Characterization of an isoform specific anticoagulant function of TFPI-alpha

 描述（由申请方提供）：组织因子途径抑制物（TFPI）是一种必需的抗凝蛋白。血浆TFPI降低与静脉和动脉血栓形成有关，正在开发阻断TFPI活性的药物以治疗血友病患者。TFPI是一种选择性剪接蛋白，所有亚型都能抑制组织因子（TF）引发的凝血。在之前的资助期间，我们发现TFPIα是内皮上的主要亚型，而TFPIα是血小板内的主要亚型。使用小鼠模型系统，我们还发现造血细胞TFPI（主要是血小板TFPIα）限制血管损伤后的血栓生长，并减弱血友病患者的止血反应。这些结果表明，TFPIα具有TFPIβ所不具有的特异性抗凝活性。我们认识到，TFPIα的碱性C-末端区域（不存在于TFPIβ中）和FV B-结构域的碱性区域具有惊人的同源性，并试图确定TFPIα如何与FV/FVa特异性相互作用。我们现在已经证明，TFPIα有效抑制与保留FV B结构域酸性区域的FVa形式组装的凝血酶原酶，例如由FXa激活或在血小板内发现的凝血酶原酶，但不抑制与去除整个B结构域的FVa形式组装的凝血酶原酶，例如由凝血酶激活的凝血酶原酶。我们的初步数据进一步表明，TFPI基因敲除小鼠的胚胎致死率是通过繁殖成PAR-4基因敲除小鼠来挽救的，这首次证明了TFPI活性和血小板功能直接相互平衡，以防止消耗性凝血病的发展。这些数据强烈表明TFPI抗凝活性由两种不同的机制介导：1）TF-FVIIa的FXa依赖性抑制，由存在于所有TFPI同种型中的Kunitz结构域1和2介导;和2）凝血酶原酶的早期形式的抑制，所述凝血酶原酶在FXa活化FV或从胶原活化的血小板释放FVa后组装。这种抑制作用需要TFPIα的碱性C-末端区域和FV B-结构域的酸性区域之间的外位点相互作用。该提案的重点是确定TFPIα抑制凝血酶原酶的生化相互作用，并确定其生理重要性。目的1将检测高度保守的LIKT氨基酸序列对TFPIα抑制凝血酶原酶的贡献。目的2将通过将表达不同形式血小板TFPI的骨髓移植到经辐照的TFPI-/-/PAR 4-/-小鼠中，使用血小板活性的离体研究和血管损伤后血栓生长的体内研究来进一步表征血小板TFPIα的抗凝功能。目的3将在FV/FVa特异性改变的小鼠模型系统中研究TFPIα介导的凝血酶原酶抑制。凝血酶原酶的抑制是一种新认识到的抗凝机制，不执行任何其他人类蛋白质。因此，预计拟议实验的结果将提供新的见解 研究了广泛的血栓形成和出血性疾病的病理生理学，并可能导致其治疗的新方法。

 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.