不同分子质量海带岩藻聚糖硫酸酯抗血栓活性及机理研究
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摘要
岩藻聚糖硫酸酯是一种富含硫酸基团的阴离子多糖，主要来源于褐藻、海参和海胆等。因其具有抗凝血、抗病毒、抗肿瘤、抑制血管增殖以及抗氧化等多种生物学活性,成为当今海洋功能性食品和药物研究的热点。
体外研究表明，不同来源的大分子岩藻聚糖硫酸酯均具有良好的抗血栓活性。但由于这些大分子岩藻聚糖硫酸酯一般分子组成复杂，溶解性低，吸收较差。若直接注射又会产生较大的毒性，进入体内后副作用较大，限制了其应用。
近年来，有报道称从泡叶藻中提取的低分子质量岩藻聚糖硫酸酯在体内同样显示出抗血栓活性。由于提取来源不同，岩藻聚糖硫酸酯的化学组分和结构会有较大差异，对于从海带中提取的低分子质量的岩藻聚糖硫酸酯是否同样具有抗血栓活性，还鲜有报道，亦未见对其抗血栓机制进行研究的相关报道。
本课题组前期应用自由基氧化法和阴离子交换法，从海带中制备纯化了具有不同分子质量和硫酸根含量的4种岩藻聚糖硫酸酯组分，分别为低分子质量的岩藻聚糖硫酸酯LF1和LF2，中分子质量的MFa和MFb。本文对这4种岩藻聚糖硫酸酯进行了抗血栓活性以及相关机理的研究，主要工作及结论如下：
1.对不同分子质量海带岩藻聚糖硫酸酯的体内抗血栓活性进行了研究
首先对不同分子质量海带岩藻聚糖硫酸酯进行急性毒性研究，并结合前期预实验得到的药效学结果，确定合理的给药剂量。在此基础上，采用Fecl3刺激法制作动脉血栓模型，采用结扎法制作下腔静脉血栓模型，对不同分子质量和硫酸根含量的岩藻聚糖硫酸酯体内抗血栓活性进行全面评价。
通过急性毒性实验发现，随着分子质量的增加，其毒性逐渐增强。其中2种LMWF的LD50相近（分别为716.0mg·kg-1和639.1mg·kg-1），而2种MMWF的LD50相近（分别为50.6mg·kg-1和45.1mg·kg-1）。
在本实验中，4种岩藻聚糖硫酸酯均具有良好的抗血栓作用。且随着分子质量增加，其血栓湿重逐渐减少，血栓生成抑制率逐渐增加，说明其抗血栓作用逐渐增强。并且这种抗血栓活性呈现明显的剂量相关性。
2.对不同分子质量海带岩藻聚糖硫酸酯的内皮保护作用及其机制进行了研究。
同时采用体内研究和体外研究的方法，探讨了不同分子质量海带岩藻聚糖硫酸酯对内皮细胞的保护作用。在体内研究中，通过制作大鼠内皮损伤模型，显微镜下观察大鼠胸主动脉内皮损伤情况，并测定内皮损伤指标血管性血友病因子（vWF）。在体外研究中，通过细胞培养，考察药物对内皮损伤的保护作用，除vWF外，本文还使用内皮微颗粒（EMPs）水平作为内皮损伤指标。
在本文中，我们发现低分子质量的岩藻聚糖硫酸酯对内皮细胞均具有良好的保护作用；而中分子质量的岩藻聚糖硫酸酯虽然对内皮细胞的损伤有所改善，但这种保护作用远远低于LMWF。同时发现，LMWF可通过降低vWF和EMPs浓度，阻断血小板与受损血管内皮中的胶原结合，从而抑制了血小板活化，发挥内皮保护作用。
3.考察了不同分子质量海带岩藻聚糖硫酸酯对血小板系统的影响及其作用机制
通过制作大鼠血管内皮损伤模型，考察了海带岩藻聚糖硫酸酯对血小板粘附率的影响；应用二磷酸腺苷（ADP）、花生四烯酸（AA）和血小板活化因子（PAF）3种诱导剂考察了海带岩藻聚糖硫酸酯对血小板聚集率的影响；检测了影响血小板聚集的重要指标TXB2和6-Keto-PGF1α和对胞浆Ca2+内流的影响。
结果显示，低分子质量的LF1和LF2均可明显降低血小板粘附率，而中分子质量的MFa和MFb对血小板粘附率没有明显影响。LF1和LF2对由ADP、AA和PAF诱导的血小板聚集均具有较好的抑制作用，而中分子质量的MFa和MFb对血小板聚集不仅没有抑制作用，反而表现出促进血小板聚集的作用。4组岩藻聚糖硫酸酯均表现出降低TXB2的作用，其中LF1和LF2组的降低作用强于MFa和MFb；另外，LF1和LF2同时具有升高6-keto-PGF1α的作用，而MFa和MFb则与模型组比较无统计学差异。此外，LMWF会对PAF诱导产生的Ca2+内流起到显著的抑制作用，而MMWF则没有这种抑制作用。
研究结果表明，LMWF可通过抑制血小板的粘附和聚集，发挥抗血栓作用；而MMWF对血小板功能影响很小，其抗血栓作用更依赖于其他途径。进一步发现，LMWF抑制血小板活化的具体作用机制是通过降低TXB2和升高6-Keto-PGF1α水平，以及抑制胞外Ca2+内流来实现的。
4.对不同分子质量海带岩藻聚糖硫酸酯的抗凝活性和相关机制进行了研究
以抗血栓活性实验中确定的岩藻聚糖硫酸酯剂量为基础，通过体内研究，首先对不同分子质量岩藻聚糖硫酸酯的抗凝血活性进行了比较。并通过出血实验，考察了不同分子质量的海带岩藻聚糖硫酸酯作为抗栓候选药的安全性。最后，通过对调节凝血过程的几种关键物质TFPI、AT-Ⅲ、PC和FIB进行检测，对其抗凝血机理进行了深入探讨。
结果显示，海带岩藻聚糖硫酸酯的抗凝活性呈现明显的剂量依赖性，并且抗凝活性随着分子质量的增加而增加。此外，出血实验显示，随着海带岩藻聚糖硫酸酯的分子质量增加，出血危险性也增加。
研究结果表明，不同分子质量的岩藻聚糖硫酸酯，发挥抗凝作用的机制也不同。对于LMWF而言，主要通过提高TFPI活性发挥作用；而MMWF除可通过TFPI途径发挥抗凝作用外，当达到一定浓度时，会通过影响纤维蛋白原浓度发挥抗凝血作用。
5.考察了不同分子质量海带岩藻聚糖硫酸酯对纤溶系统的影响及其作用机制
首先通过体内研究，考察岩藻聚糖硫酸酯对全血凝块溶解实验、优球蛋白溶解时间两个纤溶指标，对不同分子质量的海带岩藻聚糖硫酸酯纤溶活性进行了比较。并通过大鼠动脉血栓模型和静脉血栓模型，考察了调节体内纤溶系统的关键指标尿激酶型纤溶酶原活化剂（u-PA）、组织型纤溶酶原激活物(t-PA)、纤溶酶原激活物抑制物-1(PAI-1)等，以确定具体的作用机理
结果显示，海带岩藻聚糖硫酸酯的纤溶活性随着分子质量的增加而逐渐增强。同时，纤溶活性的强弱也呈现出一定的剂量相关性。说明对岩藻聚糖硫酸酯可通过激活纤溶酶原转换为纤溶酶发挥溶栓作用。从结果中还可以看出，其对纤溶酶原的激活主要是通过增强t-PA活性和抑制PAI-1活性来实现的。对u-PA活性并无影响。
6.考察了不同分子质量海带岩藻聚糖硫酸酯对血液流变学的影响
通过制作大鼠血瘀模型，考察了岩藻聚糖硫酸酯对全血黏度、血浆黏度、红细胞压积、红细胞沉降率等血液流变学参数的影响。
结果显示，LMWF对各项血液流变学指标均没有改变，而MMWF可明显降低全血黏度、血浆黏度、红细胞压积、红细胞沉降率及血沉方程K值等指标，提示通过改善血液流变学指标，可能是MMWF发挥抗血栓作用的一个重要途径。
通过本文的研究，我们证实了海带中提取的不同分子质量的岩藻聚糖硫酸酯同时具有抗凝血和溶栓的功能，还可通过抑制血小板活化而发挥良好的内皮保护作用，这是目前临床所使用的抗血栓药所不具备的，因此具有良好的开发前景。
另外我们也发现，海带岩藻聚糖硫酸酯发挥抗血栓作用的的途径随着分子质量的不同会发生改变，对于低分子质量的海带岩藻聚糖硫酸酯主要是通过抗凝血、抗血小板活化和纤溶功能发挥抗血栓作用；而对于中分子质量海带岩藻聚糖硫酸酯来说，主要通过抗凝血、纤溶作用和改善血液流变学来发挥抗血栓作用。
通过本文的研究发现，对于同一种生物功能，其作用强弱甚至是作用机制也会随着分子质量的改变而发生改变。如LMWF与MMWF均具有抗凝血和纤溶作用，但这两种作用都随着海带岩藻聚糖硫酸酯分子质量的增加而增强。同时，虽然不同分子质量岩藻聚糖硫酸酯都具有抗凝血功能，但LMWF主要通过提高TFPI活性发挥作用；而MMWF除可通过TFPI途径发挥抗凝作用外，当达到一定浓度时，会通过影响纤维蛋白原浓度发挥抗凝血作用。
通过本文的研究，我们全面考察了海带中提取的不同分子质量岩藻聚糖硫酸酯的体内抗血栓活性，并对其抗血栓机理进行了系统研究。为其下一步作为抗血栓候选药进入临床研究提供了坚实的理论基础和数据支持。
Fucoidans extracted from brown algae (Phaeophyta), sea cucumber(Echinodermata, Holothuroidea) and sea urchins (Echinodermata, Echinoidea)represent an intriguing group of natural fucose-enriched sulfated polysaccharides withpotential applications in medicine. Over the past decade, many papers havedemonstrated that fucoidan possess excellent biological properties, includinganticoagulant, antiviral, antitumor, antiangiogenic, enhance and antioxidant activities.Moreover, fucoidan from brown algae have the advantage of low contamination levelof virus and/or prions.
For the high hemorrhagic risk and poor solubility and absorption, high molecularweight fucodian was degraded by mild acid hydolysis and radical process degradation.Then two middle molecular weight fucoidan（MMW fucoidan） Mfa and MFb wereprepared by acid extraction andion-exchange chromatography. After radical processdegradation, two low molecular weight fucoidan (LMW fucoidan) LF1and LF2wereobtained.
In this article, the antithrombotic activities and related mechanisms of fourfucoidan fractions with different molecular weight and sulfate content were compared.
1. The antithrombotic activities of four fucoidan fractions with different molecularweight and sulfate content are compared in the rat model of arterial thrombosis inducedby FeCl3and venous thrombosis induced by ligation of inferior caval venous. In thisexperiment, all the four fucoidan fractions exhibited effective inhibitory effect on theformation of arterial thrombosis and venous thrombosis. After injection of fucoidan,the thrombotic weight decreased and inhibitory rate increased with the increasing of molecular weight. The results indicate that the fucoidan fractions with the highermolecular weight had the stronger antithrombotic activities. Furthermore, theantithrombotic activities of fucoidan increased in a dose-dependent manner.
