Regulation of Human Platelet Prothrombinase

人血小板凝血酶原酶的调节

• 批准号: 7328152
• 负责人: Paula Babiarz Tracy
• 金额: $ 33.53万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7328152
• 关键词: Age; Arts; Atherosclerosis; Binding; Binding Sites; Biological Models; Biotin; Blood Platelets; Characteristics; Competitive Binding; Complex; Coupled; Factor Va; Factor Xa; Generations; Goals; Hematopoietic; Hemostatic function; Human; Kinetics; Label; Link; Megakaryocytes; Membrane; Membrane Proteins; Modeling; Peptides; Phospholipids; Plasma; Platelet Activation; Proteins; Proteomics; Prothrombin; Reagent; Regulation; Role; Serine Protease; Surface; Techniques; Testing; Thrombin; Thromboplastin; Thrombosis; Venous; Vesicle; cofactor; crosslink; factor IXa-factor VIIIa; prothrombinase complex; receptor; therapeutic target

The ability of platelets to regulate thrombin generation at their membrane surfaceis central to their role in hemostasis, thrombosis, and atherosclerosis. Thrombin generation is effectedthrough the assembly and function of the enzymatic complex, Prothrombinase, consisting of a Ca^-dependent, membrane-bound complex of the cofactorfactorVa and the serine protease factorXa. Subsequent to platelet activation, platelet-derived factor Va and/or plasma-derived factor Va are expressed on or bind to the platelet membrane surfaceand in so doing form at least part of the receptorfor factorXa. Thus, several protein/protein and protein/membrane interactions participate in and regulate complex assembly. The major goal of this project is to define how platelets actively participate in and regulate Prothrombinase assembly and function. The following hypotheses have been formulated regarding unique mechanisms by which platelets regulate thrombin formation, catalyzed by Prothrombinase, and will be tested. 1) Platelets regulate thrombin generation through the expression of three discrete platelet subpopulations. One binds both factors Va and Xa to effect Prothrombinase assembly and function; one binds only factor Va, whereas one subpopulation is incapable of binding either protein. 2) Unique membrane proteins regulate Prothrombinase assembly and function on the activated platelet surface.3) Platelets release a cofactor molecule, factor Va, which is functionally and physically unique when compared to plasma-derived factor Va. 4) Physical and functional characteristics of Prothrombinase assembled on defined phospholipid vesicles are not mimicked by complex assembly on the activated platelet membrane. State of the art arterial and venous flow models will be used to characterizethe recruitment of the procoagulant platelet subpopulations to thrombogenic surfaces. Hematopoietic regulation will be determined using CD34+"derived megakaryocytes in flow cytometric analyses. Factor Xa will be labeled with a trrfunctionalcross-linking reagent that will result inthe transfer of a biotin handle to activated platelet membrane proteins, other than factorVa, with which it interacts to effect its function. State of the art mass spectrometric analyses will be used to identify the isolated biotinylated proteins. Functional and physical differences between plasma and platelet-derived factor Va will be assessed using analyses of kinetic of prothrombin activation coupled to mass spectrometric techniques. Definition of important factor Va interactions with factor Xa and prothrombin, which regulate Prothrombinase assembly and function on the activated platelet surface will require the synthesis of select peptides mimicking important regions in both proteins for use in both competitive binding and functional analyses. Successful completion of these goals will demonstrate the mechanisms by which platelets actively and uniquely regulate the generation of thrombin attheir membranesurface. These studies will identify several different mechanisms essential for thrombin generation at the activated platelet membrane, all of which will be potential therapeutic targets in hypercoagulable and thrombotic states.

血小板调节其膜表面凝血酶生成的能力是其止血作用的核心。 血栓形成和动脉粥样硬化。凝血酶的生成是通过酶的组装和功能来实现的 复合体，凝血酶原酶，由辅因子Va和钙依赖的膜结合复合体组成 丝氨酸蛋白酶因子Xa。在血小板激活后，血小板衍生因子Va和/或血浆衍生因子Va 表达在血小板膜表面或与血小板膜表面结合，在此过程中形成至少部分凝血因子Xa受体。 因此，几种蛋白质/蛋白质和蛋白质/膜相互作用参与并调节复杂的组装。这个 这个项目的主要目标是确定血小板如何积极参与和调节凝血酶原酶的组装和 功能。关于血小板调节的独特机制，提出了以下假设 凝血酶形成，由凝血酶原酶催化，将进行测试。1)血小板通过调节凝血酶生成 三个离散的血小板亚群的表达。一种结合两种因子Va和Xa以影响凝血酶原酶 组装和功能；一个亚群只结合因子Va，而一个亚群不能结合任何一种蛋白质。2) 独特的膜蛋白调节激活的血小板表面凝血酶原酶的组装和功能。3)血小板 释放辅因子分子，因子Va，与血浆来源的相比，它在功能和物理上都是独一无二的 因子Va。4)固定在磷脂小泡上的凝血酶原酶的物理和功能特性如下 而不是通过激活的血小板膜上的复杂组装来模仿。最先进的动脉和静脉血流 模型将被用来表征促凝血剂血小板亚群在血栓形成表面的募集。 在流式细胞仪分析中，将使用CD34+来源的巨核细胞来确定造血调节。 将用三功能交联剂标记，该交联剂将导致生物素手柄转移到激活的血小板上 膜蛋白，而不是因子Va，它与膜蛋白相互作用以影响其功能。最新的质谱学 分析将用于鉴定分离的生物素化蛋白。血浆在功能和生理上的差异 将通过凝血酶原激活与质量耦合的动力学分析来评估血小板衍生因子Va 光谱技术。重要因子Va与调节因子Xa和凝血酶原相互作用的定义 凝血酶原酶在活化的血小板表面的组装和功能需要合成选定的多肽 模仿这两种蛋白质中的重要区域，用于竞争结合和功能分析。成功 这些目标的完成将展示血小板主动和独特地调节 在膜表面产生凝血酶。 这些研究将确定几种不同的机制，在激活的凝血酶产生 血小板膜，所有这些都将成为高凝和血栓形成的潜在治疗靶点 各州。