Structural Correlates of (Pro)cofactor Function

项目2--项目摘要/摘要 人们普遍认为，负责限制血栓发展的主要调控机制 已经被确认了。然而，最近的研究表明，TFPIa与凝血因子V(A)和 抑制凝血酶生成对这一教条提出质疑。我实验室的工作已经为 破译可能支持TFPIa调节这些抗凝作用的机制基础。我们 认为通过分子模拟，TFPIa使用了FV B结构域的关键结构区域，这些区域仍然 在血栓形成的早期阶段，FV被部分切割。其中包括基本(BR) 以及酸性(AR)区域，它们是负责将FV保持在非活性状态的关键自身抑制元件 原辅因子状态。这项提议的目标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，并确定哪个池 FV(血浆或血小板衍生的)促成了这些效应。另一个目标是更好地理解 TFPIa调节天然形式的FV的分子碱基，包括Fv-East Texas和Fv- 阿姆斯特丹，最近在血浆中发现的剪接形式的FV缺失了很大一部分B- 域。这些关于FV激活、其生物学和调节的新角度打开了几个 未被探索的实验路线。在第一个目标中，我们将描述结构/功能 维持Fv原因子状态并确定TFPIa是否使用这些分子的决定因素 表面以抑制余因函数。新的生化和结构方法(与 项目1、3和核心B)将被用来提供对这些分子相互作用的详细见解。在 第二个目标，我们将研究缺乏BR的自然产生的FV的生化特性 并表征了它们与TFPIa的相互作用。我们假设TFPIa利用了一组不同的结构 决定因素与不同形式的Fv(A)结合，该复合体通过 多种机制。在最后一个目标中，我们将解决fvar-TFP Ia相互作用的生理相关性， 确定它可能如何调节体内血栓的发展，并研究这是否通过 血浆和/或血小板形式的FV。我们推测，部分裂解形式的FV在 发展中的血栓，它们是TFP Ia的靶标。我们推测，对这种监管的破坏 机制导致血栓生长的动力学改变。我们将使用以下工具测试这些创新想法 生化、动力学、生物物理和活体方法采用了一系列定制的FV试剂。知识 从这项提议中获得的信息将通过提供超乎寻常的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 对凝血酶生成的深刻影响，对这一应用的研究将具有重要意义 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.