio lChem. 1999;274(27):19055-19062.
59. Han J, Zhou X, Yokoyama T, Hajjar DP, Gotto AM, Jr., Nicholson AC. Pitavastatin downregulates expression of the macrophage type B scavenger receptor, CD36. Circulation.2004;109(6):790-796.
60. Fuhrman B, Koren L, Volkova N, Keidar S, Hayek T, Aviram M. Atorvastatin therapy in hypercholesterolemic patients suppresses cellular uptake of oxidized-LDL by differentiating monocytes. Atherosclerosis. 2002;164(1):179-185.
61. Ozer NK, Negis Y, Aytan N, Villacorta L, Ricciarelli R, Zingg JM, Azzi A. Vitamin E inhibits CD36 scavenger receptor expression in hypercholesterolemic rabbits. Atherosclerosis.2006; 184(1):15-20.
62. Ghosh A, Li W, Febbraio M, Espinola RG, McCrae KR, Cockrell E, Silverstein RL. Platelet CD36 mediates interactions with endothelial cell-derived microparticles and contributes to thrombosis in mice. J Clin Invest. 2008;118(5):1934-1943.
63. Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell. 2000;103(2):239-252.
64. Wu TW, Zeng LH, Fung KP, Wu J, Pang H, Grey AA, Weisel RD, Wang JY. Effect of sodium tanshinone ⅡA sulfonate in the rabbit myocardium and on human cardiomyocytes and vascular endothelial cells. Biochem Pharmcol. 1993;46(12):2327-2332.
65. Takahashi K, Ouyang X, Komatsu K, Nakamura N, Hattori M, Baba A, Azuma J. Sodium tanshinone ⅡA sulfonate derived from Danshen (Salvia miltiorrhiza) attenuates hypertrophy induced by angiotensin Ⅱ in cultured neonatal rat cardiac cells. Biochem Pharmcol.2002;64(4):745-749.
66. Lin HC, Ding HY, Chang WL. Two new fatty diterpenoids from Salvia miltiorrhiza. JNa t Prod.2001;64(5):648-650.
67. Wang N, Luo HW, Niwa M, Ji J. A new platelet aggregation inhibitor from Salvia miltiorrhiza. Planta Med.1989;55(4):390-391.
1. Wattigney WA, Mensah GA, Croft JB. Increasing trends in hospitalization for atrial fibrillation in the United States,1985 through 1999:implications for primary prevention. Circulation.2003;108(6):711-716.
2. Bornstein N, Corea F, Galllai V, Parnetti L. Heart-brain relationship:atrial fibrillation and stroke. Clin Exp Hypertens.2002;24(7-8):493-499.
3. Wolf PA, Abbott RD, Kannel WB. Atrial fibrillation:a major contributor to stroke in the elderly. The Framingham Study. Arch Intern Med. 1987;147(9):1561-1564.
4.张学义,张薇,张运.非瓣膜性心房颤动缺血性脑卒中危险因素的超声研究.中华超声影像学杂志,2005；14(2)：96-98.
5. Watson T, Shantsila E, Lip GY. Mechanisms of thrombogenesis in atrial fibrillation:Virchow's triad revisited. Lancet.2009;373(9658):155-166.
6. Caldeira D, David C, Sampaio C. Rate versus rhythm control in atrial fibrillation and clinical outcomes:updated systematic review and meta-analysis of randomized controlled trials. Arch Cardiovasc Dis. 2012;105(4):226-238.
7. Raposeiras-Roubin S, Rodino-Janeiro BK, Grigorian-Shamagian L, Seoane-Blanco A, Moure-Gonzalez M, Varela-Roman A, Alvarez E, Gonzalez-Juanatey JR. Evidence for a role of advanced glycation end products in atrial fibrillation. Int J Cardiol.2012;157(3):397-402.
8. Ardoin SP, Shanahan JC, Pisetsky DS. The role of microparticles in inflammation and thrombosis. Scand J Immunol.2007;66(2-3):159-165.
9. Janiszewski M, Do Carmo AO, Pedro MA, Silva E, Knobel E, Laurindo FR. Platelet-derived exosomes of septic individuals possess proapoptotic NAD(P)H oxidase activity:A novel vascular redox pathway. Criti Care Med. 2004;32(3):818-825.
10. Benjamin EJ, Wolf PA, D' Agostino RB, Silbershatz H, Kannel WB, Levy D. Impact of atrial fibrillation on the risk of death:the Framingham Heart Study. Circulation.1998;98(10):946-952.