2. The protective action on endotheliocytes and related mechanisms of four fucoidanfractions with different molecular weight were investigated in vivo and in vitro.
In the rat model of endotheliocytes injury, the significant endotheliocytes protectionof the four fucoidan fractions could be observed visually under the optical microscopeand the protection effect of LMW fucoidans are stronger than MMW fucoidans. OnlyLMW fucoidans could decrease the content of von willebrand factor (vWF) in plasma.Furthermore, endothelial cell culture was used to determine the effect of fucoidan oncontent of vWF in vitro, and the results showed that LMW fucoidan fractions havesignificant improvement on vWF and EMPs while MMW fucoidan had no any effect.The results indicate that the LMW fucoidan fractions had good protective action onendothelial cells by decrease the contents of vWF and EMPs. The protective effect ofLMW fucoidans was significantly stronger than MMW fucoidans. The result alsoreveals that the protective action on endotheliocytes of LMW fucoidans relies ondecreasing the concentration of EMPs and vWF, and thus inhibitingplatelet activation indirectly.
3. The effect of fucoidan on the platelet adhesion was measured in rat models withendotheliocytes injury. The effect on the platelet aggregation induced by adenosinediphosphate (ADP), arachidonic acid (AA) and platelet activating factor (PAF),furthermore, the TXB2,6-Keto-PGF1αlevel and the effect on cytoplasmic free calciumlevel in platelets were measured in vivo. The results show that only LMW fucoidanscan significantly decrease the platelet adhesion ratio induced by ADP, AA and PAF.On the contrary，MMW fucoidan can promote the platelet aggregation. All the fourfucoidan fractions can decrese the level of TXB2and LMW fucoidans show strongereffect than MMW fucoidans. Furthermore, only LMW fucoidans can increase the levelof6-keto-PGF1αand inhibit the Ca2+influxing in platelets induced byplatelet active factor.
The results indicated that inhibiting the adhesion and aggregation of platelet was avery important pathway for anti-thrombotic action of LMW fucoidan，while MMWfucoidan had no the same effect. For the first time, we revealed the detailed mechanismof LMW fucoidans on inhibition platelet activation is through promption of TXB2level,reduction of6-Keto-PGFlαand Ca2+influxing levels in platelets.
4. The anticoagulant activitives in vivo of the four fucoidans was measured at the doseof antithrombotic activity. Bleeding time, the level of TFPI, ATⅢ, PC and FIB, whichare all the key indicators on the regulating mechanism of coagulation. The results showthat molecular weight played a more important role on the anticoagulant activities andbleeding time of fucoidans than sulfated ester content. For the first time, we findfucoidans with different molecular weight have different mechanism to anticoagulant.All the four fucoidan fractions can improve the TFPI concentration in plasma and haveno effect on ATⅢ and PC. Only MMW fucoidans with high dose can improve the FIBconcentration in plasma.
5. To compare the fibrinolysis Activity of fucoidans with different molecur weight,twofibrinolytic system indices are investigated including the systemic clots dissolution andenglobulin lysis time. To examine the mechanism of thrombolytic activities, thearterial thrombosis models and venous thrombosis models are used to detect the effectof fucoidan fractions on fibrinolytic parameters including u-PA，t-PA and PAI-1,which were all the key indicators on regulating fibrinolytic system in vivo. Theresults showed that fucoidan fractions have good fibrinolytic activities. All the fourfucoidan fractions with different molecules could significantly improve t-PA andt-PA/PAI-1, meanwhile, decrease PAI-1. Furthermore, the effect on fibrinolytic systemof fucoidan fractions significantly enhanced with the increase of molecular weight.
6. Hemorheologic indices were observed in rats with stagnation of blood. The resultsshowed that LMW fucoidan had no effect on the hemorheologic indice, while MMWfucoidan could significantly improve all the parameters of hemorheology such aswhole blood viscosity, plasma viscosity, hematocrit and erythrocyte sedimentation rate. It indicated MMW fucoidan can inhibit venous thrombosis by improvinghemorrheology of blood.
Through the studies, we prove all the four fucoidan fractions with differentmolecular weight both have anticoagulant activites and fibrinolytic activities. Not onlythat, they also have the protective action on endotheliocytes. Any other antithromboticdrug has no this properties, so fucoidans have a good prospect of development.
In addition, we find the antithrombotic mechanisms are different for thefucoidans with different molecular weight. For LMW fucoidans, they extertantithrombotic activities by， inhibiting platelet activation and, while for MMWfucoidans, the main acting way including anticoagulant, fibrinolytic activities andimproving hemorrheology of blood.
The level of bioactivities can suffer from changes in molecular weight. Forexample, both LMW fucoidans and MMW fucoidans have anticoagulant andfibrinolytic activities, but the fucoidan fractions with the higher molecular weight havethe stronger bioactivities. All the four fucoidan fractions can improve the TFPIconcentration in plasma and MMW fucoidans with high dose can also improve the FIBconcentration in plasma.
This experiment shows that the influence of molecur weight for the antithromboticmechanisms far greater than sulfate content
体外研究表明，不同来源的大分子岩藻聚糖硫酸酯均具有良好的抗血栓活性。但由于这些大分子岩藻聚糖硫酸酯一般分子组成复杂，溶解性低，吸收较差。若直接注射又会产生较大的毒性，进入体内后副作用较大，限制了其应用。
近年来，有报道称从泡叶藻中提取的低分子质量岩藻聚糖硫酸酯在体内同样显示出抗血栓活性。由于提取来源不同，岩藻聚糖硫酸酯的化学组分和结构会有较大差异，对于从海带中提取的低分子质量的岩藻聚糖硫酸酯是否同样具有抗血栓活性，还鲜有报道，亦未见对其抗血栓机制进行研究的相关报道。
本课题组前期应用自由基氧化法和阴离子交换法，从海带中制备纯化了具有不同分子质量和硫酸根含量的4种岩藻聚糖硫酸酯组分，分别为低分子质量的岩藻聚糖硫酸酯LF1和LF2，中分子质量的MFa和MFb。本文对这4种岩藻聚糖硫酸酯进行了抗血栓活性以及相关机理的研究，主要工作及结论如下：
1.对不同分子质量海带岩藻聚糖硫酸酯的体内抗血栓活性进行了研究
首先对不同分子质量海带岩藻聚糖硫酸酯进行急性毒性研究，并结合前期预实验得到的药效学结果，确定合理的给药剂量。在此基础上，采用Fecl3刺激法制作动脉血栓模型，采用结扎法制作下腔静脉血栓模型，对不同分子质量和硫酸根含量的岩藻聚糖硫酸酯体内抗血栓活性进行全面评价。
通过急性毒性实验发现，随着分子质量的增加，其毒性逐渐增强。其中2种LMWF的LD50相近（分别为716.0mg·kg-1和639.1mg·kg-1），而2种MMWF的LD50相近（分别为50.6mg·kg-1和45.1mg·kg-1）。
在本实验中，4种岩藻聚糖硫酸酯均具有良好的抗血栓作用。且随着分子质量增加，其血栓湿重逐渐减少，血栓生成抑制率逐渐增加，说明其抗血栓作用逐渐增强。并且这种抗血栓活性呈现明显的剂量相关性。
2.对不同分子质量海带岩藻聚糖硫酸酯的内皮保护作用及其机制进行了研究。
同时采用体内研究和体外研究的方法，探讨了不同分子质量海带岩藻聚糖硫酸酯对内皮细胞的保护作用。在体内研究中，通过制作大鼠内皮损伤模型，显微镜下观察大鼠胸主动脉内皮损伤情况，并测定内皮损伤指标血管性血友病因子（vWF）。在体外研究中，通过细胞培养，考察药物对内皮损伤的保护作用，除vWF外，本文还使用内皮微颗粒（EMPs）水平作为内皮损伤指标。
在本文中，我们发现低分子质量的岩藻聚糖硫酸酯对内皮细胞均具有良好的保护作用；而中分子质量的岩藻聚糖硫酸酯虽然对内皮细胞的损伤有所改善，但这种保护作用远远低于LMWF。同时发现，LMWF可通过降低vWF和EMPs浓度，阻断血小板与受损血管内皮中的胶原结合，从而抑制了血小板活化，发挥内皮保护作用。
3.考察了不同分子质量海带岩藻聚糖硫酸酯对血小板系统的影响及其作用机制
通过制作大鼠血管内皮损伤模型，考察了海带岩藻聚糖硫酸酯对血小板粘附率的影响；应用二磷酸腺苷（ADP）、花生四烯酸（AA）和血小板活化因子（PAF）3种诱导剂考察了海带岩藻聚糖硫酸酯对血小板聚集率的影响；检测了影响血小板聚集的重要指标TXB2和6-Keto-PGF1α和对胞浆Ca2+内流的影响。
结果显示，低分子质量的LF1和LF2均可明显降低血小板粘附率，而中分子质量的MFa和MFb对血小板粘附率没有明显影响。LF1和LF2对由ADP、AA和PAF诱导的血小板聚集均具有较好的抑制作用，而中分子质量的MFa和MFb对血小板聚集不仅没有抑制作用，反而表现出促进血小板聚集的作用。4组岩藻聚糖硫酸酯均表现出降低TXB2的作用，其中LF1和LF2组的降低作用强于MFa和MFb；另外，LF1和LF2同时具有升高6-keto-PGF1α的作用，而MFa和MFb则与模型组比较无统计学差异。此外，LMWF会对PAF诱导产生的Ca2+内流起到显著的抑制作用，而MMWF则没有这种抑制作用。
研究结果表明，LMWF可通过抑制血小板的粘附和聚集，发挥抗血栓作用；而MMWF对血小板功能影响很小，其抗血栓作用更依赖于其他途径。进一步发现，LMWF抑制血小板活化的具体作用机制是通过降低TXB2和升高6-Keto-PGF1α水平，以及抑制胞外Ca2+内流来实现的。
4.对不同分子质量海带岩藻聚糖硫酸酯的抗凝活性和相关机制进行了研究
以抗血栓活性实验中确定的岩藻聚糖硫酸酯剂量为基础，通过体内研究，首先对不同分子质量岩藻聚糖硫酸酯的抗凝血活性进行了比较。并通过出血实验，考察了不同分子质量的海带岩藻聚糖硫酸酯作为抗栓候选药的安全性。最后，通过对调节凝血过程的几种关键物质TFPI、AT-Ⅲ、PC和FIB进行检测，对其抗凝血机理进行了深入探讨。
结果显示，海带岩藻聚糖硫酸酯的抗凝活性呈现明显的剂量依赖性，并且抗凝活性随着分子质量的增加而增加。此外，出血实验显示，随着海带岩藻聚糖硫酸酯的分子质量增加，出血危险性也增加。
研究结果表明，不同分子质量的岩藻聚糖硫酸酯，发挥抗凝作用的机制也不同。对于LMWF而言，主要通过提高TFPI活性发挥作用；而MMWF除可通过TFPI途径发挥抗凝作用外，当达到一定浓度时，会通过影响纤维蛋白原浓度发挥抗凝血作用。
5.考察了不同分子质量海带岩藻聚糖硫酸酯对纤溶系统的影响及其作用机制
首先通过体内研究，考察岩藻聚糖硫酸酯对全血凝块溶解实验、优球蛋白溶解时间两个纤溶指标，对不同分子质量的海带岩藻聚糖硫酸酯纤溶活性进行了比较。并通过大鼠动脉血栓模型和静脉血栓模型，考察了调节体内纤溶系统的关键指标尿激酶型纤溶酶原活化剂（u-PA）、组织型纤溶酶原激活物(t-PA)、纤溶酶原激活物抑制物-1(PAI-1)等，以确定具体的作用机理
结果显示，海带岩藻聚糖硫酸酯的纤溶活性随着分子质量的增加而逐渐增强。同时，纤溶活性的强弱也呈现出一定的剂量相关性。说明对岩藻聚糖硫酸酯可通过激活纤溶酶原转换为纤溶酶发挥溶栓作用。从结果中还可以看出，其对纤溶酶原的激活主要是通过增强t-PA活性和抑制PAI-1活性来实现的。对u-PA活性并无影响。
