Purification and Characterization of a Platelet Aggregation Inducer from Bothrops jararaca

• Main Authors: Shu,Han-Lin, 許翰琳
• Other Authors: Huang,T.F.
• Format: Others
• Language: zh-TW
• Published: 1993
• Online Access: http://ndltd.ncl.edu.tw/handle/01097520641079254823

碩士 === 國立臺灣大學 === 藥理學研究所 === 81 === Bothrops jararaca 蛇毒經 Sephadex G-75, DEAE-Sephadex A-50,以及 Sephadex G-75 純化後，得到一個純化且具有引起血小板凝集作用的蛇毒 蛋白。由 SDS-PAGE 可知不論還原態或非還原態下，其皆呈單一物質而分 子量約為55,000 daltons，簡稱此成分為蛇毒凝集素(Venom inceder此蛇 毒凝集素在兔子血小板懸浮液中能引起血小板凝集和ATP釋放隨著濃度的 增加，其引發凝集之潛伏期跟著縮短.另 外蛇毒凝集素能引起細胞內鈣離 子的增加。 Thrombin抑制劑：hirudin (5 U/ml), heparin (5和 antithrombinⅢ (0.2 U/ml), TAMe (2 mM) 能幾乎完全抑制蛇毒凝集素 在血小板懸浮液中引發之血小板凝集和細胞內鈣離子的增加。在血小板懸 浮液中，低濃度(0.02μg/ml)的蛇毒凝集素並不能引起血小板凝集。隨著 外加 prothrombin (0.02∼0.2 U/ml)，則低濃度之蛇毒凝集素可引起血 小板凝集。隨著外加prothrombin濃度的增加，其引發凝集之潛伏期也隨 著縮短，但凝集程度則相近。Indomethacin (10μM) 和 PAF 拮抗劑- BN 52021 (20μM)不能抑制蛇毒凝集素在血小板懸浮液中所引起的血板凝 集和細胞內鈣離子的增 加。CP/CPK (10 mM/16 U/ml) 則可部分抑制蛇毒 凝集素所引起的血小板凝集 ，但對細胞內鈣離子的增加，則沒有影響。 EDTA (5 mM)能完全抑制蛇毒凝集素或thrombin在血小板懸浮液中所引起 的血小板凝集和 ATP 釋放。但TMB-8 (50μM) 不能抑制蛇毒凝集素和 thrombin 所引起的血小板凝集和細胞內鈣離子的增加。PGE1 (1μM)能抑 制蛇毒凝集素和 thrombin 所引起的血小板凝集和細胞內鈣離子的增加 。 Sodium nitroprusside (100μM)對於蛇毒凝集素 和 thrombin 所引 起的血小板凝集和細胞內鈣離子的增加沒有影響。PLC抑制劑－ neomycin(5 mM)能完全抑制由蛇毒凝集素和thrombin所引起的血小板凝集 和細胞內鈣離子的增加。PLA2抑制劑－mepacrine (50μM)或PKC抑制劑－ staurosporine ( 300nM)未有上述反應.在indomethacin (10μM)存在下 ，蛇毒凝集素或 thrombin都能引起phosphoinositides轉換代謝。而IP生 成量之增加能為 neomycin ( 5 mM)所抑制，而mepacrine (50μM)則無法 抑制其增加量。綜合上述結果，此蛇毒凝集素可能為一個prothrombin activator,可經由水解附著於血小板細胞膜上之prothrombin成thrombin 而間接造成血小板凝集；其活化血小板之途徑主要乃經由活化 PLC ，造 成phosphatidyl- inositides之轉換，引發細胞內游離鈣離子之增加而活 化血小板造成凝集 A platelet aggregation inducer was purified from Bothrops jararaca venom.Judged by SDS-PAGE,its molecular weight was esti- mated to be 55,000 daltons.This venom inducer caused platelet aggregation and ATP release of rabbit washed platelets. Fura-2 AM loaded platelets,venom inducer increased the free cytoslic Ca2+. Platelet aggregation and the rise of free intracellular Ca2+ caused by venom inducer were significantly inhibited by thrombin inhibitor, e.g.heparin plus thrombin III hirudin and TAMe. Low concentration of venom inducer (0.02μg/ ml) did not induce platelet aggregation.However,venom inducer at this concentration induced platelet aggregation and ATP release if exogenous prothrombin was present in the medium. Neither cyclooxygenase inhibitor indomethacin nor PAF antagonist BN 52021 affected platelet aggregation and the rise of intracellular Ca2+ caused by venom inducer . In contrast, ADP scavenger CP/CPK (10mM/16U/ml) partially reduced platelet aggregation without affecting the rise of intracellular Ca2+. Platelet aggregations caused by venom inducer or thrombin were significantly inhibited by EDTA (5mM)and PGE1(1μM).Neomycin (5mM),a PLC inhibitor, markedly suppressed platelet aggregation and rise of free intracellular Ca2+ caused by venom inducer and thrombin. On the other hand, mepacrine(50μM),a PLA2 inhibitor and staurosporine , a PKC inhibitor exerted no effected.In presence of indomethacin, venom inducer and thrombin still caused phosphoinositides breakdown. The increase in IP formation was suppressed by neomycin but not by mepacrine . In conclusion,the purified inducer may act as a prothrombin activator . It activates platelets indirectly by converting prothrombin into thrombin,which in turn activates platelets mainly through the PLC activation pathway ,causing phosphatidyl- inositides turnover ,increasing the cytosolic free Ca2+, subsequently leading to release reaction and aggregation.