Liu L, Freedman J, Hornstein A, Fenton J W, Ofosu F A
Canadian Red Cross Society Blood Services, Hamilton, Ontario, Canada.
Br J Haematol. 1994 Nov;88(3):592-600. doi: 10.1111/j.1365-2141.1994.tb05078.x.
The interactions of alpha-thrombin with platelets are critical in haemostasis and arterial thrombosis. This study established methods for characterizing the binding of alpha-thrombin to platelets and some of its consequences in platelet-rich plasma. The binding of alpha-thrombin to platelets and the subsequent platelet activation were quantified by flow cytometry, using affinity purified polyclonal antibodies to human alpha-thrombin and a monoclonal antibody to GMP-140, respectively. Dose-dependent binding of alpha-thrombin to platelets and their activation occurred in parallel, both reaching the maxima for each enzyme concentration within 10s after > or = 1.0 nM alpha-thrombin was added to recalcified PRP containing 1 microM recombinant tick anticoagulant peptide. The tick anticoagulant peptide abrogated prothrombin activation in the platelet-rich plasma. alpha-Thrombin binding to platelets, and their activation, were abrogated by a monoclonal antibody to the hirudin tail-like domain of the seven transmembrane thrombin receptor on platelets. Therefore this receptor represents an important site for alpha-thrombin binding to platelets suspended in plasma. D-Phe-Pro-ArgCH2-alpha-thrombin only bound to platelets when its concentration was > or = 100 nM, and it did so without inhibiting platelet activation by alpha-thrombin. Whereas concentrations of hirudin equimolar to those of alpha-thrombin failed to abrogate alpha-thrombin-mediated activation of platelets, a 10-fold molar excesses of hirudin over alpha-thrombin abrogated alpha-thrombin binding to platelets. The demonstration that > or = 1.0 nM alpha-thrombin can bind to platelets and initiate their activation raises the possibility that the levels of thrombin generated in venous and arterial thrombosis contribute to platelet activation in vivo.
α-凝血酶与血小板的相互作用在止血和动脉血栓形成过程中至关重要。本研究建立了表征α-凝血酶与血小板结合的方法及其在富血小板血浆中的一些后果。通过流式细胞术分别使用针对人α-凝血酶的亲和纯化多克隆抗体和针对GMP-140的单克隆抗体，对α-凝血酶与血小板的结合以及随后的血小板活化进行定量。α-凝血酶与血小板的剂量依赖性结合及其活化同时发生，在向含有1μM重组蜱抗凝肽的再钙化富血小板血浆中加入≥1.0 nMα-凝血酶后10秒内，两者均达到每种酶浓度的最大值。蜱抗凝肽可消除富血小板血浆中的凝血酶原激活。血小板上七跨膜凝血酶受体的水蛭素尾样结构域的单克隆抗体可消除α-凝血酶与血小板的结合及其活化。因此，该受体是α-凝血酶与悬浮在血浆中的血小板结合的重要位点。仅当D-苯丙氨酸-脯氨酸-精氨酸-CH2-α-凝血酶的浓度≥100 nM时，它才与血小板结合，并且这样做不会抑制α-凝血酶对血小板的活化。虽然与α-凝血酶等摩尔浓度的水蛭素浓度未能消除α-凝血酶介导的血小板活化，但水蛭素相对于α-凝血酶10倍的摩尔过量可消除α-凝血酶与血小板的结合。≥1.0 nMα-凝血酶可与血小板结合并启动其活化的证明增加了静脉和动脉血栓形成中产生的凝血酶水平在体内促成血小板活化的可能性。
