Characterization of an isoform specific anticoagulant function of TFPI-alpha

 描述(申请人提供)：组织因子途径抑制物(TFPI)是一种重要的抗凝血蛋白。血浆TFPI降低与静脉和动脉血栓形成有关，正在开发阻断TFPI活性的药物来治疗血友病患者。TFPI是一种选择性剪接蛋白，所有的异构体都能够抑制组织因子(TF)启动的凝血。在之前的资助期间，我们发现TFP Iα是内皮细胞的主要异构体，而TFP Iα是血小板的主要异构体。在小鼠模型系统中，我们还发现，造血细胞TFP I，主要是血小板TFP Iα，可以限制血管损伤后血栓的生长，并减弱血友病的止血反应。这些结果表明，TFP Iα具有TFP Iβ所不具有的特异性抗凝血活性。我们认识到TFP Iα的基本C末端区域与Fv B结构域的基本区域具有显著的同源性，并试图定义TFP Iβ如何与Fv/Fva特异性地相互作用。我们现在已经证明，TFP Iα有效地抑制凝血酶原酶，这些凝血酶原酶与保留FV B结构域酸性区域的FVA形式组装在一起，例如由FXA激活的或在血小板中发现的，但不与去除整个B结构域的FVA形式组装在一起，例如由凝血酶激活的FVA。我们的初步数据进一步证明，通过培育PAR-4缺失小鼠，TFPI缺失小鼠的胚胎致死性得以挽救，这首次证明了TFPI活性和血小板功能直接相互抵消，以防止消耗性凝血障碍的发展。这些数据有力地表明，TFPI的抗凝活性是通过两种不同的机制介导的：1)FXA依赖的抑制TF-FVIIa的作用，这是由所有TFPI亚型中存在的ukitz结构域1和2介导的；2)FXA激活FV或从胶原激活的血小板释放FVA后，抑制凝血酶原酶早期形式的组装。这种抑制需要TFP Iα的碱性C-末端区域和FV的B-结构域的酸性区域之间的外位相互作用。这一建议的重点是确定允许TFP Iα抑制凝血酶原酶的生化相互作用并确定其生理意义。目的1研究一个保守的LIKT氨基酸序列在TFP Iα抑制凝血酶原酶中的作用。目的2通过体外血小板活性研究和血管损伤后血栓生长的体内研究，将表达不同形式的血小板α的骨髓移植到受照射的TFP-/-/PAR4-/-小鼠体内，进一步研究其抗凝功能。目的3将在Fv/FVA特异性改变的小鼠模型系统中研究TFP Iα介导的凝血酶原酶抑制作用。抑制凝血酶原酶是一种新发现的抗凝机制，任何其他人类蛋白质都不会执行。因此，预计拟议实验的结果将提供新的见解。 研究范围广泛的血栓性和出血性疾病的病理生理学，并可能导致治疗它们的新方法。

 DESCRIPTION (provided by applicant): Tissue factor pathway inhibitor (TFPI) is an essential anticoagulant protein. Decreased plasma TFPI is associated with venous and arterial thrombosis and pharmaceutical agents that block TFPI activity are being developed to treat patients with hemophilia. TFPI is an alternatively spliced protein and all isoforms are capable of inhibiting tissue factor (TF) initiated blood coagulation. During the previous funding period we found that TFPIα is the primary isoform on endothelium, while TFPIα is the primary isoform within platelets. Using murine model systems we also found that hematopoietic cell TFPI, which is primarily platelet TFPIα, limits thrombus growth following vascular injury and weakens the hemostatic response in hemophilia. These results suggested that TFPIα has a specific anticoagulant activity that is not performed by TFPIβ. We recognized that the basic C-terminal region of TFPIα, which is not present in TFPIβ, and the basic region of the FV B-domain have striking homology and sought to define how TFPIα may specifically interact with FV/FVa. We have now demonstrated that TFPIα effectively inhibits prothrombinase assembled with forms of FVa that retain the acidic region of the FV B-domain, such as that activated by FXa or found within platelets, but not with forms of FVa that have the entire B-domain removed, such as that activated by thrombin. Our preliminary data further demonstrate that the embryonic lethality of TFPI null mice is rescued by breeding into PAR-4 null mice, providing the first demonstration that TFPI activity and platelet function directly counterbalance each other to prevent development of consumptive coagulopathy. These data strongly suggest that TFPI anticoagulant activity is mediated by two separate mechanisms: 1) FXa-dependent inhibition of TF-FVIIa, mediated by Kunitz domains 1 and 2 that are present in all TFPI isoforms; and 2) The inhibition of early forms of prothrombinase that assemble following activation of FV by FXa or release of FVa from collagen-activated platelets. This inhibition requires an exosite interaction between the basic C-terminal region of TFPIα and the acidic region of the B-domain of FV. This proposal is focused on identifying the biochemical interactions that allow for inhibition of prothrombinase by TFPIα and defining its physiological importance. Aim 1 will examine the contribution of a well-conserved LIKT amino acid sequence to the inhibition of prothrombinase by TFPIα. Aim 2 will further characterize the anticoagulant function of platelet TFPIα by transplanting bone marrow expressing different forms of platelet TFPI into irradiated TFPI-/-/PAR4-/- mice using ex vivo studies of platelet activity and in vivo studies of thrombus growth following vascular injury. Aim 3 will investigate TFPIα-mediated inhibition of prothrombinase in murine model systems in which FV/FVa are specifically altered. Inhibition of prothrombinase is a newly recognized anticoagulant mechanism not performed by any other human protein. Therefore, it is anticipated that the results of the proposed experiments will provide new insights into the pathophysiology of a wide range of thrombotic and bleeding disorders and potentially lead to new approaches for their treatment.