6.考察了不同分子质量海带岩藻聚糖硫酸酯对血液流变学的影响
通过制作大鼠血瘀模型，考察了岩藻聚糖硫酸酯对全血黏度、血浆黏度、红细胞压积、红细胞沉降率等血液流变学参数的影响。
结果显示，LMWF对各项血液流变学指标均没有改变，而MMWF可明显降低全血黏度、血浆黏度、红细胞压积、红细胞沉降率及血沉方程K值等指标，提示通过改善血液流变学指标，可能是MMWF发挥抗血栓作用的一个重要途径。
通过本文的研究，我们证实了海带中提取的不同分子质量的岩藻聚糖硫酸酯同时具有抗凝血和溶栓的功能，还可通过抑制血小板活化而发挥良好的内皮保护作用，这是目前临床所使用的抗血栓药所不具备的，因此具有良好的开发前景。
另外我们也发现，海带岩藻聚糖硫酸酯发挥抗血栓作用的的途径随着分子质量的不同会发生改变，对于低分子质量的海带岩藻聚糖硫酸酯主要是通过抗凝血、抗血小板活化和纤溶功能发挥抗血栓作用；而对于中分子质量海带岩藻聚糖硫酸酯来说，主要通过抗凝血、纤溶作用和改善血液流变学来发挥抗血栓作用。
通过本文的研究发现，对于同一种生物功能，其作用强弱甚至是作用机制也会随着分子质量的改变而发生改变。如LMWF与MMWF均具有抗凝血和纤溶作用，但这两种作用都随着海带岩藻聚糖硫酸酯分子质量的增加而增强。同时，虽然不同分子质量岩藻聚糖硫酸酯都具有抗凝血功能，但LMWF主要通过提高TFPI活性发挥作用；而MMWF除可通过TFPI途径发挥抗凝作用外，当达到一定浓度时，会通过影响纤维蛋白原浓度发挥抗凝血作用。
通过本文的研究，我们全面考察了海带中提取的不同分子质量岩藻聚糖硫酸酯的体内抗血栓活性，并对其抗血栓机理进行了系统研究。为其下一步作为抗血栓候选药进入临床研究提供了坚实的理论基础和数据支持。
Fucoidans extracted from brown algae (Phaeophyta), sea cucumber(Echinodermata, Holothuroidea) and sea urchins (Echinodermata, Echinoidea)represent an intriguing group of natural fucose-enriched sulfated polysaccharides withpotential applications in medicine. Over the past decade, many papers havedemonstrated that fucoidan possess excellent biological properties, includinganticoagulant, antiviral, antitumor, antiangiogenic, enhance and antioxidant activities.Moreover, fucoidan from brown algae have the advantage of low contamination levelof virus and/or prions.
For the high hemorrhagic risk and poor solubility and absorption, high molecularweight fucodian was degraded by mild acid hydolysis and radical process degradation.Then two middle molecular weight fucoidan（MMW fucoidan） Mfa and MFb wereprepared by acid extraction andion-exchange chromatography. After radical processdegradation, two low molecular weight fucoidan (LMW fucoidan) LF1and LF2wereobtained.
In this article, the antithrombotic activities and related mechanisms of fourfucoidan fractions with different molecular weight and sulfate content were compared.
1. The antithrombotic activities of four fucoidan fractions with different molecularweight and sulfate content are compared in the rat model of arterial thrombosis inducedby FeCl3and venous thrombosis induced by ligation of inferior caval venous. In thisexperiment, all the four fucoidan fractions exhibited effective inhibitory effect on theformation of arterial thrombosis and venous thrombosis. After injection of fucoidan,the thrombotic weight decreased and inhibitory rate increased with the increasing of molecular weight. The results indicate that the fucoidan fractions with the highermolecular weight had the stronger antithrombotic activities. Furthermore, theantithrombotic activities of fucoidan increased in a dose-dependent manner.
2. The protective action on endotheliocytes and related mechanisms of four fucoidanfractions with different molecular weight were investigated in vivo and in vitro.
In the rat model of endotheliocytes injury, the significant endotheliocytes protectionof the four fucoidan fractions could be observed visually under the optical microscopeand the protection effect of LMW fucoidans are stronger than MMW fucoidans. OnlyLMW fucoidans could decrease the content of von willebrand factor (vWF) in plasma.Furthermore, endothelial cell culture was used to determine the effect of fucoidan oncontent of vWF in vitro, and the results showed that LMW fucoidan fractions havesignificant improvement on vWF and EMPs while MMW fucoidan had no any effect.The results indicate that the LMW fucoidan fractions had good protective action onendothelial cells by decrease the contents of vWF and EMPs. The protective effect ofLMW fucoidans was significantly stronger than MMW fucoidans. The result alsoreveals that the protective action on endotheliocytes of LMW fucoidans relies ondecreasing the concentration of EMPs and vWF, and thus inhibitingplatelet activation indirectly.
3. The effect of fucoidan on the platelet adhesion was measured in rat models withendotheliocytes injury. The effect on the platelet aggregation induced by adenosinediphosphate (ADP), arachidonic acid (AA) and platelet activating factor (PAF),furthermore, the TXB2,6-Keto-PGF1αlevel and the effect on cytoplasmic free calciumlevel in platelets were measured in vivo. The results show that only LMW fucoidanscan significantly decrease the platelet adhesion ratio induced by ADP, AA and PAF.On the contrary，MMW fucoidan can promote the platelet aggregation. All the fourfucoidan fractions can decrese the level of TXB2and LMW fucoidans show strongereffect than MMW fucoidans. Furthermore, only LMW fucoidans can increase the levelof6-keto-PGF1αand inhibit the Ca2+influxing in platelets induced byplatelet active factor.
The results indicated that inhibiting the adhesion and aggregation of platelet was avery important pathway for anti-thrombotic action of LMW fucoidan，while MMWfucoidan had no the same effect. For the first time, we revealed the detailed mechanismof LMW fucoidans on inhibition platelet activation is through promption of TXB2level,reduction of6-Keto-PGFlαand Ca2+influxing levels in platelets.
4. The anticoagulant activitives in vivo of the four fucoidans was measured at the doseof antithrombotic activity. Bleeding time, the level of TFPI, ATⅢ, PC and FIB, whichare all the key indicators on the regulating mechanism of coagulation. The results showthat molecular weight played a more important role on the anticoagulant activities andbleeding time of fucoidans than sulfated ester content. For the first time, we findfucoidans with different molecular weight have different mechanism to anticoagulant.All the four fucoidan fractions can improve the TFPI concentration in plasma and haveno effect on ATⅢ and PC. Only MMW fucoidans with high dose can improve the FIBconcentration in plasma.
5. To compare the fibrinolysis Activity of fucoidans with different molecur weight,twofibrinolytic system indices are investigated including the systemic clots dissolution andenglobulin lysis time. To examine the mechanism of thrombolytic activities, thearterial thrombosis models and venous thrombosis models are used to detect the effectof fucoidan fractions on fibrinolytic parameters including u-PA，t-PA and PAI-1,which were all the key indicators on regulating fibrinolytic system in vivo. Theresults showed that fucoidan fractions have good fibrinolytic activities. All the fourfucoidan fractions with different molecules could significantly improve t-PA andt-PA/PAI-1, meanwhile, decrease PAI-1. Furthermore, the effect on fibrinolytic systemof fucoidan fractions significantly enhanced with the increase of molecular weight.
6. Hemorheologic indices were observed in rats with stagnation of blood. The resultsshowed that LMW fucoidan had no effect on the hemorheologic indice, while MMWfucoidan could significantly improve all the parameters of hemorheology such aswhole blood viscosity, plasma viscosity, hematocrit and erythrocyte sedimentation rate. It indicated MMW fucoidan can inhibit venous thrombosis by improvinghemorrheology of blood.
Through the studies, we prove all the four fucoidan fractions with differentmolecular weight both have anticoagulant activites and fibrinolytic activities. Not onlythat, they also have the protective action on endotheliocytes. Any other antithromboticdrug has no this properties, so fucoidans have a good prospect of development.
In addition, we find the antithrombotic mechanisms are different for thefucoidans with different molecular weight. For LMW fucoidans, they extertantithrombotic activities by， inhibiting platelet activation and, while for MMWfucoidans, the main acting way including anticoagulant, fibrinolytic activities andimproving hemorrheology of blood.
The level of bioactivities can suffer from changes in molecular weight. Forexample, both LMW fucoidans and MMW fucoidans have anticoagulant andfibrinolytic activities, but the fucoidan fractions with the higher molecular weight havethe stronger bioactivities. All the four fucoidan fractions can improve the TFPIconcentration in plasma and MMW fucoidans with high dose can also improve the FIBconcentration in plasma.
