日本龜殼花蛇毒蛋白Disintegrins,TFV-1和TFV-3的抗血栓活性與作用機轉探討

Two disintegrins, TFV-1 and TFV-3, purified from Trimeresurus flavoviridis snake venom, exhibit distinct effects on platelet activation

潘姿瑜(Tzu-Yu Pan)

指導教授 ： 黃德富

國立臺灣大學/醫學院/藥理學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.11358

摘要

Disintegrins是從蛇毒蛋白中發現的強效小分子血小板凝集抑制劑，其作用機轉為抑制血小板上的integrin αIIbβ3，並被認為具有開發成為抗血栓作用劑的潛力。然而，現行臨床上使用的αIIbβ3拮抗劑普遍存在著增加出血風險及血小板低下等危及生命的副作用。在本篇研究中，從日本龜殼花 (Trimeresurus flavoviridis) 蛇毒蛋白的原毒中純化得到兩個特性不同的disintegrin，分別命名為TFV-1及TFV-3。本篇的主要研究目的是探討並比較TFV-1及TFV-3這兩個dsintegrin的抗血栓作用及機轉不同之處。 經由MALDI-TOF測得TFV-1及TFV-3的分子量分別為7310及7646 Da；活性方面，TFV-1及TFV-3都呈現藥物濃度相關性地有效抑制在人類富含血小板的血漿和人類血小板懸浮液中所引發的凝集反應。TFV-1及TFV-3對於ADP (20 μM)，thrombin (0.1 U/mL) 和collagen (10 μg/mL) 所引起血小板凝集反應的IC50分別為99 nM及17 nM，64 nM及39 nM和73 nM及28 nM。 藉由流式細胞儀的分析，觀察到TFV-3會明顯地抑制integrin αIIbβ3單株抗體7E3結合到integrin αIIbβ3上，但TFV-1卻不會影響7E3結合到integrin αIIbβ3上。另一方面，在integrin αIIbβ3單株抗體AP2存在下，TFV-3會活化integrin αIIbβ3並將FcγRII吸引過來，引起血小板的凝集反應且引發下游一連串訊息分子的磷酸化，但TFV-1觀察不到此現象。從clot retraction試驗和thromboelastography結果顯示，TFV-3會明顯地影響血液凝固的生成和凝血的過程，而TFV-1則較少影響。此外，於動物實驗的體外試驗中，TFV-1及TFV-3也都呈現藥物濃度相關性地有效抑制collagen (10 μg/mL) 在老鼠富含血小板的血漿中所引發的血小板凝集反應。另外，在尾靜脈給予抗血栓劑量下，TFV-3相對於TFV-1會明顯地延長尾部出血時間，然而，TFV-1及TFV-3兩者皆不會影響血小板的數目。總結上述，TFV-1及TFV-3對於integrin αIIbβ3有不同的結合位置並且對血小板活化的過程有不同的影響，由本篇研究得知，TFV-1有較小的出血傾向且較不會影響到正常的生理止血功能，因此，藉由研究TFV-1和TFV-3之間的結構差異，可以提供未來在研發較不會造成αIIbβ3構型改變及較少出血風險的新一代integrin αIIbβ3拮抗劑一些有價值的資訊。

關鍵字

蛇毒蛋白 ； 整合蛋白 ； 血小板 ； 血小板活化 ； 血小板低下症

並列摘要

Disintegrins are potent small-mass platelet inhibitors found in snake venoms, which are integrin αIIbβ3 antagonists and potential antithrombotic agents. However, current integrin αIIbβ3 antagonists have the life-threatening adverse effect of causing thrombocytopenia and bleeding. In this study, two disintegrins, TFV-1 and TFV-3, were purified from Trimeresurus flavoviridis snake venom, with different properties. The molecular weight of TFV-1 and TFV-3 were determined as 7310 Da and 7646 Da by MALDI-TOF, respectively. Both TFV-1 and TFV-3 concentration-dependently inhibited platelet aggregation in human platelet-rich plasma and washed platelet suspension. The IC50 values of TFV-1 and TFV-3 for platelet aggregation induced by ADP (20 μM), thrombin (0.1 U/ml) and collagen (10 μg/ml) were estimated to be 99 and 17 nM，64 and 39 nM, and 73 and 28 nM, respectively. In the indirect binding assay, TFV-3 significantly inhibited 7E3, a mAb raised against integrin αIIbβ3, binding to αIIbβ3, but TFV-1 did not. On the other hand, TFV-3 induced platelet aggregation and triggered time-dependent phosphorylation of signal molecules in the presence of AP2, an integrin αIIbβ3 monoclonal antibody, due to FcγRII recruitment, but TFV-1 did not. As shown in clot retraction assay and thromboelastography, TFV-3 significantly affected the clot formation and the coagulation process, however, TFV-1 showed little effects on clot formation. Moreover, TFV-1 and TFV-3 concentration-dependently inhibited platelet aggregation in mice PRP and dose-dependently inhibited platelet aggregation of PRP in response to collagen in ex vivo model. Furthermore, TFV-3 prolonged the tail bleeding time more significantly than TFV-1 as they were intravenously administered at antithrombotic doses. However, both TFV-1 and TFV-3 did not alter the platelet counts. In summary, TFV-1 and TFV-3, two disintegrins exhibit distinct binding epitopes on integrin αIIbβ3 and different effects on platelet activation process. TFV-1 has less tendency in causing bleeding with minimal effect on normal physiological hemostasis. Therefore, the structural difference between TFV-1 and TFV-3 may provide valuable information for the future development of new integrin αIIbβ3 inhibitors with minimal alteration of integrin αIIbβ3 conformation and bleeding.

並列關鍵字

Snake venom ； Integrin ； Platelet ； Platelet activation ； Thrombocytopenia

參考文獻

1. Ruggeri Z M. Platelets in atherothrombosis. Nat Med. 2002; 8(11):1227-34.

2. Michelson A D. Antiplatelet therapies for the treatment of cardiovascular disease. Nat Rev Drug Discov. 2010; 9(2):154-69.

3. Gibbins J M. Platelet adhesion signalling and the regulation of thrombus formation. J Cell Sci. 2004; 117(Pt 16):3415-25.

4. Jackson S P and Schoenwaelder S M. Antiplatelet therapy: in search of the 'magic bullet'. Nat Rev Drug Discov. 2003; 2(10):775-89.

5. Montalescot G. Platelet biology and implications for antiplatelet therapy in atherothrombotic disease. Clin Appl Thromb Hemost. 2011; 17(4):371-80.