Mechanisms Regulating Factor V Activation and Function

V 因子激活和功能的调节机制

• 批准号: 9080092
• 负责人: Rodney M Camire
• 金额: $ 42万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9080092
• 关键词: Acidic Region; Address; Antibodies; Anticoagulants; Antisense Oligonucleotides; Binding; Binding Sites; Biochemical; Biology; Blood Coagulation Factor; Blood Platelets; Blood Vessels; Blood coagulation; C-terminal; Cells; Cleaved cell; Coagulation Process; Collaborations; Custom; Data; Development; Elements; Event; Excision; Factor V; Generations; Goals; Growth; Hemophilia A; Hemorrhage; Hemostatic Agents; Hemostatic function; In Vitro; Injury; Investigation; Kinetics; Knowledge; Laboratories; Length; Ligands; Mediating; Molecular; Molecular Mimicry; Mus; Phase; Physiological; Plasma; Play; Process; Property; Proteins; Proteolysis; RNA Splicing; Reagent; Regulation; Role; Series; Site; Surface; Testing; Texas; Therapeutic; Thinking; Thrombin; Thromboplastin; Thrombosis; Thrombus; Work; activated Protein C; aptamer; base; biophysical analysis; cofactor; crosslink; driving force; in vivo; innovation; insight; intravital microscopy; mouse model; novel; public health relevance; tool

 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.

 描述(由申请人提供)：负责限制血栓形成的调节机制对维持正常止血至关重要。新的发现表明，TFP Iα结合凝血因子V(A)并抑制凝血酶原酶组装，这突显了人们对凝血启动的理解正在演变。我实验室的工作已经为破译TFP Iα介导这些抗凝作用的机制基础奠定了基础。我们假设，通过分子模拟，TFP Iα使用Fv B结构域的关键结构区域，这些区域在血栓发展的早期阶段部分切割原因子后保留下来。这些区域包括碱性(BR)和酸性(AR)区域，它们是负责将FV保持在非活性原因子状态的关键自我抑制元件。这项提议的目的是破译这些分子过程，研究它们如何影响Fv(A)的激活和功能，提供其生物学相关性的证据，并确定哪些Fv池(血浆或血小板来源)对这些影响起作用。一个 进一步的目标是更好地了解TFP Iα调制自然出现的FV形式的分子基础，包括FV-东德克萨斯州和FV-阿姆斯特丹，最近在血浆中发现的缺失很大一部分B结构域的FV剪接形式。这些关于FV激活、其生物学和调节的新角度开辟了几条未被探索的实验路线。在第一个目标中，我们将描述结构/功能决定因素 维持Fv原因子状态，并确定TFP Iα是否利用这些分子表面来抑制辅因子功能。将寻求新的生化和结构方法(通过合作)，以提供对这些分子相互作用的详细见解。在第二个目标中，我们将研究缺乏BR的自然产生的FV的生化性质，并表征它们与TFP Iα的相互作用。我们假设TFP Iα利用一组不同的结构决定因素来结合不同形式的Fv(A)，并且该复合体通过多种机制调节其抗凝作用。在最后一个目标中，我们将解决Fvar-TFP Iα相互作用的生理学相关性，确定它如何调控体内血栓的发展，并研究这是否由血浆和/或血小板形式的FV介导。我们推测部分裂解形式的FV在血栓形成部位大量存在，并且它们是TFP Iα的靶标。我们推测，这种调节机制的破坏会导致血栓生长动力学的改变。我们将使用生化、动力学、生物物理、结构和体内方法测试这些创新想法，并使用一系列定制的FV试剂。从这项提议中获得的知识将通过提供关于FV在血管损伤部位释放的结构和功能的特殊水平的分子细节来推动这一领域的发展。由于Fv/Va对凝血酶的生成有如此深远的影响，对这一应用的研究将对开发新的止血调节方法以控制出血或限制血栓形成具有重要意义。