This experiment shows that the influence of molecur weight for the antithromboticmechanisms far greater than sulfate content
引文
[1] RibeiroAC, Vieira RP, Mourao PAS, et al.Asulfated-α-L-fucan from sea cucumber.Carbohydr. Res.,1994,255(3):225~240
[2] Barbara M,Ana-Cristina R,Ana-Paula, et al. Sulfated fucans from echinoderms have a regulartetrasaccharide repeating unit defined by specific patterns of sulfation at the O-2and O-4positions. J. Biol. Chem.,1994,269(35):22113~22123
[3]林洪，江洁，薛长湖，等.褐藻岩藻聚糖结构和活性研究进展.中国海洋药物，2002，90（6）：42~47
[4]李波，许时璎.羊栖菜中褐藻糖胶的提取纯化研究.食品工业，2004，2：40~42
[5]邢杰，刘守涛，高君，等.海带多糖的提取及组份L1的分离与鉴定.抚顺石油学院学报，1998，18(1)：8~9
[6]尤瑜敏，许时璎.羊栖菜中褐藻糖胶的提取工艺.无锡轻工大学学报，2002，5(3)：233~239
[7]何云海，汪秋宽，刘红丹.用复合酶酶解提取海带岩藻聚糖硫酸酯的工艺研究.上海水产大学学报，2006，21（1）：55~59
[8]郭亚贞，王慥.海带中褐藻糖胶的提取与纯化.上海水产大学学报.2000，9(3)：276~280
[9] Ozawa T, Yamamoto J, Yamagishi T. Two fucoidans in the holdfast of cultivated Laminariajaponica. Nature Medicine,2006,60(6):236~239
[10]陈蕾，吴皓.多糖降解方法的研究进展.中华中医学刊，2008，26(1)：133~135
[11] Shinichi Furukawa, Tatsuo Fujikawa, Daizo Koga, et al. Production of fucoidan degradingenzymes, fucoidan, and fucoidan sulfatase by Vibro sp1N25. Nippon Suisan Gakkaishi,1992,58(8):1499~1503
[12] Takcshi Sakai, Hitomi Kimura, Ikunoshin Kato. A Marine strain of flavobacteriaccac utilizesbrown seaweed fucoidan.Mar Biothechnol,2002,(4):399~405
[13]吴茜茜，蔡敬民，吴克，等.海洋真菌LD8岩藻多糖酶的固态发酵条件研究.海洋科学，2004，28(6)：12~27
[14]吴克，刘斌，吴茜茜，等.海洋真菌Dendryphiella arenaria TM94产岩藻多糖酶发酵及酶学性质.生物学杂志，2003，20(2)：14~16
[15] Kitamura K, Matsuo M, Yasui T. Enzymatic degradation of fucoidan by fucoidanase from thehepatopancreas of Patinopecten yessoensis. Biosic Biotech Biochem,1992,56(11):1829~1834
[16] Régis Daniel, Olivier Berteau, Jacqueline Jozefonvicz,et al. Degradation of algal(Ascophyllum nodosum) fucoidan by an enzymatic activity contained in digestive glands ofthe marine mollusc Pecten maximus. Carbohydrate Research.1999,322(3-4):291~297
[17] Takashi ohshiro, Yoshiyuki ohmoto, Yoshihisa ono, et al. Isolation and Characterization of aNovel Fucoidan-Degrading Microorganism.Bioscience, Biotechnology, andBiochemistry.2010,74(8):1729~1732
[18] I Iu Bakunina, O I Nedashkovskaia, S A Alekseeva, et al. Degradation of fucoidan by themarine proteobacterium seudoalteromonas citrea. Mikrobiologiia.2002,71(1):49~55
[19] Kim WJ, Kim SM, Lee YH, et al. Isolation and Characterization of Marine Bacterial StrainDegrading Fucoidan from Korean Undaria pinnatifida Sporophylls. Microbiology andBiotechnology,2008,18(4):616~623
[20]陈惠源，蔡俊鹏，刘江涛.岩藻多糖降解酶产生菌的筛选及鉴定.现代食品科技，2005，21(3)：42~44
[21] Kyong Duk Oh. degradation of brown alga-derived fucoidan. US.20090270607A1,2009-9-27
[22]武晓琳.分子量对海参岩藻聚糖硫酸酯活性和消化吸收的影响研究.[硕士论文].山东：中国海洋大学，2011
[23] Vitor H. Pomin, Ana Paula Valente, Mariana S. Pereira, et al. Mild acid hydrolysis of sulfatedfucans: a selective2-desulfation reaction and an alternative approach for preparing tailoredsulfated oligosaccharides. Glycobiology,2005,15(12):1376~1385
[24] Yun Hou, Jing Wang, Weihua Jin, et al. Degradation of Laminaria japonica fucoidanby hydrogen peroxide and antioxidant activities of the degradation products ofdifferent molecular weights. Carbohydrate Polymers,2012,87(1):153~159
[25] Luyt CE, Meddahi-Pellé A, Ho-Tin-Noe B, et al. Low Molecular Weight Fucoidan PromotesTherapeutic Revascularization in a Rat Model of Critical Hindlimb Ischemia. J Pharmacol ExpTher,2003,305(1):24~30
[26]富士川龙郎，中島克子.褐藻におけるフコイタソ様多糖分佈.日本濃雲化學雜誌，1975，49(9):455~461
[27] Nishide E, Anzai H, Uchida, et al. Sugar constituents of fucose-containing polysaccharidesfrom various Japanese brown algae. Hydrobiologia,1990,204/205(1):573~576
[28] Nishino T, Nishioka C, Ura H et al. Isolation and partial characterization of a novel aminosugar containing fucan sulfate from commerical Fucus vericulosus fucoidan. Carbohydr. RES.,1994,255(3):213~224
[29] Conchie J, Percival EGV. Fucoidin. Part Ⅱ：The hydrolysis of a methylated fucoidan preparedfrom fucus vesiculosis. J. Chem. Soc.,1950:827
[30] Patankar MS, Oehninger S, Barnett T, et al. A revised structure for fucoidan may explain someof its biological activities. J Biol Chem,1993,268(29):21770~21776
[31] Nishino T, Aizu Y, Nagumo T. Antithrombin activity of a fucan sulfate from the brownseaweed Ecklonia kurome. Thromb. Res.,1991,62(6):765~773
[32] Nishino T, Nagumo T, Kiyohara H. et al. Strutural characterization of a new anticoagulantfucan sulfate from the brown seaweed Ecklonia kurome. Carbohydr. Res.,1991,211(1):77~90
[33] Chizhov OA, Dell A, Morris RH, et al. A study of fucoidan from the brown seaweed Chordafilum. Carbohydr. Res.,1999,320(1-2):108~119
[34] Daniel R, Berteau O, Chevolot L, et al. Regioselective desulfation of sulfatedL-fucopyranoside by a new sulfoesterase from the marine mollusk Pecten maximus:application to the structural study of algal fucoidan (Ascophyllum nodosum). Eur J Biochem.2001,268(21):5617~5626
[35] Elizabeth P, Rahman MA. Chemistry of the polysaccarfides of the brown seaweed dictyprerisPlagiogramma. Phytochemistry,1981,20(7):1579
[36] Pereira MS. Mulloy B, Mourao PAS. Structure and anticoagulant activity of sulfated fucans:comparison between the regular repetitive, and linear fucans from echinoderms with the moreheterogeneous and branched polymers from brown algae. J. Biol. Chem.,1999,274(12):7656~7667
[37] Chevolot L, Foucault A, Chaubet F, et al. Further data on the structure of brown seaweedfucans: relations hipswith anticoagulant activity. Carbohydr Res.,1999,319(1-4):154~165
[38] Springer G F, Wurzel H A, Mcsneal G M,et al. Isolation of anticoagulant fractions from crudefucoidan. Proc Soc Exp Biol Med,1957,94:404~409
[39] Nishino T, Fukuda T, Nagumo T, et al. Inhibition of the generation of thrombin and factor Xaby a fucoidan from brown seaweed Ecklonia Kurome. Thromb Res,1999,96(1):37~49
[40] Nishino T, Kiyohara H, Yamada H, et al. An anticoagulant fucoidan from the brown seaweedEcklonia Kurome.Phytochemistry,1991,30(2):535~539
[41] Nishino T, Aizu Y, Nagumo T. The influence of sulf ate content and molecular weight of afucan sulfate from the brown seaweed Ecklonia Kurome on its antithromb in activity. ThrombRes.,1991,64(6):723~731
[42] Woo-Jung Kim, Sung-Min Kim, Hyun Guell Kim, et al. Purification and AnticoagulantActivity of a Fucoidan from Korean Undaria pinnatifida Sporophyll. Algae.2007,22(3):247~252
[43] Ushakova NA, Morozevich GE, Ustiuzhanina NE, et al. Anticoagulant activity of fucoidansfrom brown algae Biomed Khim.2008,54(5):597~606
[44] Albana Cumashi, Natalia A. Ushakova, Marina E. Preobrazhenskaya, et al. A comparativestudy of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities ofnine different fucoidans from brown seaweeds. Glycobiology.2007,17(5):541~552
[45] Grauffel V, Kloareg B, Mabeau S, et al. New natural polysaccharides with potent antithrombicactivity-fucans from brown-algae. Biomaterials.1989,10(6):363~368
[46] Mauray S, DE Raucourt E, Chaubet F, et al. Comparative anticoagulant activity and influenceon thrombin generation of dextran derivatives and of a fucoidan fraction. J Biomater SciPolym Ed.,1998,9(4):373~387
[47] Colliec S,Fischer AM,Tapon-Berlaudiere J,et al．Anticoagulant properties of a fucoidan.Fraction Thromb Res,,1991,64（2）:143~154
[48] Mauray S, Sternberg C, Theveniaux J, et al. Venous antithrombotic and anticoagulantactivities of a fucoidan fraction.Thromb Haemost.1995,74(5):1280~1285
[49] Durand E, Helley D, Ayman HZ. Effect of low molecular weight fucoidan and low molecularweight heparin in a rabbit model of arterial thrombosis. J Vasc Res.,2008,45(6):529~537
[50]刘承颖，王维民.半叶马尾藻中岩藻聚糖硫酸酯的提取纯化及抗氧化研究.食品研究与开发，2008，29(11)：71~75
[51] Costa, LS, Fidelis, GP, Cordeiro, SL,et al. Biological activities of sulfated polysaccharidesfrom tropical seaweeds. Biomed. Pharmacother.2010,64（1）,21~28
[52] Leandro Silva Costa, Gabriel Pereira Fidelis, Cinthia Beatrice Silva Telles, et al. Antioxidantand Antiproliferative Activities of Heterofucans from the Seaweed Sargassum filipendula. Mar.Drugs.2011,9:952~966
[53] Zhang Z, Wang F, Wang X,et al. Extraction of the polysaccharides from five algae and theirpotential antioxidant activity in vitro. Carbohydr. Polym.2010,82,118~121
[54] Wang J, Zhang Q, Zhang Z, et al. Potential antioxidant and anticoagulant capacity of lowmolecular weight fucoidan fractions extracted from Laminaria japonica. Int. J. Biol. Macromol.2010,46（1）:6~12
[55] Zhao X, Xue C H, Li B F. Study of antioxidant activities of sulfated polysaccharides fromLaminaria japonica. J App Phyco,2008,20（4）:431~436
[56] JAE GUN HAN, ABDUL QADIR SYED, M INCHUL KWON, et al. Antioxidant,immunomodulatory and anticancer activity of fucoidan isolated from Fucus vesiculosus.Journal of Biotechnology,2008,7:1346
[57]赵雪，薛长湖，王静凤，等.海带岩藻聚糖硫酸酯低聚糖对小鼠肝损伤的保护作用.营养学报，2003，25(3)：286~289
[58] Saito A, Yoneda M, Yokohama S, et al. Fucoidan prevents concanavalin A-induced liver injurythrough induction of endogenous IL-10in mice. Herpetology Research,2006,35(3):190~198
[59] SHINJI HAYASHI, AYANO ITOH, KATSUH IRO ISODA, et al.Fucoidan partly preventsCCl4induced liver fibrosis. European Journal of Ph armacology,2008,580(3):380~384
[60] Okaiy, Okai KH, Ishizaka S, et al. Possible immunomodulating activities in a extraction ofedible seaweed. Hizikia fusiforme (Hijiki). J Sci Food Agri,1996,72(4):455~460
[61]詹林盛，张新生，吴小红，等.褐藻多糖对正常小鼠淋巴细胞功能和细胞因子产生的影响.营养学报，2001，23（2）：122~125
[62]杨晓林，孙菊云，许汉年，等.褐藻糖酵的免疫调节作用.中国海洋药物，1995，14（3）：9~13
[63]赵雪．海带岩藻聚糖硫酸酯的化学组成与活性的研究．[博士学位论文].山东：中国海洋大学，2004
