龜殼花蛇毒蛋白Disintegrin抗血栓活性與作用機轉之探討

Abstract

Disintegrins是小分子量的抗血栓蛋白，最早由蛇毒蛋白中所發現，並有著強效的血小板凝集抑制活性。Disintegrins的作用機轉是抑制血小板上的GPIIb/IIIa，被認為具有開發為抗血栓抑制劑的潛力。龜殼花 (Trimeresurus mucrosquamatus) 蛇毒蛋白原毒粉末依序經由CM-Sephadex C-50離子交換層析法、Sephadex G-75 膠質過濾法、FPLC Superdex-75膠質過濾法，最後再以逆向高效液相色譜分析法進行最後純化分離，純化得到的兩種disintegrin分別為TMV-2和TMV-7。在本篇研究中，主要的目的是探討並比較TMV-2和TMV-7抗血栓作用和機轉不同之處。TMV-2和TMV-7有著不一樣的等電點，TMV-2的等電點約為4.5而TMV-7的等電點約為5-7。TMV-2和TMV-7都是單鏈的小分子，分子量經由MALDI-TOF測定分別為7663 和7672 Da。由LC-MS/MS所鑑定出的部分序列顯示，TMV-2有24%的序列相似於batroxostatin (由Bothrops atrox蛇毒蛋白中分離出的disintegrin)；而TMV-7也有24%的序列相似於batroxostatin，但有高達79%的序列相似於cotiarin (由Bothrops cotiara蛇毒蛋白中分離出的disintegrin)。TMV-2和TMV-7都能有效抑制在人類富含血小板的血漿、人類血小板懸浮液以及經elastase處理的人類血小板懸浮液中所引發的凝集反應，其抑制的程度會隨著濃度上升而增大。TMV-2和TMV-7都不會影響thromboxane A2的生成，但都會增強由collagen和thrombin所引起的P-selectin的表現。利用流式細胞儀的分析，可觀察到TMV-2對於GPIIb/IIIa的單株抗體7E3結合到GPIIb/IIIa上有明顯抑制作用，然而TMV-7卻不會抑制單株抗體7E3結合到GPIIb/IIIa上。而TMV-2和TMV-7對於另一種GPIIb/IIIa的單株抗體10E5的影響卻是會加強此單株抗體結合到GPIIb/IIIa上。在動物實驗方面，TMV-2和TMV-7也能抑制老鼠富含血小板的血漿所中所引起的血小板凝集反應，其抑制的程度也能隨著濃度上升而增大。在給予老鼠0.25μg/g的劑量之下，TMV-2會顯著的延長出血時間，然而TMV-7則不會延長出血時間。另外，TMV-2和TMV-7兩者都不會減少血小板的數量。綜合上述結果可知，TMV-2和TMV-7這兩個等電點不相同的disintegrin有著不一樣的特性。TMV-2比起TMV-7有著更強效的血小板凝集抑制效果，而且TMV-7結合到GPIIb/IIIa上的位置顯然和TMV-2及單株抗體7E3不同。另外，我們發現當TMV-2或TMV-7和單株抗體10E5一起作用時反而會引發血小板的活化。而給予老鼠0.25μg/g的劑量之下，TMV-7的副作用和TMV-2比起來顯得較少。由此我們認為TMV-2和TMV-7這兩種disintegrin之間的差異研究可利於新一代GPIIb/IIIa拮抗劑的研發，而且由TMV-2和TMV-7作用於GPIIb/IIIa的不同之處或許可以提供訊息來探討GPIIb/IIIa拮抗劑所引發血小板減少症的機轉。

Disintegrins are small moleculars and potent platelet inhibitors found in the snake venom. Disintegrins are GPIIb/IIIa antagonists and potential antithrombotic agents. By means of CM-Sephadex C-50, Sephadex G-75 gel filtration, FPLC Superdex 75 gel filtration and reverse phase HPLC, two disintegrins, TMV-2 and TMV-7, were purified from Trimeresurus mucrosquamatus snake venom. In this study, we investigated and compared the difference between TMV-2 and TMV-7. TMV-2 and TMV-7 had different isoelectric point (pI). The pI of TMV-2 was estimated to be around 4.5, whereas that of TMV-7 was estimated to be around 5-7. TMV-2 and TMV-7 were shown to be a single peptide chain. By MALDI-TOF, the molecular weigh of TMV-2 and TMV-7 were determined as 7663 and 7672 Da, respectively. The sequence of TMV-2 was 24% identical to batroxostatin, a disintegrin purified from the snake venom of Bothrops atrox, whereas the sequence of TMV-7 was 24% identical to batroxostatin and 79% identical to cotiarin, a disintegrin purified from the snake venom of Bothrops cotiara. Both TMV-2 and TMV-7 concentration-dependently inhibited platelet aggregation in human platelet-rich plasma, washed human platelet suspension and elastase-treated human platelets. Also, TMV-2 and TMV-7 did not interfere with the formation of thromboxane A2. However, both TMV-2 and TMV-7 enhanced the P-selectin expression induced by collagen and thrombin. In the indirect binding assay, TMV-2 significantly inhibited 7E3, a mAb raised against GPIIb/IIIa, binding to GPIIb/IIIa, but TMV-7 did not. Both TMV-2 and TMV-7 enhanced 10E5, a mAb raised against GPIIb/IIIa, binding to GPIIb/IIIa. In the animal models, TMV-2 and TMV-7 dose-dependently inhibited platelet aggregation in mice PRP. Furthemore, TMV-2 (0.25μg/g) prolonged the bleeding time more significantly than TMV-7 (0.25μg/g) as they were intravenously administered. However, both TMV-2 and TMV-7 did not alter the platelet counts. In conclusion, TMV-2 and TMV-7, two disintegrins with different isoelectric point, have the different characters. Although both TMV-2 and TMV-7 are GPIIb/IIIa inhibitor, the inhibitory effect of TMV-2 was more potent than TMV-7. Also, our data revealed that the binding site of TMV-7 on GPIIb/IIIa was different from TMV-2 and mAb 7E3. Moreover, combination of TMV-2 or TMV-7 with mAb 10E5 could lead to platelet activation. Finally, we found that TMV-7 had fewer side effects than TMV-2 at the same dosage. Therefore, the difference between TMV-2 and TMV-7 may provide valuable information for the development of GPIIb/IIIa inhibitors and the mechanisms involved in GPIIb/IIIa inhibitor-induced thrombocytopenia.