蛇毒蛋白Trowaglerix之精製及其引發血小板 凝集機轉之探討

Abstract

出血性蛇毒蛋白能影響血液凝固和血小板凝集活性。在本實驗中，將Tropidolaemus wagleri原毒經由FPLC（fast performance liquid chromatography）之Mono S陽離子交換層析法以及Superdex-G200膠質過濾法進行純化分離，得到一具有引發血小板凝集之成分，命名為trowaglerix。以SDS-PAGE分析，發現在非還原狀態下，其分子量大於175 kDa；以膠質過濾法測定native分子量為212 kDa；在2D PAGE還原情形下，可見大約接近15 kDa的蛋白次單元。 Trowaglerix在人類血小板懸浮液（PS）和富含血小板血漿（PRP），皆會呈現濃度相關性地引起血小板凝集，其EC50分別為2.56±0.49和2.63±0.21 ng/ml。同樣地，Trowaglerix會劑量相關性地造成TxB2的產生、細胞內鈣離子的增加，以及血小板表面P-selectin的表現，且都在濃度為10 ng/ml時達飽和情形。PGE1、EDTA和BAPTA/AM會抑制trowaglerix引起的血小板凝集，但heparin、hirudin以及aprotinin卻不影響其作用，表示trowaglerix造成血小板凝集現象，並不是來自於thrombin或serine protease產生所引起的。Cytochalasin D會部分抑制trowaglerix引起的凝集（33.3±4.9 % inhibition），而indomethacin和CP/CPK只有些微的抑制作用。MEK抑制劑-PD98059不影響trowaglerix引起的凝集，PLC抑制劑-U73122和tyrosine kinase抑制劑-genistein有些微的抑制作用，而PI3-K抑制劑-LY294002和Syk抑制劑-piceatannol有較明顯的抑制情形；Src family kinase抑制劑-PP2會明顯地延長trowaglerix所造成的shape change，並有顯著的抑制trowaglerix所造成的凝集作用。 單獨使用GPIa/IIa拮抗劑（6F1）或GPIb拮抗劑（6D1）只能些微抑制trowaglerix引起的血小板凝集，即使兩者同時使用時也沒有太大的加成作用。Trowaglerix不會在福馬林固定之血小板上引起凝聚（agglutination）的情形。以流式細胞儀分析，GPIa/IIa單株抗體（6F1）、GPIb單株抗體（6D1、AP1）以及GPVI單株抗體（11A12）皆會部分抑制FITC-trowaglerix結合到血小板上；但利用biotinylated trowaglerix來觀察，發現它會專一性地結合到血小板表面上的GPVI上。此外，trowaglerix引起血小板訊息分子tyrosine磷酸化的情形類似convulxin所引起的，包括FcRγ chain、Src、Syk、LAT以及PLCγ2，當使用了Src family kinase抑制劑-PP2會全面性地抑制trowaglerix引起的訊息傳遞。將trowaglerix以靜脈注射方式打入ICR小鼠體內，發現會劑量相關性延長其尾巴出血時間，經測量小鼠血中血小板數目，發現trowaglerix同樣具有劑量相關性減少其體內的血小板數目。 綜合上述結果，Trowaglerix是一個具有高分子量的蛇毒蛋白，會專一性結合到血小板GPVI上，活化血小板後引起許多訊息分子的磷酸化，包括PLC γ2、PI3K、Syk、Src、LAT以及Fcγ，胞內游離鈣離子之增加和P-selectin表現，最後引發功能性αIIbβ3的表現，造成Ca2+依賴性的血小板凝集。由於trowaglerix的專一性，可以用來研究其與已知作用在GPVI的C-type lectins（i.e. convulxin、ophioluxin和stejnulxin）之間的差異，探討它們與GPVI分子層次之交互作用，提供GPVI拮抗劑之研發。亦可當作研究血小板上GPVI與其他受體（GPIb與GPIa/IIa）間之相關性的工具。