[64] Berteau O, Mulloy B. Sulfated fucans, fresh perspectives:structures, functions, and biologicalproperties of sulfated fucans and an overview of enzymes active toward this class ofpolysaccharides. Glycobiology.2003,13:29R~40R
[65] Joo-Woong Park, Hyun-Hyo Suh, Kyung-Bok Lee, et al. Immunomodulating Activity of aFucoidan Isolated from Korean Undaria pinnatifida Sporophyll. Algae.2007,22(4):333~338
[66] Choi EM, Kim A, Kim YO, et al. Immunostimulating activity of arabinogalactan and fucoidanin vitro. J. Med. Food.2005,8(4):446~453
[67] Maruyama H, Tamauchi H, Hashimoto M, et al. Antitumor activity and immune response ofMekabu fucoidan extracted from Sporophyll of Undaria pinnatifida. In vivo.2003,17(3):245~249
[68] Yamamoto I, Nagumo T, Takahashi M, et al. Antitumor effect of seaweeds Ⅲ. Antitumoreffect of an extract from Sargassum Kjellmanianum. Japan J ExP Med,1981,51(3):187~189
[69] Yamamoto I, Takahashi M, Tamura E, et al. Antitumor activity of crude extracts from ediblemarine algae against L-1210Leukemia. Botancia Marina,1982,25:455~457
[70] Yamamoto I, Takahashi M, Suzuki I, et al. Antitumor effect of seaweeds Ⅳ. Enhancement ofantitumor activity by sulfation of a crude fucoidan fraction from Sargassum Kjellmanianum.Japan J ExP Med,1984,54(4):143~151
[71]宋剑秋，徐誉泰，张华坤，等.海带硫酸多糖对小鼠腹腔巨噬细胞的免疫调节作用.中国免疫学杂志，2000，16（2）：70~72
[72] Zhuang C, Itoh H, Mizuno T, et al. Antitumor active fucoidan from the brown seaweed,Umitoranoo(Sargassum thunbergii).Biosci Biothech BioChem,1995,59(4):563~567
[73] Itoch H, Noda H, Amno H, et al. Antitumor activity and immunological properties of marinealgae Polysaccharides, especially fucoidan, prepared from Sargassum thunbergii of PhaeoPhyceae. Anticancer Res.,1993,13(6A):2045~2052
[74] Itoh H, Noda H, Amno H, et al. Immuological analysis of inhibition of lung metastases byfucoidan(GIV-A) prepared from brown seaweed Sargassum thunbergii. Anticancer Res.,1995,15(SB):1937~1947
[75] Usuui T, Asari K, Mizuno T. Isolation of Highly Purified “fucoidan” from Eisenia bicyclis andits anticoagulant and antitumor activities. Agric Biol Chem,1980,44（8）:1965~1966
[76]王庭欣，秦淑贞，赵文，等.海带多糖对环磷酰胺诱小鼠骨位细胞微核率的抑制作用.癌变·畸变·突变，1999，11(2)：106~107
[77] Ye J, Li Y, Teruya K, Katakura Y, et al. Enzyme-digested Fucoidan Extracts Derived fromSeaweed Mozuku of Cladosiphon novae-caledoniae kylin Inhibit Invasion and Angiogenesisof Tumor Cells. Cytotechnology.2005,47(1-3):117~126
[78] Alekseyenko TV, Zhanayeva SY, Venediktova AA, et al. Antitumor and antimetastatic activityof fucoidan, a sulfated polysaccharide isolated from the Okhotsk Sea Fucus evanescens brownalga. Bull Exp Biol Med.2007,143(6):730~732
[79] Aisa Y,Miyakawa Y,Nakazato T,et al.Fucoidan induces apoptosis of human HS-sultan cellsaccompanied by activation of caspase-3and down-regulation of ERK pathways.Am JHematol,2005,78(1):7~14
[80] Koyanagi S,Tanigawa N,Nakagawa H,et al.Oversulfation of fucoidan enhances itsanti-angiogenic and antitumor activities.Biochem Pharmacol,2003,65(2):173~179
[81] Bernardi G，Springer GF．Properties of highly purified fucan．Biol Chem，1962，237:75~77
[82] Colliee S, Fiseher AM, Tapon-BerlaudiereJ, et al.Anticoagulant properties of a fucoidanfraction. Trombosis Research,1991,64(2):143～154
[83] Sandrine M,Clauds S,Theveniaux J,et al．Venous Antithrombotic and anticoagulant activitiesof a fucoidan fraction．Thromb Haemost.,1995,74(5):1280～1285
[84] Soeda S, Sakaquchi S, Shimeno H, et al. Fibrinolytic and anticoagulant activities of highlysulfated fucoidan.Biochem Pharma,1992,43(8):1853~1858
[85] Millet J, Jauoult SC, Mauray S, et al. Antithrombotic and anticoagulant activities of a lowmolecular weight fucoidan by the subcuataneous route. Thromb Haemost,1999,81(3):391~395
[86] Doctor VM，Hill C，Jackson GJ，et al. Effect of fucoidan during activation of humanplasminogen.Throm Res.,1995,79(3):237~247
[87]薛长湖，李兆杰，辛梅，等.岩藻聚糖硫酸酯的制备方法.中国，专利号：ZL98110253.0.，1998
[88] Glable CG, Yednock T, Rosen SD, et al. Reversible disruption of cultured endothelialmonolayers by sulfated fucans. J Cell Sci,1983,61(5):475~490
[89]辛现良，耿美玉，李桂玲，等.海洋硫酸多糖911体外对HIV-1作用的研究.中国海洋药物，2000(19)：8~11
[90]李凡，田同春，石艳春，等.岩藻糖胶体外抗病毒作用的研究.白求恩医科大学学报，1995，21(3)：18~22
[91]李德远，徐战，黄利民，等.海带岩藻糖胶对大鼠饮食性高血脂症的影响.食品科学，2001，22(2)：92~95
[92] Chabut D, Fischer A M, Colliec-Jouault S, et al. Low molecular weight fucoidan and heparinenhance the basic fibroblast grow th factor-induced tube form ation of endothelia l cellsthrough heparan sulfate-dependent alpha6over expression. Mol Pharmacol.2003,64(3):696~702
[93] Sabine Matou, Dominique Helley, Delphine Chabut. et al. Effect of fucoidan on fibroblastgrowth factor-2-induced angiogenesis in vitro. Thrombosis Research.2002，106(4-5):213~221
[94] C. Boisson-Vidal, F. Zemani, G. Caligiuri, et al. Neoangiogenesis induced by progenitorendothelial cells: effect of fucoidan from marine algae. Cardiovascular&HematologicalAgents in Medicinal Chemistry,2007,5(1):67~77
[95] Faouzia Z, Danielle B, Isabelle GF, et al. Low molecular weight fucoidan enhances theproangiogenic phenotype of endothelial progenitor cells. Biochemical Pharmacology.2005,70(8):1167~1175
[96]谌素华，王维民.褐藻岩藻聚糖硫酸酯生物活性研究进展.食品工业科技，2009，30（6）：371~374
[97] American Heart Association. Statistical supplement. Dallas:American Heart Association,1997
[98]薛家驹，薛家芹.血栓病.西安：陕西科学技术出版社，1997.308~340
[99]李家增.血管血栓性疾病的发病机制和防治.基础医学与临床，2006，26（6）：561~565
[100]石英.血栓与血栓性疾病的治疗进展.新疆医学，1989，19（4）：231~233
[101]沈同，王镜岩.生物化学.北京：高等教育出版社，1993.131~133
[102]邹和昌.溶栓剂的发展与研究.中国药学杂志，1997，32（5）：263~267
[103]刘诚.纳豆激酶液体发酵条件及其酶学稳定性的研究：[硕士学位论文].南京：南京农业大学，2000
[104]武汉医学院.病理学—病理解剖学分册，北京：人民卫生出版社，1979.36~37
[105]殷侃、许白.抗血栓药物研究进展.中国医药情报，1995，1（3），153~160
[106]上海第一医学院.人体生理学.北京：人民卫生出版社，1978，39~105
[107]许芳、杨艳燕、阁达中.溶栓药物的研究进展，现代商贸工业，2003（7）：45~46
[108]杜平中.溶栓药物研究新进展.中国新药杂志.2001，10（8）：573~577
[109]彭波，许实波，许东晖，赵金华．褐藻多糖硫酸酯的抗凝和纤溶活性．中草药，2001，32（11）：1015~1018
[110] Barroso EM, Costa LS, Medeiros VP, et al.Anon-anticoagulant heterofucan hasantithrombotic activity in vivo. Planta Med.,2008,74:712~718
[111]黎七雄，江晖，肖清秋，等.半数致死量(LD50)Bliss法的评价及计算.数理医药学杂志，1995，8（4）：318~320
[112]徐叔云，卞如濂，陈修主编.药理实验方法学.北京：人民卫生出版社，1991.201~209
[113]王杰，王成梁，马淑兰，等.血栓康对大鼠实验性静脉血栓形成的影响.中国中医科技，2006，13(2)：93~94
[114] Kurz K D, Main B W, Sandusky G E.Rat model of arterial thrombosis induced by ferricchloride. Thromb Res,1990,60(4):269~280
[115]刘煜，常晓丹，吴梧桐.重组水蛭素Ⅲ乳化微粒口服的药效学.中国药学杂志，2009，44(7)：502~505
[116]张芳林，郭晟，朱令元，等.毛冬青酸抗血栓实验研究及机制探讨[J].江西医学院学报，2003，43(3)：33~37
[117]徐秋霞，王松，赵永志，等.血栓栓塞性疾病.北京:中国医药科技出版社，2004.12
[118]谢露．广西海带多糖L01提取纯化与抗血栓作用机制研究．[博士学位论文].广西：广西医科大学，2002
[119]赵建宏,林琳,高继发.SD大鼠血管内皮细胞损伤模型的制作方法.中国临床药理学与治疗学.1999,4（4）:276~278
[120]薛俊丽,冯波.人脐静脉内皮细胞体外培养方法.同济大学学报(医学版),2007,28（5）:110~113
[121]王皓,张静生.人脐静脉内皮细胞的体外培养.中西医结合心脑血管病杂志,2008,6(3):368~369
[122]王妍，陶军，杨震，等.肿瘤坏死因子促进内皮细胞微颗粒释放的实验研究.中华心血管病杂志，2005，33（12）：1137~1140
[123] Berckmas RJ, Nieuwland R, BoingAN, et al. Cel-l derived microparticles irculate in hea thyhuman s and support low grade thrombin generation. Thromb Haemost,2001,85(4):639~646
[124]王丽娜，郭旭昌，邓树勋.血管紧张素II与运动功能性异常.中国临床复，2003，7（27）:3786
[125] Sakariassen KS, Bolhuis PA, Sixma JJ. Human blood platelet adhesion to arterysubendothelium is mediated by factor VIII-von Willebrand factor bound to subendothelium.Nature.1979,279(5174):636~638
[126] Coller BS, Peerschke EI, Scudder LE, et al. Studies with a murine monoclonal antibody thatabolishes ristocetin-induced binding of von Willebrand factor to platelets: additionalevidence in support of GPIb as a platelet receptor for von Willebrand factor.Blood.1983,61(1):99~110
[127] Combes V, Simon AC, Grau GE, et al. In vitro generation of endothelial microparticles andpossible prothrombotic activity in patients with lupus anticoagulant. Clin invest,1999,104(1):93~102
[128] Jimenez J, Jy W, Mauro LM, et al. Elevated endothelial microparticles in thromboticthrombocytopenic purpura: findings from brain and renal microvascular cell culture andpatients with active disease. Br J Haematol,2001,112(1):81~90
[129] Brodsky SV, Malinowski K, Golightly M, et al. Plasminogen activator inhibitor promotesformation of endothelial microparticles with procoagulant potential. Circulation2002,106(18):2372~2378
[130] Wang JM, Wang Y, Huang JY, et al. C-reactive protein-induced endothelial microparticlegeneration in HUVECs is related t o BH4-dependen t NO formation. J Vasc Res,2007,44(3):241~248
[131] Horstman LL, Jy W, Jimenez JJ, et al. Endothelial microparticles as markers of endothelialdysfunct ion. Front Biosci,2004,9(5-1):1118~1135
[132] Abid HM, Boing AN, Biro E, et al. Phospholipid composition of in vitro endothelialmicroparticles and their in vivo thrombogenic propertie. Thromb Res,2008,121(6):865~871
[133]史旭波,胡大一.血小板与抗血小板治疗.临床荟萃，2008,23（4）：229~231
[134] Zhu Z, ZhangQ, Chen L, et al. Higher specificity of the activity of low molecular weightfucoidan for thrombin-induced platelet aggregation. Thromb Res.2010;125(5):419~426
[135] Durig J, Bruhn T, Zurborn K H, et al. Anticoagulant fucoidan fractions from Fucusvesiculosus induce platelet activation in vitro. J Na t Prod,1993,56(4):478~488
[136]徐叔云，卞如濂，陈修主编.药理实验方法学.北京：人民卫生出版社，2001.1271~1280
[137]杨蕾，修春，王宁生.冰片对大鼠血小板胞浆内游离钙离子浓度的影响.时珍国医国药.2010，21(1):1~3
[138]谢笑龙，龚其海，陆远富，等.钩藤碱对家兔血小板聚集及胞浆游离钙离子浓度的影响.中国药理学与毒理学杂志.2011，25(1):68~71
[139]庄明明，温奕欣，刘少列，等.流式细胞仪测定血小板胞浆游离钙浓度.西安交通大学学报(医学版).2005，26(5):508~510
[140] Merhi Y, Provost P, Guidoin R, et al. Importance of platelets in neutrophil adhesion andvasoconstriction after deep carotid arterial injury by angioplasty in pigs. ArteriosclerThromb Vasc. Biol.1997,17(6):1185~1191
[141]杨丽川，胡建林，陈鹏，等.β环糊精包合血竭抗PAF诱导血小板聚集作用机理探讨.中药药理与临床，2008，24(2)：37~38
[142]汪钟，郑植荃.现代血栓病学.北京：北京医科大学中国协和医科大学联合出版社，1996.212~223
[143]王宏涛，靳洪涛，孙建宁，等.异亚丙基莽草酸抗血栓作用的实验研究.药学学报，2002，37(4)：245~247.
