烙铁头蛇毒C-型凝集素样蛋白的生物活性及其作用机制研究
文献类型：学位论文
| 作者 | 台虹 |
| 学位类别 | 博士 |
| 答辩日期 | 2004-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 熊郁良 ; 张云 |
| 关键词 | 烙铁头蛇毒 C-型凝集素样蛋白 血小板聚集 血小板减少 抗血栓 补体 IX因子结合蛋白 |
| 其他题名 | Studies on biological activity and mechanism of a C-type lectin-like protein from trimeresurus mucrosquamatus venom |
| 中文摘要 | 蛇毒C一型凝集素样蛋白一般是由两个相似亚基组成的异构二聚体或由多个异构二聚体组成的多聚体。烙铁头蛇毒血小板活化素TMVA是一个高分子量的C一型凝集素样蛋白。本文研究了烙铁头蛇毒C一型凝聚素样蛋白的生物活性及其作用机制。同时还从烙铁头蛇毒中分离到一个抗凝蛋白（mucroqucetin）。TMVA呈量效关系地诱导P好和洗涤血小板聚集，提示其活化血小板并不调节v从下或不依赖于vWF。抗人血小板糖蛋白（GP）Ib单克隆抗体HIPI呈剂量依赖性特异性抑制TMVA诱导的血小板聚集。抗人GPllb单克隆抗体PZ也抑制血小板聚集。单克隆抗体和流式细胞术分析显示，FITC一TMVA特异性地、剂量依赖性地结合到福尔马林固定的人血小板上，在FIIC-TMVA4O砚浓度时达到饱和性结合状态，这种结合被HIPI特异性地抑制，并呈剂量依赖性。TMVA不结合血小板GPIX、GPllb、GPllla、GPIa、GPlla和GPIV。Mocarhagin仅能部分阻断TMVA诱导的血小板聚集，然而TMVA却能诱导mocarhagin阻断的血小板聚集。以上结果显示TMVA在GPIbQ上的主要受体位于富含亮氨酸第二次重复区氨基酸残基59一81，除此之外，TMVA可能还有其他结合位点。小鼠体内首次给予TMVA后，在短时间内（15min-30min）引起循环血小板数及网织血小板百分率暂时性减低、血小板膜表面P-seleetin的表达暂时性增加，但循环血液中血小板一白细胞复合物未增加；抗鼠血小板单克隆抗体P一selectin、GPllb、GPlll。免疫组化染色显示脾和肺的组织巨噬细胞呈阴性反应，提示TMVA导致循环血小板减少不是巨噬细胞系统对血小板的清除所致。可能是由于TMVA．通过GPIb活化血小板后使P-selectin暴露于胞浆膜表面，这些脱颗粒的血小板在补体的参与下自身溶破，而导致快速的暂时性血小板减少。给予TMVA．后组织病理学显示，肝、脾、肺、肾、胰、大肠及小肠血管未血栓形成；TMVA不影响内源性和外源性凝血系统；TMVA呈剂量依赖性延长小鼠的出血时间，但在TMvA25p眺g时小鼠的出血时间未延长、仍小于5分钟，是一个安全的剂量。以上结果充分证明TMVA具有体内抗血栓作用，血小板自身溶破导致血小板减少在体内抗血栓中可能也起到重要的作用。体内外实验显示TMVA在动物体内外都具有抗补体活性作用。TMVA上调血小板上DAF和CD59的表达，不同程度地上调白细胞上D胚和CDS夕的表达。TMVA在补体系统中的作用可能与其在异种器宫移植中抗IIAR的发生相关。Mucroqucetin是一个由Q链和p链组成的异二聚体蛋白质，其分子量为25KD。。N一末端氨基酸序列与其它该类蛇毒C一型凝集素样蛋白有很高的同源性。Mucroqucetin呈量效关系延长部分凝血活酶时间（APTT），几乎不影响凝血酶原时间（PT）。因子IX和X的抑制试验显示，该纯化蛋白呈量效关系抑制工X因子的凝血活性，不影响X因子，提示mucroqucetin的抗凝活性主要通过结合FIX而实现，是一个血液凝固IX因子结合蛋白。 |
| 英文摘要 | C-type lectin-like protein of snake venoms are generally composed of two a{3-heterodimers and (aP)n complex. TMVA is a C-type lectin-like protein with potent platelet activating and with high molecular weights from Trimeresurus mucrosquamatus venom. Its biochemical activity and mechanism were investigated. In addition, an anticoagulant protein, mucroqucetin, was isolated from Trimeresurus mucrosquamatus venom. In the absence and presence of von Willebrand factor (vWF), TMVA dose-dependently induced aggregation in both washed platelets and PRP. The similar aggregation activities of TMVA in both PRP and washed platelet suggested that TMVA did not modulate vWF. Anti-GP Ib monoclonal antibodies (mAbs), HIP1, specifically inhibited TMVA-induced aggregation in a dose-dependent manner. The aggregation also was inhibited by mAb P2 (an anti-GP lib mAb). Flow cytometric analysis revealed that FITC-TMVA bound to human formalin-fixed platelets in a saturable manner, and its binding was specifically blocked by HIP1 in a dose-dependent manner. Flow cytometric analysis also showed that TMVA did not bind to platelet GPIX, GPIIb, GPIIIa, GPIa, GPIIa and GPIV. Moreover, the aggregation induced by TMVA was partially inhibited when platelet was pretreated with mocarhagin, a snake venom protease that cleaves human GPIb. However, TMVA might induce platelet aggregation blocked by mocarhagin. These results taken together suggest that TMVA is a strong platelet agonist via the second leucine-rich repeat domaim of residues 59-81 specific to GPIb