Characterization of an isoform specific anticoagulant function of TFPI-alpha

摘要 组织因子途径抑制物(TFPI)是一种重要的抗凝血蛋白。血浆TFPI降低是 与静脉和动脉血栓形成有关，以及阻断TFPI活性的药物正在 开发用于治疗血友病患者。TFPI是一种选择性剪接蛋白，所有的亚型都是 能够抑制组织因子(TF)启动的凝血。我们的实验室鉴定出TFP Iβ是 内皮细胞上的主要异构体，而血管内皮生长因子Iα是血小板内的主要异构体。使用小鼠模型 系统，我们证明了血小板转移因子抑制因子α抑制血管损伤后血栓的生长并减弱。 血友病患者的止血反应。这些结果表明tfpiα具有特异的抗凝血剂。 不是由TFP Iβ执行的活动。我们认识到TFP Iα的基本C-末端区域和基本的 Fv B结构域的区域具有显著的同源性，并试图定义TFP Iα可能如何特异性地相互作用 FV/FVA。我们的生化研究描述了tfpiα是如何有效地抑制凝血酶原酶装配的 保留Fv B结构域酸性区域的FVA形式，例如被FXA激活的形式或在 血小板，但不包括去除了整个B结构域的FVA形式，例如被凝血酶激活的FVA。 在上一个资助期，我们致力于进一步描述TFP Iα-FV的相互作用，发现 TFP Iα抑制与Fv Leiden(Fv Leiden)组装的凝血酶原酶的能力降低，LIKT TFP IαC-末端的氨基酸是抑制活性所必需的，但对亲和力没有贡献 FVA的TFP Iα。我们还建立了几个新的小鼠模型来研究血管紧张素转换酶的生理作用。 α-Fv在体内的相互作用。初步研究结果表明，通过调节改变TFP Iα-Fv相互作用 TFP Iα或Fv在血小板中的表达显著改变TFP I-K1缺失小鼠的胚胎存活。另外， 我们发现，过度表达可激活小鼠蛋白C的转基因改变了胚胎的存活 TFPI-K1基因缺失小鼠。一些存活下来的小鼠可以活到成年，但随后往往会出现严重的脑积水。 拟议的目标旨在进一步研究这些耐人寻味的初步发现，以定义TFP I- 胚胎发育中的相关生物学。目标1将研究如何改变TFP Iα和TFP I的表达 FV一般或特定于血小板内，调节胚胎发育。《目标2》将描述 小鼠大脑以及TFPI缺乏如何调节其发育。抑制凝血酶原酶原的α是一种 最近发现的抗凝机制是任何其他人类蛋白质所不具备的。因此，它是 预计拟议的实验结果将描述凝血和 TFPI调控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.