[144]丁水平，方淑贤.吲哚布芬治疗血栓性疾病研究进展.医药导报，2006，25(10)：1039~1041
[145]史旭波，胡大一.血栓形成与凝血机制及调节.临床荟萃，2007，22（14）：989~991
[146] McClure MO,Whitby D,Patience C,et al．Dextran sulfate and fucoidan are potent inhibitorsof HIV infection in vitro．Antiviral Chem Chemother．1991,2:19~26
[147]曲香芝，崔弘，陈正爱.不同炮制法的淫羊藿提取物对小鼠出血和凝血时间的影响.时珍国医国药，2007，18(4)：118
[148] Giesen PL, Rauch U, Bohrmann B, et al. Blood-borne tissue factor: another view ofthrombosis. Proc Natl Acad Sci USA,1999,96(5):2311~2315
[149] Tedgui A, Mallat Z. Apoptosis as a determinant of atherothrombosis. Thromb Haemost.2001,86(1):420~426
[150] Moons AH, Levi M, Peters RJ. Tissue factor and coronary artery disease. Cardiovasc Res,2002,53(2):313~325
[151] Ragni M, Golino P, Cirillo P, et al. Endogenous tissue factor pathway inhibitor modulatesthrombus formation in an in vivo model of rabbit carotid artery stenosis and endothelialinjury. Circulation,2000,102(1):113~117
[152]谢露，陈蒙华，黎静，等．海带多糖L01对实验性动物血液凝固和血小板活性的影响．中医康复研究，2005，9(5)：124~125
[153]李萍，弥曼，杜洪霞，等.阿司匹林与硒合用对血瘀症大鼠血液流变学的影响.实用医学杂志，2010，26(16)：2911~2913
[154]王巧云，刘永云，徐理华，等.银杏达莫注射液抑制大鼠静脉血栓形成及作用机制探讨.中国新药杂志，2007，16(20)：1679~1681
[2] Barbara M,Ana-Cristina R,Ana-Paula, et al. Sulfated fucans from echinoderms have a regulartetrasaccharide repeating unit defined by specific patterns of sulfation at the O-2and O-4positions. J. Biol. Chem.,1994,269(35):22113~22123
[3]林洪，江洁，薛长湖，等.褐藻岩藻聚糖结构和活性研究进展.中国海洋药物，2002，90（6）：42~47
[4]李波，许时璎.羊栖菜中褐藻糖胶的提取纯化研究.食品工业，2004，2：40~42
[5]邢杰，刘守涛，高君，等.海带多糖的提取及组份L1的分离与鉴定.抚顺石油学院学报，1998，18(1)：8~9
[6]尤瑜敏，许时璎.羊栖菜中褐藻糖胶的提取工艺.无锡轻工大学学报，2002，5(3)：233~239
[7]何云海，汪秋宽，刘红丹.用复合酶酶解提取海带岩藻聚糖硫酸酯的工艺研究.上海水产大学学报，2006，21（1）：55~59
[8]郭亚贞，王慥.海带中褐藻糖胶的提取与纯化.上海水产大学学报.2000，9(3)：276~280
[9] Ozawa T, Yamamoto J, Yamagishi T. Two fucoidans in the holdfast of cultivated Laminariajaponica. Nature Medicine,2006,60(6):236~239
[10]陈蕾，吴皓.多糖降解方法的研究进展.中华中医学刊，2008，26(1)：133~135
[11] Shinichi Furukawa, Tatsuo Fujikawa, Daizo Koga, et al. Production of fucoidan degradingenzymes, fucoidan, and fucoidan sulfatase by Vibro sp1N25. Nippon Suisan Gakkaishi,1992,58(8):1499~1503
[12] Takcshi Sakai, Hitomi Kimura, Ikunoshin Kato. A Marine strain of flavobacteriaccac utilizesbrown seaweed fucoidan.Mar Biothechnol,2002,(4):399~405
[13]吴茜茜，蔡敬民，吴克，等.海洋真菌LD8岩藻多糖酶的固态发酵条件研究.海洋科学，2004，28(6)：12~27
[14]吴克，刘斌，吴茜茜，等.海洋真菌Dendryphiella arenaria TM94产岩藻多糖酶发酵及酶学性质.生物学杂志，2003，20(2)：14~16
[15] Kitamura K, Matsuo M, Yasui T. Enzymatic degradation of fucoidan by fucoidanase from thehepatopancreas of Patinopecten yessoensis. Biosic Biotech Biochem,1992,56(11):1829~1834
[16] Régis Daniel, Olivier Berteau, Jacqueline Jozefonvicz,et al. Degradation of algal(Ascophyllum nodosum) fucoidan by an enzymatic activity contained in digestive glands ofthe marine mollusc Pecten maximus. Carbohydrate Research.1999,322(3-4):291~297
[17] Takashi ohshiro, Yoshiyuki ohmoto, Yoshihisa ono, et al. Isolation and Characterization of aNovel Fucoidan-Degrading Microorganism.Bioscience, Biotechnology, andBiochemistry.2010,74(8):1729~1732
[18] I Iu Bakunina, O I Nedashkovskaia, S A Alekseeva, et al. Degradation of fucoidan by themarine proteobacterium seudoalteromonas citrea. Mikrobiologiia.2002,71(1):49~55
[19] Kim WJ, Kim SM, Lee YH, et al. Isolation and Characterization of Marine Bacterial StrainDegrading Fucoidan from Korean Undaria pinnatifida Sporophylls. Microbiology andBiotechnology,2008,18(4):616~623
[20]陈惠源，蔡俊鹏，刘江涛.岩藻多糖降解酶产生菌的筛选及鉴定.现代食品科技，2005，21(3)：42~44
[21] Kyong Duk Oh. degradation of brown alga-derived fucoidan. US.20090270607A1,2009-9-27
[22]武晓琳.分子量对海参岩藻聚糖硫酸酯活性和消化吸收的影响研究.[硕士论文].山东：中国海洋大学，2011
[23] Vitor H. Pomin, Ana Paula Valente, Mariana S. Pereira, et al. Mild acid hydrolysis of sulfatedfucans: a selective2-desulfation reaction and an alternative approach for preparing tailoredsulfated oligosaccharides. Glycobiology,2005,15(12):1376~1385
[24] Yun Hou, Jing Wang, Weihua Jin, et al. Degradation of Laminaria japonica fucoidanby hydrogen peroxide and antioxidant activities of the degradation products ofdifferent molecular weights. Carbohydrate Polymers,2012,87(1):153~159
[25] Luyt CE, Meddahi-Pellé A, Ho-Tin-Noe B, et al. Low Molecular Weight Fucoidan PromotesTherapeutic Revascularization in a Rat Model of Critical Hindlimb Ischemia. J Pharmacol ExpTher,2003,305(1):24~30
[26]富士川龙郎，中島克子.褐藻におけるフコイタソ様多糖分佈.日本濃雲化學雜誌，1975，49(9):455~461
[27] Nishide E, Anzai H, Uchida, et al. Sugar constituents of fucose-containing polysaccharidesfrom various Japanese brown algae. Hydrobiologia,1990,204/205(1):573~576
[28] Nishino T, Nishioka C, Ura H et al. Isolation and partial characterization of a novel aminosugar containing fucan sulfate from commerical Fucus vericulosus fucoidan. Carbohydr. RES.,1994,255(3):213~224
[29] Conchie J, Percival EGV. Fucoidin. Part Ⅱ：The hydrolysis of a methylated fucoidan preparedfrom fucus vesiculosis. J. Chem. Soc.,1950:827
[30] Patankar MS, Oehninger S, Barnett T, et al. A revised structure for fucoidan may explain someof its biological activities. J Biol Chem,1993,268(29):21770~21776
[31] Nishino T, Aizu Y, Nagumo T. Antithrombin activity of a fucan sulfate from the brownseaweed Ecklonia kurome. Thromb. Res.,1991,62(6):765~773
[32] Nishino T, Nagumo T, Kiyohara H. et al. Strutural characterization of a new anticoagulantfucan sulfate from the brown seaweed Ecklonia kurome. Carbohydr. Res.,1991,211(1):77~90
[33] Chizhov OA, Dell A, Morris RH, et al. A study of fucoidan from the brown seaweed Chordafilum. Carbohydr. Res.,1999,320(1-2):108~119
[34] Daniel R, Berteau O, Chevolot L, et al. Regioselective desulfation of sulfatedL-fucopyranoside by a new sulfoesterase from the marine mollusk Pecten maximus:application to the structural study of algal fucoidan (Ascophyllum nodosum). Eur J Biochem.2001,268(21):5617~5626
[35] Elizabeth P, Rahman MA. Chemistry of the polysaccarfides of the brown seaweed dictyprerisPlagiogramma. Phytochemistry,1981,20(7):1579
[36] Pereira MS. Mulloy B, Mourao PAS. Structure and anticoagulant activity of sulfated fucans:comparison between the regular repetitive, and linear fucans from echinoderms with the moreheterogeneous and branched polymers from brown algae. J. Biol. Chem.,1999,274(12):7656~7667
[37] Chevolot L, Foucault A, Chaubet F, et al. Further data on the structure of brown seaweedfucans: relations hipswith anticoagulant activity. Carbohydr Res.,1999,319(1-4):154~165
[38] Springer G F, Wurzel H A, Mcsneal G M,et al. Isolation of anticoagulant fractions from crudefucoidan. Proc Soc Exp Biol Med,1957,94:404~409
[39] Nishino T, Fukuda T, Nagumo T, et al. Inhibition of the generation of thrombin and factor Xaby a fucoidan from brown seaweed Ecklonia Kurome. Thromb Res,1999,96(1):37~49
[40] Nishino T, Kiyohara H, Yamada H, et al. An anticoagulant fucoidan from the brown seaweedEcklonia Kurome.Phytochemistry,1991,30(2):535~539
[41] Nishino T, Aizu Y, Nagumo T. The influence of sulf ate content and molecular weight of afucan sulfate from the brown seaweed Ecklonia Kurome on its antithromb in activity. ThrombRes.,1991,64(6):723~731
