Active Site-labeled Prothrombin Inhibits Prothrombinase in Vitro and Thrombosis in Vivo
Active Site-labeled Prothrombin Inhibits Prothrombinase in Vitro and Thrombosis in Vivo
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DOI：
10.1074/jbc.m111.230292
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发表时间：
2011-07-01
影响因子：
4.8
通讯作者：
Bock, Paul E.
中科院分区：
文献类型：
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作者：
Kroh, Heather K.;Panizzi, Peter;Bock, Paul E.
用D-Phe-Pro-Arg-CH 2Cl（FPR-ProT）特异性标记的小鼠和人凝血酶原（ProT）活性位点抑制富血小板和贫血小板小鼠和人血浆中组织因子引发的凝血酶生成。FPR-凝血酶前体1（Pre 1）、片段1（F1）、片段1.2（F1.2）和FPR-凝血酶未产生显著抑制，证明需要所有三个ProT结构域。由失活的ProT（S195 A）突变体抑制ProT激活的动力学与作为替代非生产性底物的竞争性抑制相容，尽管FPR-ProT偏离了这种机制，暗示了一个更复杂的过程。FPR-ProT表现出与ProT（S195 A）相比类似的10倍更有效的抗凝活性，这是由于ProT催化结构域中的构象变化在FPR标记后诱导更蛋白酶样构象。与ProT和ProT（S195 A）不同，凝血酶原酶切割FPR-ProT的途径从甲藻凝血酶重定向至FPR-凝血酶前体2（Pre 2）.F1.2抑制性中间体的形成。在真实的时间宽视野和共聚焦荧光显微镜中，在小鼠小动脉中激光诱导血栓形成期间，通过FPR-CH 2Cl（[AF 660]FPR-ProT）栓系的Alexa Fluor（R）660标记的ProT的定位被检查。[AF 660]FPR-ProT在血小板聚集之前迅速结合至损伤部位的血管壁，随后结合至血管壁近端但非远端的血栓。[AF 660]FPR-ProT抑制血栓生长，而缺乏F1膜结合结构域的[AF 660]FPR-Pre 1不结合或抑制。标记的F1.2定位类似于[AF 660]FPR-ProT，表明与富含磷脂酰丝氨酸的膜结合，但不抑制血栓形成。这些研究为体内和体外ProT激活机制提供了新的见解，以及独特的外切位点导向凝血酶原酶抑制剂的特性。
Mouse and human prothrombin (ProT) active site specifically labeled with D-Phe-Pro-Arg-CH2Cl (FPR-ProT) inhibited tissue factor-initiated thrombin generation in platelet-rich and platelet-poor mouse and human plasmas. FPR-prethrombin 1 (Pre 1), fragment 1 (F1), fragment 1.2 (F1.2), and FPR-thrombin produced no significant inhibition, demonstrating the requirement for all three ProT domains. Kinetics of inhibition of ProT activation by the inactive ProT(S195A) mutant were compatible with competitive inhibition as an alternate nonproductive substrate, although FPR-ProT deviated from this mechanism, implicating a more complex process. FPR-ProT exhibited similar to 10-fold more potent anticoagulant activity compared with ProT(S195A) as a result of conformational changes in the ProT catalytic domain that induce a more proteinase-like conformation upon FPR labeling. Unlike ProT and ProT(S195A), the pathway of FPR-ProT cleavage by prothrombinase was redirected from meizothrombin toward formation of the FPR-prethrombin 2 (Pre 2).F1.2 inhibitory intermediate. Localization of ProT labeled with Alexa Fluor (R) 660 tethered through FPR-CH2Cl ([AF660]FPR-ProT) during laser-induced thrombus formation in vivo in murine arterioles was examined in real time wide-field and confocal fluorescence microscopy. [AF660]FPR-ProT bound rapidly to the vessel wall at the site of injury, preceding platelet accumulation, and subsequently to the thrombus proximal, but not distal, to the vessel wall. [AF660]FPR-ProT inhibited thrombus growth, whereas [AF660]FPR-Pre 1, lacking the F1 membrane-binding domain did not bind or inhibit. Labeled F1.2 localized similarly to [AF660]FPR-ProT, indicating binding to phosphatidylserine-rich membranes, but did not inhibit thrombosis. The studies provide new insight into the mechanism of ProT activation in vivo and in vitro, and the properties of a unique exosite-directed prothrombinase inhibitor.