摘要
血小板是血液中最小的、具有多种功能的细胞,它在血栓形成、动脉粥样硬化等病理过程中扮演了重要的角色。蛇毒中广泛存在抑制血小板聚集的活性组分,它们的生化特性及作用机理均不尽相同。近年来,已有文献报道了从不同的种属的蛇毒,特别是从蝰科蝮亚科毒蛇所分泌的蛇毒中提取及分离出多种血小板聚集抑制剂,并对它们的活性进行了分析。关于尖吻蝮蛇毒这方面的研究较少,本课题拟从尖吻蝮蛇蛇毒(Agkistrodon acutus venom,AAV)中分离纯化出具有的抗血小板聚集作用组分,测定其理化性质,观察其体内外抗血小板聚集活性,为进一步研究开发提供实验依据。
1.抗血小板聚集组分的分离纯化
应用Superdex 75凝胶过滤色谱,从尖吻蝮蛇粗毒中分离得到6个蛋白峰;其中蛋白峰Ⅳ抑制二磷酸腺苷(ADP)诱导的血小板聚集活性最强,该组分再经Sephadex G-25凝胶色谱、DEAE Sepharose Fast Flow离子交换色谱和Lichrospher C18反相色谱等纯化得一个电泳纯蛋白组分,暂定名为AAV-IV3-2。SDS—PAGE蛋白电泳鉴定发现,AAV-IV3-2呈单一条蛋白区带。
2. AAV-IV3-2部分理化性质测定根据AAV-IV3-2和Marker在凝胶上的相对位置,利用Image Master VDS凝胶成像系统中的软件计算得出AAV-IV3-2的分子量大约为15.3kDa。
3.AAV-IV3-2对血小板聚集的影响
采用经典的Born法,发现AAV-IV3-2在体外可抑制ADP和凝血酶诱导的血小板聚集,且该作用呈明显的量效关系,AAV-IV3-2对ADP、凝血酶诱导的血小板聚集的IC50分别为1.78μg/ml和5.06μg/ml。
4、AAV-IV3-2对ADP-Na诱导的小鼠急性肺栓塞的影响
采用ADP-Na诱导的小鼠急性肺栓塞模型发现, AAV-IV3-2 iv可以明显缩短小鼠呼吸窘迫时间,中高剂量组(150、200μg/kg)小鼠恢复正常呼吸、自主活动的时间(秒)分别为114.2±31.53和101.9±42.19,与生理盐水组(176.6±57.52)相比均有明显缩短(P<0.05)。提示AAV-IV3-2在体内具有抗血小板作用。
结论:应用凝胶过滤和离子交换色谱方法可以从尖吻蝮蛇毒中分离得到一个相对分子量为15.3kDa、纯度为电泳纯的蛋白组分AAV-IV3-2。AAV-IV3-2能抑制ADP和凝血酶诱导的血小板聚集,并有明显的量效依赖关系;在小鼠体内也具有抗血小板作用。
Snake venom was known to possess components affecting platelet function in various ways. Screening anti-platelet aggregation protein from snake venom becomes an intriguing work because of their potent clinical use in treating abnormal platelet activation and arterial thrombus. This study was designed to isolate and purify anti-platelet aggregation protein from Agkistrodon acutus venom(AAV) and analyse its characterization and biological activities.
1. Purification and characterization of the anti-platelet aggregation protein from Agkistrodon acutus Venom
Six fractions(Ⅰ～Ⅵ) were isolated from AAV by Gel filtration chromatography on Superdex 75 .The fractionⅣpossess the best anti-platelet aggregation activity, so it suggested as a contained the anti-platelet aggregation component. An anti-platelet aggregation protein named AAV-IV3-2 was purified from the fraction by sephadex G-25 gel filtration, DEAE Sepharose Fast Flow ion-exchange and Lichrospher C18 reverse chromatography. It was homogeneous as a single band showed on SDS-polyacrylmide gel electrophoresis (SDS-PAGE) with molecular weight 15.3kDa as calculated by Image Master VDS system.
2. The effects of AAV-IV3-2 on the human platelet aggregation.
According to the Born’s method, the effects of AAV-IV3-2 on platelet aggregation induced by ADP and thrombin were assayed. The results showed that AAV-IV3-2 dose-dependently inhibited ADP- and thrombin-induced platelet aggregation with IC50 1.78 and 5.06μg/ml respectively.
3. The effect of AAV-IV3-2 on the time of respiratory distress of acute pulmonary thrombosis in mice induced by ADP-Na.
The time of respiratory distress of acute pulmonary thrombosis in mice of middle, high dose groups(114.2±31.53、101.9±42.19,S)respectively were remarkably shorter than that of NS group (176.6±57.52,S)(P<0.05).
CONCLUSION
AAV-IV3-2 was purified from Agkistrodon acutus venom with molecular weight 15.3kDa. It was a potent platelet aggregation inhibitor.
引文
[1]阮长耿.血小板一基础与临床[M].上海:上海科学技术出版社,1994.38-41.
[2] Trikha M, De Clerck YA, Markland FS et al .Contortrostatin, a snake venom disintegrin, inhibits beta 1 integrin-mediated human metastatic melanoma cell adhesion and blocks experimental metastasis[J]. Cancer Res, 1994(18), 54: 4993-4998.
[3]沃格尔HG,沃格尔WH.药理学实验指南——新药发现和药理学评价[M].北京:科学出版社,2001,196-8.
[4] Born GVR. Aggregation of blood platelets by adenosine diphosphate and its reversal [J] .Nature, 1962,19(4):927-9.