and it might have another functional binding-sites on GPIb molecule. After 15 min-30 min TMVA firstly injected into the body of mice, platelet counts and percentage of reticulated platelets of circulation blood temporarily decreased and the expression of P-selectin on the platelets temporarily increased, but platelet/leucocyte complexes within the circulation blood were similar to that of administrated ago. Immunohistologic staining of anti-mouse GPIIb clone MWReg30, GPIIIa clone 2C9.G2 and P-selectin clone RB40.34 in spleen and lung slice of TMVA-treated mouse group showed negative reaction of the spleen macrophages and lung macrophages. These results indicated that TMVA-induced thrombocytopenia was not caused by the increasing clearance of reticuloendothelial system phagocytosing platelets, and might be the reason of TMVA activating platelets to bring about P-selectin redistributing to the cell surface upon platelet activation, and the degranulated platelets self-breaking under the complement. Histopathology showing no thrombi in spleen, lungs and so on tissue sections stained by hematoxylin and eosin (H.E) were seen one hour after TMVA was injected to abdominal cavity of mice. The antithrombotic activity was evaluated in vivo by intravenously administration of various doses of TMVA. TMVA prolonged bleeding time of mice in a dose-dependent manner. At a dosage of 25 ug/kg, TMVA effectively prevented thrombus formation in the animal model without apparently increasing the risk of bleeding. TMVA did not yet affect intrinsic and extrinsic coagulation pathway. Therefore, TMVA played an important role in antithrombotic activity. TMVA presented the activity of anticomplement in vitro and in vivo. TMVA incubated with guinea pig mixed serum for 20 min at 37°C, the total complement activity in mixed serum of guinea pig significantly decreased. After 30 min intravenous injection of various concentrations TMVA, the units of rats' serum total complement activity decreased. However, when incubated with TMVA for 30 min at 37°C, expression of DAF and CD59 on platelets and leukocyte enhanced. These results suggested that anti-xenogeneic hyperacute rejection might be related to the enhancement of DAF and CD59 and to the decrease of total complement. An anticoagulant protein, mucroqucetin, was isolated from Trimeresurus mucrosquamatus venom. On SDS-polyacrylamide gel electrophoresis, mucroqucetin showed a single band with an apparent molecular weight of 25,000 under non-reducing conditions, and a band with apparent molecular weights of 14,000 under reducing conditions. Mucroqucetin significantly prolonged the plasma clotting time in APTT assay. Coagulant factors activity assay showed that it inhibited the coagulant activity of factor IX in a dose-dependent manner, but did not affect that of factor X, suggesting that mucroqucetin is a FDC-binding protein. |
| 语种 | 中文 |
| 公开日期 | 2010-10-15 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6199]  |
| 专题 | 昆明动物研究所_其他 昆明动物研究所_动物活性蛋白多肽组学 |
| 推荐引用方式 GB/T 7714 | 台虹. 烙铁头蛇毒C-型凝集素样蛋白的生物活性及其作用机制研究[D]. 北京. 中国科学院研究生院. 2004. |
入库方式： OAI收割
来源：昆明动物研究所
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