Structural Correlates of (Pro)cofactor Function

项目2-项目概要/摘要 通常认为，负责限制血栓形成的主要调节机制 已经被确认。然而，最近的研究结果表明，TFPIa结合凝血因子V（a）， 抑制凝血酶的产生质疑这一教条。我实验室的工作为 破译可能支持TFPIa如何介导这些抗凝作用的机制基础。我们 我认为，通过分子模拟，TFPIa使用FV B结构域的关键结构区域， 在血栓形成的早期阶段FV部分裂解后。基本（BR） 和酸性（AR）区域，它们是负责保持FV处于非活性状态的关键自抑制元件。 原辅因子状态。这项提案的目的是破译这些分子过程，研究如何 它们影响FV（a）的激活和功能，提供其生物学相关性的证据，并确定哪一组 FV（血浆或血小板衍生）有助于这些作用。更进一步的目标是更好地理解 TFPIa通过其调节天然存在的FV形式的分子碱基，包括FV-东德克萨斯和FV- Amsterdam，最近鉴定了血浆中发现的FV剪接形式，其缺失了大部分B- 域这些关于FV激活、其生物学和调控的新视角开辟了几个新的思路。 未经探索的实验路线。在第一个目标中，我们将表征结构/功能 维持FV原辅因子状态并决定TFPIa是否利用这些分子的决定因素 表面以抑制辅因子功能。新的生化和结构方法（与 项目1、3和核心B）将继续进行，以提供对这些分子相互作用的详细了解。在 第二个目标，我们将研究缺乏BR的天然FV形式的生化特性 并表征它们与TFPIa的相互作用。我们假设TFPIa利用一组不同的结构， 决定簇结合不同形式的FV（a），复合物通过以下途径介导其抗凝作用： 多重机制在最后一个目标中，我们将讨论FVAR-TFPIa相互作用的生理相关性， 确定它如何调节体内血栓的形成，并研究这是否是由 血浆和/或血小板形式的FV。我们假设FV的部分裂解形式在 血栓的形成，并且它们是TFPIa的靶点。我们推测，这种监管的中断 机制导致血栓生长的动力学改变。我们将测试这些创新的想法， 生物化学、动力学、生物物理学和体内方法，采用许多定制FV试剂。知识 从这一建议中获得的将通过提供以下分子细节来推动该领域的发展： 血管损伤部位释放的FV的结构和功能调节。因为FV/Va有这样一个 对凝血酶生成的深远影响，在这方面的应用研究将有重要意义， 开发调节止血以控制出血或限制血栓形成的新方法。

Project 2-Project Summary/Abstract It is commonly assumed that major regulatory mechanisms responsible for limiting thrombus development have been identified. However, recent findings showing that TFPIa binds coagulation factor V(a) and dampens thrombin generation questions this dogma. Work from my laboratory has laid the groundwork for deciphering the mechanistic bases that may underpin how TFPIa mediates these anticoagulant effects. We think that through molecular mimicry, TFPIa uses key structural regions of the FV B-domain that remain following partial cleavage of FV during the early phases of thrombus development. These include basic (BR) and acidic (AR) regions that are key autoinhibitory 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 TFPIa 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 TFPIa employs these molecular surfaces to dampen cofactor function. New biochemical and structural approaches (in collaboration with Project 1, 3 and Core B) 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 TFPIa. We hypothesize that TFPIa utilizes a different set of structural determinants to bind to different forms of FV(a) and the complex mediates its anticoagulant effects through multiple mechanisms. In the last aim, we will address the physiologic relevance of the FVAR-TFPIa 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 TFPIa. 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, 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 functional 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.