11. Lip GY, Boos CJ. Antithrombotic treatment in atrial fibrillation. Heart. 2006;92(2):155-161.
12. Lakshminarayan K, Solid CA, Collins AJ, Anderson DC, Herzog CA. Atrial fibrillation and stroke in the general medicare population:a 10-year perspective (1992 to 2002). Stroke.2006;37(8):1969-1974.
13. Pongratz G, Brandt-Pohlmann M, Henneke KH, Pohle C, Zink D, Gehling G, Bachmann K. Platelet activation in embolic and preembolic status of patients with nonrheumatic atrial fibrillation. Chest.1997;111(4):929-933.
14. Lip GY. The prothrombotic state in atrial fibrillation:new insights, more questions, and clear answers needed. Am Heart J.2000;140(3):348-350.
15. Minamino T, Kitakaze M, Sanada S, Asanuama H, Kurotobi T, Koretsune Y, Fukunami M, Kuzuya T, Hoki N, Hori M. Increased expression of P-selectin on platelets is a risk factor for silent cerebral infarction in patients with atrial fibrillation:role of nitric oxide. Circulation.1998;98(17):1721-1727.
16. Gage BF, van Walraven C, Pearce L, Hart RG, Koudstaal PJ, Boode BS, Petersen P. Selecting patients with atrial fibrillation for anticoagulation:stroke risk stratification in patients taking aspirin. Circulation. 2004;110(16):2287-2292.
17. Inal JM, Ansa-Addo EA, Stratton D, Kholia S, Antwi-Baffour SS, Jorfi S, Lange S. Microvesicles in health and disease. Arch Immunol Ther Ex. 2012;60(2):107-121.
18. Heijnen HF, Schiel AE, Fijnheer R, Geuze HJ, Sixma JJ. Activated platelets release two types of membrane vesicles:microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood.1999;94(11):3791-3799.
19. Aatonen M, Gronholm M, Siljander PR. Platelet-derived microvesicles: multitalented participants in intercellular communication. Semin Thromb Hemost.2012;38(1):102-113.
20. Ratajczak J, Wysoczynski M, Hayek F, Janowska-Wieczorek A, Ratajczak MZ. Membrane-derived microvesicles:important and underappreciated mediators of cell-to-cell communication. Leukemia.2006;20(9):1487-1495.
21. Mackman N. On the trail of microparticles. Circ Re.2009;104(8):925-927.
22. Podrez EA, Byzova TV, Febbraio M, Salomon RG, Ma Y, Valiyaveettil M, Poliakov E, Sun M, Finton PJ, Curtis BR, Chen J, Zhang R, Silverstein RL, Hazen SL. Platelet CD36 links hyperlipidemia, oxidant stress and a prothrombotic phenotype. Nat Med. 2007; 13(9):1086-1095.
23. Ghosh A, Li W, Febbraio M, Espinola RG, McCrae KR, Cockrell E, Silverstein RL. Platelet CD36 mediates interactions with endothelial cell-derived microparticles and contributes to thrombosis in mice. J Clin Invest. 2008; 118(5):1934-1943.
24. Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell. 2000;103(2):239-252.
25. Nozaki S, Kashiwagi H, Yamashita S, Nakagawa T, Kostner B, Tomiyama Y, Nakata A, Ishigami M, Miyagawa J, Kameda-Takemura K, et al. Reduced uptake of oxidized low density Hpoproteins in monocyte-derived macrophages from CD36-deficient subjects. JClin Invest.1995;96(4):1859-1865.
26. Wu TW, Zeng LH, Fung KP, Wu J, Pang H, Grey AA, Weisel RD, Wang JY. Effect of sodium tanshinone ⅡA sulfonate in the rabbit myocardium and on human cardiomyocytes and vascular endothelial cells. Biochem pharmacol. 1993;46(12):2327-2332.
27. Takahashi K, Ouyang X, Komatsu K, Nakamura N, Hattori M, Baba A, Azuma J. Sodium tanshinone ⅡA sulfonate derived from Danshen (Salvia miltiorrhiza) attenuates hypertrophy induced by angiotensin Ⅱ in cultured neonatal rat cardiac cells. Biochem pharmacol.2002;64(4):745-749.
28. Lin HC, Ding HY, Chang WL. Two new fatty diterpenoids from Salvia miltiorrhiza. J Nat Prod.2001;64(5):648-650.
29. Wang N, Luo HW, Niwa M, Ji J. A new platelet aggregation inhibitor from Salvia miltiorrhiza. Planta Med.1989;55(4):390-391.