Mechanisms Regulating Factor V Activation and Function

 描述（由申请方提供）：负责限制血栓形成的调节机制对于维持正常止血至关重要。新的发现表明TFPIα结合凝血因子V（a）并抑制凝血酶原酶组装，这突出表明我们对凝血起始的理解正在发展。我实验室的工作为破译TFPIα如何介导这些抗凝作用的机制基础奠定了基础。我们推测，通过分子模拟，TFPIα利用了FV B结构域的关键结构区域，这些区域在血栓形成的早期阶段前辅因子部分裂解后保留下来。这些包括碱性（BR）和酸性（AR）区域，它们是负责保持FV处于无活性原辅因子状态的关键自抑制元件。本提案的目的是解读这些分子过程，检查它们如何影响FV（a）活化和功能，提供其生物学相关性的证据，并确定哪种FV池（血浆或血小板衍生）有助于这些效应。一 进一步的目标是更好地理解TFPIα调节天然存在的FV形式的分子基础，包括FV-东德克萨斯和FV-阿姆斯特丹，最近在血浆中发现的缺失大部分B结构域的FV剪接形式。这些关于FV激活、其生物学和调控的新视角开辟了几条未探索的实验路线。在第一个目标中，我们将描述结构/功能决定因素， 维持FV原辅因子状态，并确定TFPIα是否利用这些分子表面抑制辅因子功能。新的生物化学和结构方法（通过合作）将被追求，以提供详细的洞察这些分子相互作用。在第二个目标中，我们将研究缺乏BR的天然存在形式的FV的生化特性，并表征其与TFPIα的相互作用。我们推测TFPIα利用一组不同的结构决定簇结合不同形式的FV（a），复合物通过多种机制介导其抗凝作用。在最后一个目标中，我们将讨论FVAR-TFPIα相互作用的生理相关性，确定其如何调节体内血栓形成，并研究这是否由血浆和/或血小板形式的FV介导。我们假设FV的部分裂解形式在血栓形成部位丰富，并且它们是TFPIα的靶点。我们推测，这种调节机制的中断导致血栓生长动力学的改变。我们将使用生物化学，动力学，生物物理，结构和体内方法，采用大量的定制FV试剂来测试这些创新的想法。从该建议中获得的知识将通过提供关于血管损伤部位释放的FV的结构和功能调节的特殊水平的分子细节来推动该领域的发展。由于FV/Va对凝血酶生成具有如此深远的影响，因此该应用中的研究将对开发调节止血以控制出血或限制血栓形成的新方法具有重要意义。

 DESCRIPTION (provided by applicant): Regulatory mechanisms responsible for limiting thrombus development are critical for maintaining normal hemostasis. New findings showing that TFPIα binds coagulation factor V(a) and inhibits prothrombinase assembly highlights that out understanding of the initiation of coagulation is evolving. Work from my laboratory has laid the groundwork for deciphering the mechanistic bases that may underpin how TFPIα mediates these anticoagulant effects. We hypothesize that through molecular mimicry, TFPIα uses key structural regions of the FV B-domain that remain following partial cleavage of the procofactor during the early phases of thrombus development. These include basic (BR) and acidic (AR) regions that are key auto-inhibitory elements responsible for keeping FV in an inactive procofactor state. The objectives of this proposal are to decipher these molecular processes, examine how they influence FV(a) activation and function, provide evidence its biologically relevant and identify which pool of FV (plasma or platelet-derived) contributes to these effects. A further goal is to better understand the molecular bases by which TFPIα modulate naturally occurring forms of FV including FV-East Texas and FV-Amsterdam, recently identified spliced forms of FV found in plasma that are missing a large portion of the B-domain. These new angles of thinking about FV activation, its biology, and regulation opens up several unexplored lines of experimentation. In the first aim, we will characterize the structural/functional determinants that maintain the FV procofactor state and determine whether TFPIα employs these molecular surfaces to dampen cofactor function. New biochemical and structural approaches (via collaborations) will be pursued to provide detailed insight into these molecular interactions. In the second aim, we will investigate the biochemical properties of naturally occurring forms of FV that lack the BR and characterize their interaction with TFPIα. We hypothesize that TFPIα utilizes a different set of structural determinants to bind different forms of FV(a) and the comple mediates its anticoagulant effects through multiple mechanisms. In the last aim, we will address the physiologic relevance of the FVAR-TFPIα interaction, determine how it may regulate thrombus development in vivo, and investigate whether this is mediated by plasma and/or platelet forms of FV. We hypothesize partially cleaved forms of FV are abundant at the site of the developing thrombus and that they are targets for TFPIα. We speculate that disruption of this regulatory mechanism results in altered kinetics of thrombus growth. We will test these innovative ideas using biochemical, kinetic, biophysical, structural and in vivo approaches employing a host of custom FV reagents. Knowledge gained from this proposal will move the field forward by providing an exceptional level of molecular detail on the structural and functiona regulation of FV released at the site of vascular injury. Because FV/Va has such a profound impact on thrombin generation, studies in this application will have important implications in developing new ways to modulate hemostasis to control bleeding or limit thrombosis.