[42] Woo-Jung Kim, Sung-Min Kim, Hyun Guell Kim, et al. Purification and AnticoagulantActivity of a Fucoidan from Korean Undaria pinnatifida Sporophyll. Algae.2007,22(3):247~252
[43] Ushakova NA, Morozevich GE, Ustiuzhanina NE, et al. Anticoagulant activity of fucoidansfrom brown algae Biomed Khim.2008,54(5):597~606
[44] Albana Cumashi, Natalia A. Ushakova, Marina E. Preobrazhenskaya, et al. A comparativestudy of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities ofnine different fucoidans from brown seaweeds. Glycobiology.2007,17(5):541~552
[45] Grauffel V, Kloareg B, Mabeau S, et al. New natural polysaccharides with potent antithrombicactivity-fucans from brown-algae. Biomaterials.1989,10(6):363~368
[46] Mauray S, DE Raucourt E, Chaubet F, et al. Comparative anticoagulant activity and influenceon thrombin generation of dextran derivatives and of a fucoidan fraction. J Biomater SciPolym Ed.,1998,9(4):373~387
[47] Colliec S,Fischer AM,Tapon-Berlaudiere J,et al．Anticoagulant properties of a fucoidan.Fraction Thromb Res,,1991,64（2）:143~154
[48] Mauray S, Sternberg C, Theveniaux J, et al. Venous antithrombotic and anticoagulantactivities of a fucoidan fraction.Thromb Haemost.1995,74(5):1280~1285
[49] Durand E, Helley D, Ayman HZ. Effect of low molecular weight fucoidan and low molecularweight heparin in a rabbit model of arterial thrombosis. J Vasc Res.,2008,45(6):529~537
[50]刘承颖，王维民.半叶马尾藻中岩藻聚糖硫酸酯的提取纯化及抗氧化研究.食品研究与开发，2008，29(11)：71~75
[51] Costa, LS, Fidelis, GP, Cordeiro, SL,et al. Biological activities of sulfated polysaccharidesfrom tropical seaweeds. Biomed. Pharmacother.2010,64（1）,21~28
[52] Leandro Silva Costa, Gabriel Pereira Fidelis, Cinthia Beatrice Silva Telles, et al. Antioxidantand Antiproliferative Activities of Heterofucans from the Seaweed Sargassum filipendula. Mar.Drugs.2011,9:952~966
[53] Zhang Z, Wang F, Wang X,et al. Extraction of the polysaccharides from five algae and theirpotential antioxidant activity in vitro. Carbohydr. Polym.2010,82,118~121
[54] Wang J, Zhang Q, Zhang Z, et al. Potential antioxidant and anticoagulant capacity of lowmolecular weight fucoidan fractions extracted from Laminaria japonica. Int. J. Biol. Macromol.2010,46（1）:6~12
[55] Zhao X, Xue C H, Li B F. Study of antioxidant activities of sulfated polysaccharides fromLaminaria japonica. J App Phyco,2008,20（4）:431~436
[56] JAE GUN HAN, ABDUL QADIR SYED, M INCHUL KWON, et al. Antioxidant,immunomodulatory and anticancer activity of fucoidan isolated from Fucus vesiculosus.Journal of Biotechnology,2008,7:1346
[57]赵雪，薛长湖，王静凤，等.海带岩藻聚糖硫酸酯低聚糖对小鼠肝损伤的保护作用.营养学报，2003，25(3)：286~289
[58] Saito A, Yoneda M, Yokohama S, et al. Fucoidan prevents concanavalin A-induced liver injurythrough induction of endogenous IL-10in mice. Herpetology Research,2006,35(3):190~198
[59] SHINJI HAYASHI, AYANO ITOH, KATSUH IRO ISODA, et al.Fucoidan partly preventsCCl4induced liver fibrosis. European Journal of Ph armacology,2008,580(3):380~384
[60] Okaiy, Okai KH, Ishizaka S, et al. Possible immunomodulating activities in a extraction ofedible seaweed. Hizikia fusiforme (Hijiki). J Sci Food Agri,1996,72(4):455~460
[61]詹林盛，张新生，吴小红，等.褐藻多糖对正常小鼠淋巴细胞功能和细胞因子产生的影响.营养学报，2001，23（2）：122~125
[62]杨晓林，孙菊云，许汉年，等.褐藻糖酵的免疫调节作用.中国海洋药物，1995，14（3）：9~13
[63]赵雪．海带岩藻聚糖硫酸酯的化学组成与活性的研究．[博士学位论文].山东：中国海洋大学，2004
[64] Berteau O, Mulloy B. Sulfated fucans, fresh perspectives:structures, functions, and biologicalproperties of sulfated fucans and an overview of enzymes active toward this class ofpolysaccharides. Glycobiology.2003,13:29R~40R
[65] Joo-Woong Park, Hyun-Hyo Suh, Kyung-Bok Lee, et al. Immunomodulating Activity of aFucoidan Isolated from Korean Undaria pinnatifida Sporophyll. Algae.2007,22(4):333~338
[66] Choi EM, Kim A, Kim YO, et al. Immunostimulating activity of arabinogalactan and fucoidanin vitro. J. Med. Food.2005,8(4):446~453
[67] Maruyama H, Tamauchi H, Hashimoto M, et al. Antitumor activity and immune response ofMekabu fucoidan extracted from Sporophyll of Undaria pinnatifida. In vivo.2003,17(3):245~249
[68] Yamamoto I, Nagumo T, Takahashi M, et al. Antitumor effect of seaweeds Ⅲ. Antitumoreffect of an extract from Sargassum Kjellmanianum. Japan J ExP Med,1981,51(3):187~189
[69] Yamamoto I, Takahashi M, Tamura E, et al. Antitumor activity of crude extracts from ediblemarine algae against L-1210Leukemia. Botancia Marina,1982,25:455~457
[70] Yamamoto I, Takahashi M, Suzuki I, et al. Antitumor effect of seaweeds Ⅳ. Enhancement ofantitumor activity by sulfation of a crude fucoidan fraction from Sargassum Kjellmanianum.Japan J ExP Med,1984,54(4):143~151
[71]宋剑秋，徐誉泰，张华坤，等.海带硫酸多糖对小鼠腹腔巨噬细胞的免疫调节作用.中国免疫学杂志，2000，16（2）：70~72
[72] Zhuang C, Itoh H, Mizuno T, et al. Antitumor active fucoidan from the brown seaweed,Umitoranoo(Sargassum thunbergii).Biosci Biothech BioChem,1995,59(4):563~567
[73] Itoch H, Noda H, Amno H, et al. Antitumor activity and immunological properties of marinealgae Polysaccharides, especially fucoidan, prepared from Sargassum thunbergii of PhaeoPhyceae. Anticancer Res.,1993,13(6A):2045~2052
[74] Itoh H, Noda H, Amno H, et al. Immuological analysis of inhibition of lung metastases byfucoidan(GIV-A) prepared from brown seaweed Sargassum thunbergii. Anticancer Res.,1995,15(SB):1937~1947
[75] Usuui T, Asari K, Mizuno T. Isolation of Highly Purified “fucoidan” from Eisenia bicyclis andits anticoagulant and antitumor activities. Agric Biol Chem,1980,44（8）:1965~1966
[76]王庭欣，秦淑贞，赵文，等.海带多糖对环磷酰胺诱小鼠骨位细胞微核率的抑制作用.癌变·畸变·突变，1999，11(2)：106~107
[77] Ye J, Li Y, Teruya K, Katakura Y, et al. Enzyme-digested Fucoidan Extracts Derived fromSeaweed Mozuku of Cladosiphon novae-caledoniae kylin Inhibit Invasion and Angiogenesisof Tumor Cells. Cytotechnology.2005,47(1-3):117~126
[78] Alekseyenko TV, Zhanayeva SY, Venediktova AA, et al. Antitumor and antimetastatic activityof fucoidan, a sulfated polysaccharide isolated from the Okhotsk Sea Fucus evanescens brownalga. Bull Exp Biol Med.2007,143(6):730~732
[79] Aisa Y,Miyakawa Y,Nakazato T,et al.Fucoidan induces apoptosis of human HS-sultan cellsaccompanied by activation of caspase-3and down-regulation of ERK pathways.Am JHematol,2005,78(1):7~14
[80] Koyanagi S,Tanigawa N,Nakagawa H,et al.Oversulfation of fucoidan enhances itsanti-angiogenic and antitumor activities.Biochem Pharmacol,2003,65(2):173~179
[81] Bernardi G，Springer GF．Properties of highly purified fucan．Biol Chem，1962，237:75~77
[82] Colliee S, Fiseher AM, Tapon-BerlaudiereJ, et al.Anticoagulant properties of a fucoidanfraction. Trombosis Research,1991,64(2):143～154
[83] Sandrine M,Clauds S,Theveniaux J,et al．Venous Antithrombotic and anticoagulant activitiesof a fucoidan fraction．Thromb Haemost.,1995,74(5):1280～1285
[84] Soeda S, Sakaquchi S, Shimeno H, et al. Fibrinolytic and anticoagulant activities of highlysulfated fucoidan.Biochem Pharma,1992,43(8):1853~1858
[85] Millet J, Jauoult SC, Mauray S, et al. Antithrombotic and anticoagulant activities of a lowmolecular weight fucoidan by the subcuataneous route. Thromb Haemost,1999,81(3):391~395
[86] Doctor VM，Hill C，Jackson GJ，et al. Effect of fucoidan during activation of humanplasminogen.Throm Res.,1995,79(3):237~247
