Membrane Receptors Regulating Megakaryocyte Endocytosis of Factor V from Plasma

膜受体调节血浆中因子 V 的巨核细胞内吞作用

• 批准号: 8267670
• 负责人: Beth Bouchard
• 金额: $ 8.16万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-18 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8267670
• 关键词: Antibodies; Binding; Biochemistry; Biology; Blood Coagulation Factor; Blood Platelets; Blood Vessels; CD34 gene; Cell membrane; Chairperson; Chemistry; Clinical; Coagulants; Collaborations; Complex; Core Facility; Cytoplasmic Tail; Development; Endocytosis; Ensure; Ethics; Event; Factor V; Factor VIII; Factor Va; Factor Xa; Fc Receptor; Fee-for-Service Plans; Flow Cytometry; Funding; Generations; Goals; Grant; Hemostatic function; Housing; Injury; International; Journals; LDL-Receptor Related Protein 1; Laboratories; Letters; Literature; Mass Spectrum Analysis; Mediating; Medicine; Megakaryocytes; Membrane; Modeling; Peptide Synthesis; Phosphorylation; Plasma; Play; Process; Program Research Project Grants; Proteins; Research; Research Activity; Research Personnel; Research Project Grants; Resources; Role; Scientist; Serine; Serine Protease; Site; System; Techniques; Threonine; Thrombin; Thrombosis; Training; Universities; Vermont; abstracting; base; career development; cellular imaging; cofactor; college; interest; meetings; member; peptide V; platelet factor V; prothrombinase complex; receptor; receptor expression; symposium; therapeutic target

DESCRIPTION (provided by applicant): Platelet- and plasma-derived factor Va serve an essential role in thrombin generation catalyzed by the prothrombinase complex, a 1:1, Ca2+-dependent complex of the cofactor factor Va and the serine protease factor Xa bound to activated platelets adhered to sites of vascular injury. Recently, our laboratory demonstrated that the entire pool of the platelet-derived procofactor, factor V, originates from plasma through endocytosis of factor V by platelet precursors, megakaryocytes, via a two receptor system consisting of a specific, as of yet, unidentified factor V receptor and LDL receptor-related protein-1 (LRP-1). In this model, it is hypothesized that factor V binding to its specific receptor facilitates the binding of another factor V molecule to LRP-1, which then mediates its endocytosis. This system represents a unique function for LRP-1 whereby endocytosed factor V is modified to yield a functionally distinct molecule, and not destined for degradation. The major goal of this project is to continue to characterize this two-receptor system for factor V endocytosis by megakaryocytes. CMK and CD34+ ex vivo-derived megakaryocytes, isolated and separated based upon their ability to endocytose factor V, will be used in several complementary approaches including co-immunprecipitation and state-of-the-art mass spectrometric techniques to identify the specific, factor V receptor. Once identified, anti-factor V receptor antibodies will be produced and used in combination with anti-LRP-1 antibodies to delineate the fate of these receptors subsequent to factor V endocytosis. Receptor expression as a function of megakaryocyte differentiation will also be determined as the ability of megakaryocytes to endocytose factor V occurs early in their development, and is lost in the mature platelet. In other studies, the ability of factor VIII, a protein highly homologous to factor V whose clearence is mediated by LRP-1, to interact with this receptor system on ex vivo-derived megakaryocytes will be determined. Domains important for this interaction will be defined and used to synthesize complementary factqr V peptides in order to identify the factor V domains important for its interactions with both receptors. As the two receptor model suggests that receptor-receptor interactions are necessary for factor V endocytosis, and LRP-1 phosphorylation regulates its function, the phosphorylation state of serine/threonine residues in the cytoplasmic tail of LRP-1 will be correlated with factor V binding and endocytosis. Since platelet-derived factor Va plays a more important role in maintaining normal hemostasis than does its plasma counterpart, and is physically and functionally distinct, these studies will increase our understanding of how megakaryocytes acquire, process and package this critical coagulation factor. Successful completion of these goals will help to define the cell membrane events that regulate acquisition of factor V by megakaryocytes all of which will be potential therapeutic targets in hypercoagulable and thrombotic states. (End of Abstract)

描述(由申请人提供)： 血小板和血浆衍生的凝血因子Va在凝血酶原复合体催化的凝血酶生成中起重要作用，凝血酶原复合体是1：1的依赖于钙离子的辅因子Va和丝氨酸蛋白酶Xa的复合体，结合在激活的血小板上，附着在血管损伤的部位。最近，我们的实验室证明了整个血小板源因子，即因子V，是通过一个由未知的因子V受体和低密度脂蛋白受体相关蛋白-1(LRP-1)组成的双受体系统，由血小板前体巨核细胞吞噬因子V而产生的。在这个模型中，假设因子V与其特定的受体结合促进了另一个因子V分子与LRP-1的结合，然后LRP-1介导其内吞作用。这个系统代表了LRP-1的独特功能，即内吞因子V被修饰以产生功能不同的分子，而不是注定要降解的分子。该项目的主要目标是继续研究巨核细胞吞噬因子V的这一双受体系统。CMK和CD34+体外来源的巨核细胞根据其内吞因子V的能力进行分离和分离，将用于几种互补的方法，包括共同免疫沉淀和最新的质谱学技术，以识别特定的因子V受体。一旦确定，将产生抗因子V受体抗体，并与抗LRP-1抗体结合使用，以描绘这些受体在因子V内吞作用后的命运。受体的表达作为巨核细胞分化的函数也将被确定，因为巨核细胞对内吞因子V的能力发生在它们发育的早期，并在成熟的血小板中消失。在其他研究中，将确定因子VIII是一种与因子V高度同源的蛋白质，其清除由LRP-1介导，在体外来源的巨核细胞上与该受体系统相互作用的能力将被确定。对这种相互作用重要的结构域将被定义并用于合成互补的factqr V多肽，以确定对其与两种受体相互作用重要的因子V结构域。由于两种受体模型表明，受体相互作用是因子V内吞所必需的，而LRP-1的磷酸化调节其功能，因此LRP-1胞浆尾部丝氨酸/苏氨酸残基的磷酸化状态将与因子V的结合和内吞作用相关。由于血小板衍生因子Va在维持正常止血方面的作用比血浆因子更重要，而且在物理和功能上都是不同的，这些研究将增加我们对巨核细胞如何获取、处理和包装这一关键凝血因子的理解。这些目标的成功完成将有助于确定调节巨核细胞获取因子V的细胞膜事件，所有这些事件都将成为高凝和血栓状态下的潜在治疗靶点。(摘要结束)