IDENTIFICATION OF THE MEGAKARYOCYTE FACTOR V RECEPTOR BY MASS SPECTROMETRY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在血管损伤部位，血小板和血浆衍生因子Va对于凝血酶的产生是必不可少的。尽管血浆衍生因子Va足以满足正常止血，但已证明，血小板衍生因子Va在功能和生理上是不同的，并且代表与生理相关的辅因子分子。尽管有这些差异，但血小板衍生的原因子，即因子V，是通过血小板前体巨核细胞内吞血浆衍生的因子V而产生的，其来源是一个由未知的因子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。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，这表明这两个受体在细胞表面是植物相关的。因子V的内吞作用随后由LRP-1介导。这代表了LRP-1在物理和功能修饰的蛋白质的内吞作用中的新角色，而不是注定要被溶酶体降解。该项目的目标是通过与抗LRP-1抗体共免疫沉淀后，通过质谱学鉴定特定的因子V受体，来继续表征这两个受体系统。由于LRP-1的高相对分子质量(550 KDa)，早期试图通过免疫印迹检测LRP-1的配基结合域，但没有成功，因此建立了确定合适的蛋白质印迹条件的方法。用LRP-1阳性或阴性细胞的裂解产物证实印迹特异性。在巨核细胞与SBED-生物素衍生的受体相关蛋白孵育后，通过链霉亲和素和Western blotting证实，生物素部分被转移到LRP-1上。然而，使用链霉亲和素或抗LRP-1抗体来沉淀生物素标记的LRP-1的尝试一直没有成功。目前的实验正在评估使用SBED-生物素衍生的因子V轻链直接生物素化和分离特定的因子V受体。对调节因子V内吞的细胞膜事件的定义将增加我们对巨核细胞如何获得、处理和包装这一关键凝血因子的理解，并可能导致在高凝和血栓状态下开发潜在的治疗靶点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Platelet- and plasma-derived factor Va are essential for thrombin generation at sites of vascular injury. Though plasma-derived factor Va is sufficient for normal hemostasis, it has been demonstrated that platelet-derived factor Va is functionally and physically distinct, and represents the physiologically-relevant cofactor molecule. Despite these differences, the platelet-derived procofactor, factor V, originates from plasma through endocytosis of plasma-derived factor V by platelet precursors, megakaryocytes, via a two receptor system consisting of a specific, 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 binding of another factor V molecule to LRP-1 suggesting that the two receptors are physcially-associated on the cell surface. Factor V endocytosis is subsequently mediated by LRP-1. This represents a novel role for LRP-1 in endocytosis of a protein physically and functionally modified, and not destined for lysosomal degradation. The goal of this project is to continue to characterize this two receptor system by identifying the specific, factor V receptor by mass spectrometry following co-immunoprecipitation with anti-LRP-1 antibodies. As early attempts to detect the ligand binding domain of LRP-1 by western blotting of megakaryocyte cell lysates were unsucessful due to its high molecular weight (550 kDa), protocols were developed to define appropriate western blotting conditions. Blotting specificity was confirmed using lysates from LRP-1 positive or negative cells. Following incubation of megakaryocytes with SBED-biotin-derivatized receptor associated protein, which antagonizes LRP-1 ligand binding, the biotin moeity was transferred to LRP-1 as confirmed by streptavidin and western blotting. However, attempts to precipitate biotinylated LRP-1 using streptavidin or anti-LRP-1 antibodies have been unsucessful. Current experiments are assessing the the use of SBED-biotin-derivatized factor V light chain to biotinylate and isolate the specific, factor V receptor directly. Definition of the cell membrane events that regulate factor V endocytosis will increase our understanding of how megakaryocytes acquire, process and package this critical coagulation factor, and may lead to the development of potential therapeutic targets in hypercoagulable and thrombotic states.