[87]薛长湖，李兆杰，辛梅，等.岩藻聚糖硫酸酯的制备方法.中国，专利号：ZL98110253.0.，1998
[88] Glable CG, Yednock T, Rosen SD, et al. Reversible disruption of cultured endothelialmonolayers by sulfated fucans. J Cell Sci,1983,61(5):475~490
[89]辛现良，耿美玉，李桂玲，等.海洋硫酸多糖911体外对HIV-1作用的研究.中国海洋药物，2000(19)：8~11
[90]李凡，田同春，石艳春，等.岩藻糖胶体外抗病毒作用的研究.白求恩医科大学学报，1995，21(3)：18~22
[91]李德远，徐战，黄利民，等.海带岩藻糖胶对大鼠饮食性高血脂症的影响.食品科学，2001，22(2)：92~95
[92] Chabut D, Fischer A M, Colliec-Jouault S, et al. Low molecular weight fucoidan and heparinenhance the basic fibroblast grow th factor-induced tube form ation of endothelia l cellsthrough heparan sulfate-dependent alpha6over expression. Mol Pharmacol.2003,64(3):696~702
[93] Sabine Matou, Dominique Helley, Delphine Chabut. et al. Effect of fucoidan on fibroblastgrowth factor-2-induced angiogenesis in vitro. Thrombosis Research.2002，106(4-5):213~221
[94] C. Boisson-Vidal, F. Zemani, G. Caligiuri, et al. Neoangiogenesis induced by progenitorendothelial cells: effect of fucoidan from marine algae. Cardiovascular&HematologicalAgents in Medicinal Chemistry,2007,5(1):67~77
[95] Faouzia Z, Danielle B, Isabelle GF, et al. Low molecular weight fucoidan enhances theproangiogenic phenotype of endothelial progenitor cells. Biochemical Pharmacology.2005,70(8):1167~1175
[96]谌素华，王维民.褐藻岩藻聚糖硫酸酯生物活性研究进展.食品工业科技，2009，30（6）：371~374
[97] American Heart Association. Statistical supplement. Dallas:American Heart Association,1997
[98]薛家驹，薛家芹.血栓病.西安：陕西科学技术出版社，1997.308~340
[99]李家增.血管血栓性疾病的发病机制和防治.基础医学与临床，2006，26（6）：561~565
[100]石英.血栓与血栓性疾病的治疗进展.新疆医学，1989，19（4）：231~233
[101]沈同，王镜岩.生物化学.北京：高等教育出版社，1993.131~133
[102]邹和昌.溶栓剂的发展与研究.中国药学杂志，1997，32（5）：263~267
[103]刘诚.纳豆激酶液体发酵条件及其酶学稳定性的研究：[硕士学位论文].南京：南京农业大学，2000
[104]武汉医学院.病理学—病理解剖学分册，北京：人民卫生出版社，1979.36~37
[105]殷侃、许白.抗血栓药物研究进展.中国医药情报，1995，1（3），153~160
[106]上海第一医学院.人体生理学.北京：人民卫生出版社，1978，39~105
[107]许芳、杨艳燕、阁达中.溶栓药物的研究进展，现代商贸工业，2003（7）：45~46
[108]杜平中.溶栓药物研究新进展.中国新药杂志.2001，10（8）：573~577
[109]彭波，许实波，许东晖，赵金华．褐藻多糖硫酸酯的抗凝和纤溶活性．中草药，2001，32（11）：1015~1018
[110] Barroso EM, Costa LS, Medeiros VP, et al.Anon-anticoagulant heterofucan hasantithrombotic activity in vivo. Planta Med.,2008,74:712~718
[111]黎七雄，江晖，肖清秋，等.半数致死量(LD50)Bliss法的评价及计算.数理医药学杂志，1995，8（4）：318~320
[112]徐叔云，卞如濂，陈修主编.药理实验方法学.北京：人民卫生出版社，1991.201~209
[113]王杰，王成梁，马淑兰，等.血栓康对大鼠实验性静脉血栓形成的影响.中国中医科技，2006，13(2)：93~94
[114] Kurz K D, Main B W, Sandusky G E.Rat model of arterial thrombosis induced by ferricchloride. Thromb Res,1990,60(4):269~280
[115]刘煜，常晓丹，吴梧桐.重组水蛭素Ⅲ乳化微粒口服的药效学.中国药学杂志，2009，44(7)：502~505
[116]张芳林，郭晟，朱令元，等.毛冬青酸抗血栓实验研究及机制探讨[J].江西医学院学报，2003，43(3)：33~37
[117]徐秋霞，王松，赵永志，等.血栓栓塞性疾病.北京:中国医药科技出版社，2004.12
[118]谢露．广西海带多糖L01提取纯化与抗血栓作用机制研究．[博士学位论文].广西：广西医科大学，2002
[119]赵建宏,林琳,高继发.SD大鼠血管内皮细胞损伤模型的制作方法.中国临床药理学与治疗学.1999,4（4）:276~278
[120]薛俊丽,冯波.人脐静脉内皮细胞体外培养方法.同济大学学报(医学版),2007,28（5）:110~113
[121]王皓,张静生.人脐静脉内皮细胞的体外培养.中西医结合心脑血管病杂志,2008,6(3):368~369
[122]王妍，陶军，杨震，等.肿瘤坏死因子促进内皮细胞微颗粒释放的实验研究.中华心血管病杂志，2005，33（12）：1137~1140
[123] Berckmas RJ, Nieuwland R, BoingAN, et al. Cel-l derived microparticles irculate in hea thyhuman s and support low grade thrombin generation. Thromb Haemost,2001,85(4):639~646
[124]王丽娜，郭旭昌，邓树勋.血管紧张素II与运动功能性异常.中国临床复，2003，7（27）:3786
[125] Sakariassen KS, Bolhuis PA, Sixma JJ. Human blood platelet adhesion to arterysubendothelium is mediated by factor VIII-von Willebrand factor bound to subendothelium.Nature.1979,279(5174):636~638
[126] Coller BS, Peerschke EI, Scudder LE, et al. Studies with a murine monoclonal antibody thatabolishes ristocetin-induced binding of von Willebrand factor to platelets: additionalevidence in support of GPIb as a platelet receptor for von Willebrand factor.Blood.1983,61(1):99~110
[127] Combes V, Simon AC, Grau GE, et al. In vitro generation of endothelial microparticles andpossible prothrombotic activity in patients with lupus anticoagulant. Clin invest,1999,104(1):93~102
[128] Jimenez J, Jy W, Mauro LM, et al. Elevated endothelial microparticles in thromboticthrombocytopenic purpura: findings from brain and renal microvascular cell culture andpatients with active disease. Br J Haematol,2001,112(1):81~90
[129] Brodsky SV, Malinowski K, Golightly M, et al. Plasminogen activator inhibitor promotesformation of endothelial microparticles with procoagulant potential. Circulation2002,106(18):2372~2378
[130] Wang JM, Wang Y, Huang JY, et al. C-reactive protein-induced endothelial microparticlegeneration in HUVECs is related t o BH4-dependen t NO formation. J Vasc Res,2007,44(3):241~248
[131] Horstman LL, Jy W, Jimenez JJ, et al. Endothelial microparticles as markers of endothelialdysfunct ion. Front Biosci,2004,9(5-1):1118~1135
[132] Abid HM, Boing AN, Biro E, et al. Phospholipid composition of in vitro endothelialmicroparticles and their in vivo thrombogenic propertie. Thromb Res,2008,121(6):865~871
[133]史旭波,胡大一.血小板与抗血小板治疗.临床荟萃，2008,23（4）：229~231
[134] Zhu Z, ZhangQ, Chen L, et al. Higher specificity of the activity of low molecular weightfucoidan for thrombin-induced platelet aggregation. Thromb Res.2010;125(5):419~426
[135] Durig J, Bruhn T, Zurborn K H, et al. Anticoagulant fucoidan fractions from Fucusvesiculosus induce platelet activation in vitro. J Na t Prod,1993,56(4):478~488
[136]徐叔云，卞如濂，陈修主编.药理实验方法学.北京：人民卫生出版社，2001.1271~1280
[137]杨蕾，修春，王宁生.冰片对大鼠血小板胞浆内游离钙离子浓度的影响.时珍国医国药.2010，21(1):1~3
[138]谢笑龙，龚其海，陆远富，等.钩藤碱对家兔血小板聚集及胞浆游离钙离子浓度的影响.中国药理学与毒理学杂志.2011，25(1):68~71
[139]庄明明，温奕欣，刘少列，等.流式细胞仪测定血小板胞浆游离钙浓度.西安交通大学学报(医学版).2005，26(5):508~510
[140] Merhi Y, Provost P, Guidoin R, et al. Importance of platelets in neutrophil adhesion andvasoconstriction after deep carotid arterial injury by angioplasty in pigs. ArteriosclerThromb Vasc. Biol.1997,17(6):1185~1191
[141]杨丽川，胡建林，陈鹏，等.β环糊精包合血竭抗PAF诱导血小板聚集作用机理探讨.中药药理与临床，2008，24(2)：37~38
[142]汪钟，郑植荃.现代血栓病学.北京：北京医科大学中国协和医科大学联合出版社，1996.212~223
[143]王宏涛，靳洪涛，孙建宁，等.异亚丙基莽草酸抗血栓作用的实验研究.药学学报，2002，37(4)：245~247.
[144]丁水平，方淑贤.吲哚布芬治疗血栓性疾病研究进展.医药导报，2006，25(10)：1039~1041
[145]史旭波，胡大一.血栓形成与凝血机制及调节.临床荟萃，2007，22（14）：989~991
[146] McClure MO,Whitby D,Patience C,et al．Dextran sulfate and fucoidan are potent inhibitorsof HIV infection in vitro．Antiviral Chem Chemother．1991,2:19~26
[147]曲香芝，崔弘，陈正爱.不同炮制法的淫羊藿提取物对小鼠出血和凝血时间的影响.时珍国医国药，2007，18(4)：118
[148] Giesen PL, Rauch U, Bohrmann B, et al. Blood-borne tissue factor: another view ofthrombosis. Proc Natl Acad Sci USA,1999,96(5):2311~2315
[149] Tedgui A, Mallat Z. Apoptosis as a determinant of atherothrombosis. Thromb Haemost.2001,86(1):420~426
[150] Moons AH, Levi M, Peters RJ. Tissue factor and coronary artery disease. Cardiovasc Res,2002,53(2):313~325
[151] Ragni M, Golino P, Cirillo P, et al. Endogenous tissue factor pathway inhibitor modulatesthrombus formation in an in vivo model of rabbit carotid artery stenosis and endothelialinjury. Circulation,2000,102(1):113~117
[152]谢露，陈蒙华，黎静，等．海带多糖L01对实验性动物血液凝固和血小板活性的影响．中医康复研究，2005，9(5)：124~125
[153]李萍，弥曼，杜洪霞，等.阿司匹林与硒合用对血瘀症大鼠血液流变学的影响.实用医学杂志，2010，26(16)：2911~2913
[154]王巧云，刘永云，徐理华，等.银杏达莫注射液抑制大鼠静脉血栓形成及作用机制探讨.中国新药杂志，2007，16(20)：1679~1681
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