Snake venoms profoundly affect platelet aggregation and blood coagulation. By using column chromatography of Mono-S cationic exchanger and Superdex-G200 gel filtration, a novel snake venom protein, trowaglerix, was purified from Tropidolaemus waglerix snake venom. Under non-reducing conditions, it migrates as a protein with a mass higher than 175 kDa on SDS-PAGE. The apparent molecular weight of native trowaglerix was estimated to be 212 kDa by gel filtration on FPLC/Superdex 200 column. Under reduction on 2D PAGE, it exhibits three subunits with similar masses close to 15 kDa. Trowaglerix induced platelet aggregation of human washed platelets and platelet-rich plasma in a concentration-dependent manner with EC50, 2.56±0.49 and 2.63±0.21 ng/ml, respectively. Trowaglerix also triggered thromboxane B2 formation and the elevation of intracellular Ca2+ and the expression of P-selectin in a concentration-dependent and saturable manner. PGE1, EDTA, and BAPTA/AM completely inhibited trowaglerix-induced platelet aggregation. However, pretreatment of heparin, hirudin, or aprotinin did not significantly affect trowaglerix-induced platelet aggregation, indicating that trowaglerix-induced aggregation is independent of thrombin or serine protease formation. Cytochalasin D patially inhibited trowaglerix-induced platelet aggregation (33.3±4.9 % inhibition). ADP release reaction and thromboxane A2 formation had minor role in trowaglerix-induced platelet aggregation, because indomethacin and CP/CPK only had slight effect with 5.4±2.7 and 11.2±0.3 % inhibition, respectively. MEK inhibitor, PD98059 did not affect trowaglerix-induced platelet aggregation. PLC inhibitor, U73122 and tyrosine kinase inhibitor, genistein slightly inhibited trowaglerix-induced platelet aggregation. PI3-K inhibitor, LY294002 and Syk inhibitor, piceatannol had higher inhibitory effect. Src kinase inhibitor, PP2 significantly delayed the shape change and inhibited the aggregation induced by trowaglerix. Anti-GPIb mAb 6D1 and anti-GPIa/IIa mAb 6F1 only slightly inhibited platelet aggregation caused by trowaglerix. Even in combination, no additive inhibitory effect was observed. Trowaglerix did not elicit agglutination of fixed platelets. By flow cytometric analysis, we found that anti-GPIa/IIa mAb (6F1), anti-GPIb mAbs (6D1, AP1) and anti-GPVI mAb (11A12) displayed only a slight inhibition on the binding of FITC-trowaglerix to washed platelets. However, biotinylated trowaglerix specifically bound to GPVI, but not to GPIb or α2. In addition, trowaglerix induced a time-dependent tyrosine phosphorylation of a number of proteins similar to those activated by convulxin, including FcR γ chain, Src, Syk, LAT, and phospholipase Cγ2. PP2, a Src kinase inhibitor completely inhibited the tyrosine phosphorylation induced by trowaglerix. Intravenous injection of trowaglerix dose-dependently prolonged the bleeding time of ICR mice. Meanwhile, we also found that trowaglerix significantly decreased platelet count in a dose-dependent manner. Taken together, trowaglerix, a high molecular weight multimer of three subunits, activates platelets mainly through GPVI, leading to phosphorylation of many signal molecules, including PLCγ2, PI3K, Syk, Src, LAT and Fcγ, elevation of cytoplasmic Ca2+, P-selectin upregulation, and finally inducing the exposure of functional αIIbβ3 and Ca2+ -dependent platelet aggregation. This novel snake venom protein may provide an useful tool for studies of GPVI and signaling mechanisms involved as well as for comparative study with other C-type lectin GPVI agonists, like convulxin, ophioluxin and stejnulxin, and its association of platelet receptors, such as GPIb and GPIa/IIa. These studies would provide new insights regarding the molecular interaction of these GPVI agonists and GPVI, providing clues for the design of GPVI antagonists, a new class of antithrombotic agent.