[5]马世平,刘保林,周素娣,等。西红花总苷的药理学研究Ⅱ:对血凝,血小板聚集及血栓形成的影响[j].中草药,1999,30(3):197
[6] Ouyang C,Huang TF.Potent platelet aggregation inhibitor from Trimeresus gramineus snake venom[J].Biochim Biophys Acta,1983; 757(3):332-41.
[7] Hong SY,Koh YS,Chung KH et al.Snake venom disintegrin, saxatilin, inhibits platelet aggregation, human umbilical vein endothelial cell proliferation, and smooth muscle cell migration[J].Thromb Res,2002, 105(1):79-86.
[8] Dennis MS,Henzel WJ,Pitti RM et al.Platelet glycoprotein IIb-IIIa protein antagonists from snake venoms:Evidence for a family of platelet-aggregation inhibitors[J].Proc Natl Acad Sci USA,1989,87 (7):2471-5 .
[9]陈可冀,防治血栓形成[M].上海:上海科学技术出版社,1990:446.
[10] Fry B,Ritter MR,Markland FS et al. .Inhibitory effects of a snake venom protein on the binding of breast cancer cells to extracelluar matrix components[J] .Proc Am Assoc Cancer Res,1996,37:65-71.
[11] Danen EH,Marcinkiewicz C,Cornelissen IM et al.The disintegrin Eristostatin interferes with integrin alpha 4 beta 1 function and with experimental metastasis of human melanoma cells[J] .Exp Cell Res,1998,238(1):1
[1] Teng,C.-M. and Ko. F.-N. Comparison of the platelet aggregation induced by three thrombin-like enzymes of snake venoms and thrombin. Thromb.Haemost. 59,304-309.
[2] Read MS,et a1.Blood,l989.74:1031.
[3] Kini RM ,Evan s HI.Correlation between the enzymatic activity,anticoagulant and antiplatelet effects of phospholipase A2 imenzymes from Naja nigricoUis venom.Thromb Haeruost,1988;60:170-173.
[4] Hamako J,M atsui T,Suzuki M.et a1 Purification and character1zation of bitiscetin,a noval von Wiflebrand factor modulator protein from Bitisarietans snake venom.Biechem Biophys Res Cornmum,1996.226(1):273～279.
[5] Peng M,Lu W ,Kirby EP,et a1.Alboaggregin-B:a new platelet agonist that binds to platelet membrane glycoprotein Ib.Biochemistry,1991,30:l1529～l1536.
[6] Yoshida E,Fujimura Y,Miura S,et a1.Alboaggregin-B and Botrocetin:two snake venom proteins with highly homologous amio acid sequences but totally distinct functions on vWF binding to platelets. Biochem Biophys Res Commun,1993,191(3):1389～1392.
[7] Andrews RK,Kroll MH,Ward CM,et a1.Binding of a novel 50-kilodalton alboaggregin from Trimeresurus albolabris and related viper venom proteins to the platelet membrane slycoprotein Ib-IX-V complex . Efect on platelet aggregation and slycoprotein Ib-mediated platelet activation. Biochemistry,1996,35(38):12629～12639.
[8] Taniuchi Y,Kawasaki T,Fujimura Y,et a1.Flavocetin-A and—B two high molecular mass glycoprotein Ib hinding protein with high athinity purified from trimeresurus flavoriridis venom inhibit platelet aggregation at high shear stress.Biochem et Biophys Acta ,1995,1244(2-3):331～338.
[9] Savage B.Applaggin and Echistatin hind to platelet GpⅡb/Ⅲa.J Biol Chem ,1990,265:ll766.
[10] Paz Moreno-Murciano M,Monleon D,Calvete JJ, et al. Amino acid sequence and homology modeling of obtustatin, a novel non-RGD-containing short disintegrin isolated from the venom of Vipera lebetina obtusa [J].Protein Sci,2003,12(2):366-71.
[11] Hong SY,Sohn YD,Chung KH,Kim DS. Structural and functional significance of disulfide bonds in saxatilin, a 7.7 kDa disintegrin[J].Biochem Biophys Res Commun,2002,293(1):530-6.
[12] Sutcliffe MJ,Jaseja M,Hyde EI.Thee-dimensional structure of the RGD—containing neurotoxln homologue dend roaspln.NatStruct Bio1.1 9 .1(11):8O2-807.
[13] Huang TF.Ouyang CH.Mechanism of action of the platelet aggregation inhibit or purified from Agkistrodon halys snake venom.Toxicon .1984,22(2):243.
[14] Teng CM. Biphasic effect on platelet ag regation by phospholipase A2 purified from Vipera rttssellii snake venom.Biochlm Biophys Acta.1984,722-393.
[15] Kurt F Stocker.Medlcal us@of snake venom proteins.Florida}CRC,1990.19l～2l1.
[16] Ouyang C, Huang, TF1 Inh ibit ion of p latelet aggregation by 5’- nucleo t idase purified from T rim eresu rusg ram ineus snake venom. Toxicon , 1983, 21: 491.
[17] Ouyang C,Huangt TF.Platelet aggregation inhibhors rom Agkistrodon n snake venom.Toxicon.1986。24:1099.
[18]云南省动物研究所第四室昆明.蛇毒的研究和利用:I、我国几种常见毒蛇的蛇毒酶活力的测定.生物化学与生物物理学报,1976,8(2):151.
[19] Huang TF,Chang MC,Teng CM.Antiplatelet protease,kistomin, selectively cleaves human platlet glycoprotein Ib . Biochim Biophys Acta ,1993,1158(3):293.