一種拮抗Glycoprotein Ⅰb的百步蛇毒蛋白之抗血栓作用之探討

Abstract

出血性蛇毒蛋白具有影響血液凝固和血小板凝集的活性。在本實驗中，將Deinagkistrodon acutus 原毒經由Sephadex G-75、FPLC Superdex75 10/30 GL、FPLC Mono-S 5/50進行純化分析得到P2F4。在15% SDS-PAGE，得知P2F4在非還原狀態下，其分子量為22kDa；若以β-mercaptoethanol還原後，則發現由兩個次單元組成，分子量在10kDa與11kDa。在定序方面，P2F4的序列和目前已知C-type lectin蛇毒蛋白 N端部分有部分相似，N端序列和Botrocetin (α50%；β35%)與Bitiscetin (α50%；β50%)這類vWF刺激劑相似度非常高，然而對於C-type lectin N端保留序列（conserve sequence）CXXXWXXXXXXC並不相符合。 P2F4在血小板懸浮液和富含血小板血漿中，都會有濃度相關性抑制由Ristocetin引發之血小板凝集。在血小板懸浮液，大約45nM即可完全抑制，而在富含血小板血漿中約2.3nM即可達完全抑制，而P2F4在富含血小板血漿有較高抑制活性，但其機轉目前仍不明瞭。但P2F4在高濃度（920nM）下對於collagen（10μg/ml）下會有些微抑制現象產生。但對Thrombin（0.03U/ml）與 ADP（10μM）引發之凝集反應不具抑制作用。利用流式細胞儀分析觀察P2F4和血小板上受體間的關係，發現P2F4可以明顯減少GPⅠb專一性抗體SZ2與AP1對血小板的結合作用，但對α2β1專一性抗體6F1，GPⅥ專一性抗體204.11及6B12， αⅡbβ3專一性抗體7E3對血小板之結合作用卻皆不會有影響。P2F4（4.6nM）可抑制ristocetin所引發之TXA2合成（約50%），且抑制agglucetin引發之P-Selectin表現，因此可以推知P2F4藉由和GPⅠb結合來降低血小板活化。當以靜脈注射時，會延長出血時間，但不引起血小板下降之現象。 由於P2F4是一個在人類富含血小板血漿中具有相當高活性類似C-type lectin的蛇毒蛋白，因此將可進一步在動物模式中評估抗血栓活性，且定出全部序列並和目前已知作用於GPⅠb蛇毒蛋白序列比較，找出活性相關序列，以供學術研究與藥物開發參考。

By using column chromatography of Sephadex G-75, FPLC Superdex75 10/30 GL, and FPLC Mono-S 5/50 GL in sequence, a novel snake venom protein ,P2F4 was purified form Deinagkistrodon acutus venom. It can inhibit ristocetin-induced aggregation in human platelet suspension and human platelet- rich plasma in a concentration dependent manner. P2F4 contains two subunits with molecular weight 10kDa and 11kDa,respectively. Its native molecular was estimated to be 21kDa by gel filtration. The reduced and alkylated α and β subunits were obtained and sequenced, showing the N-terminal sequence of P2F4 was partially similar to those of C-type lectin. However, the N-terminal sequence of P2F4 differed from the conserved sequence (CXXXWXXXXXXC ) of known C-type lectins. The N-terminal sequence of P2F4 was similar to Botrocetin (α50%；β35%)and Bitiscetin (α50%；β50%) two vWF-mediating agonist. P2F4 exhibits high potency in blocking ristoction-induced aggregation. Its IC50 was estimated to be 2.3nM and 23nM in human platelet-rich plasma and in platelet suspension, respectively. P2F4 inhibited agglucetin-induced aggregation with higher concentration required in blocking ristocetin-induced aggregation. P2F4 can not reverse agglucetin-induced aggregation but can prevent further progression of platelet aggregation. On the other hand, P2F4 can reverse ristocetin- induced aggregation. P2F4 did not inhibit platelet aggregation induced by ADP, or thrombin, P2F4 at higher concentration (920nM) had slight inhibition on collagen-induced aggregation . In flow cytometric assay, P2F4 specifically interrupted GPⅠb mab ( AP1 and SZ2) binding to GPⅠb, while P2F4 did not interfere with the binding of other receptor specific antibody such as 11A12(GPⅥ), 204.11(GPⅥ), 6F1(α2β1), or 7E3(αⅡbβ3). P2F4(4.6nM) reduced TxA2 formation caused by ristocetin about 50% and reduced agglucetin-induced P-selectin expression. As P2F4 was administered intravenously it prolonged the tail bleeding time, and did not cause thrombocytopenia syndrome within 30 min. Taken together, P2F4 is a highly specific GPⅠb antagonist. It is very unique that it exhibits a higher activity in platelet-rich plasma than in platelet suspension. Whether it is a potent antithrombotic agent in vivo needs further investigation.