Characterization of an isoform specific anticoagulant function of TFPI-alpha

摘要 组织因子途径抑制物（TFPI）是一种重要的抗凝蛋白。血浆TFPI降低是 与静脉和动脉血栓形成相关，阻断TFPI活性的药物正在被研究。 是用来治疗血友病的TFPI是一种选择性剪接蛋白，所有同种型都是 能够抑制组织因子（TF）引发的血液凝固。我们的实验室确定TFPIβ为 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，我们证明了血小板TFPIα限制血管损伤后血栓的生长，并减弱了血栓形成的可能性。 血友病的止血反应提示TFPIα具有特异性抗凝作用 TFPIβ不具有的活性。我们认为TFPIα的碱性C-末端区域和TFPIα的碱性C-末端区域与TFPIα的碱性C-末端区域是同源的。 FV B结构域的区域具有惊人的同源性，并试图确定TFPIα如何特异性相互作用 FV/FVa。我们的生化研究描述了TFPIα如何有效抑制凝血酶原酶与 保留FV B-结构域的酸性区域的FVa形式，例如由FXa激活的或在FVa B-结构域中发现的FVa形式。 血小板，但不与具有整个B结构域被去除的FVa形式，如由凝血酶激活的FVa形式。 在上一个资助期间，我们致力于进一步表征TFPIα-FV相互作用，发现 TFPIα抑制与FV Leiden（FVL）组装的凝血酶原酶的能力降低， TFPIα C-末端内的氨基酸是抑制活性所必需的，但对亲和力没有贡献。 FVa的TFPIα。我们还建立了几种新的小鼠模型，以研究 体内TFPIα-FV相互作用。初步研究结果表明，通过调节TFPIα-FV的相互作用， 血小板中TFPIα或FV的表达显著改变了TFPI-K1缺失小鼠的胚胎存活。此外，本发明还 我们发现过表达高活化小鼠蛋白C的转基因改变了 TFPI-K1缺失小鼠。一些存活的小鼠活到成年，但随后往往发展成严重的脑积水。 拟议的目标旨在进一步追求这些有趣的初步发现，以定义TFPI- 在胚胎发育过程中的生物学。目的1将研究改变TFPIα的表达， FV通常或特异性地在血小板内调节胚胎发育。目标2将描述 小鼠大脑和TFPI缺乏如何调节其发展。TFPIα对凝血酶原酶的抑制作用是一种 最近认识到的抗凝机制不执行任何其他人类蛋白质。因此有 预期拟议实验的结果将描述凝血的病理生理学， TFPI调节的凝血介导的细胞信号发育过程。

Abstract 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. Our laboratory identified TFPIβ as the primary isoform on endothelium, while TFPIα is the primary isoform within platelets. Using murine model systems, we demonstrated that 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 and the basic region of the FV B-domain have striking homology, and sought to define how TFPIα may specifically interact with FV/FVa. Our biochemical studies described how 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. During the previous funding period, we have worked to further characterize the TFPIα-FV interaction, finding that TFPIα has reduced ability to inhibit prothrombinase assembled with FV Leiden (FVL) and that the LIKT amino acids within the TFPIα C-terminus are required for inhibitory activity, but do not contribute to the affinity of TFPIα for FVa. We also generated several new murine models to investigate the physiological role of the TFPIα-FV interaction in vivo. Preliminary findings indicate that altering the TFPIα-FV interaction by modulating expression of TFPIα or FV in platelets dramatically alters embryonic survival of TFPI-K1 null mice. Additionally, we found that a transgene over-expressing hyperactivatable mouse protein C alters embryonic survival of TFPI-K1 null mice. Some of the surviving mice live to adulthood, but then often develop severe hydrocephalus. The proposed aims are designed to further pursue these intriguing preliminary findings to define TFPI- associated biology during embryonic development. Aim 1 will examine how altering expression of TFPIα and FV, in general or specifically within platelets, modulates embryonic development. Aim 2 will characterize murine brain and how TFPI deficiency modulates its development. Inhibition of prothrombinase by TFPIα is a recently recognized anticoagulant mechanism not performed by any other human protein. Therefore, it is anticipated that the results of the proposed experiments will describe the pathophysiology of coagulation and the coagulation-mediated cellular signaling developmental processes modulated by TFPI.