PLATELET HIGH MOLECULAR WEIGHT KININOGEN

血小板高分子量激肽原

• 批准号: 3349539
• 负责人: ALVIN H SCHMAIER
• 金额: $ 20.67万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3349539
• 关键词: bradykinin; calpain; cell membrane; crosslink; genetic library; human subject; kallikreins; membrane proteins; monoclonal antibody; platelets; protein purification; protein transport; receptor; recombinant DNA; synthetic peptide; western blottings

The general hypothesis this proposal addresses is that the cell membrane expression of the plasma kininogens [high molecular weight kininogen (HK) and low molecular weight kininogen (LK)] is essential for bradykinin (BK) delivery to cells. In order to describe how the kininogens deliver BK to cells the objectives of this proposal are to characterize the epitopes on HK and LK that bind to platelet membranes and to determine the platelet receptor(s) for the kininogens. The specific aims of this proposal in order to achieve these objectives are as follows: (1) We will determine the epitopes on the kininogens that bind to platelets. Purified domains from each of the kininogens will be used to indirect and direct platelet binding experiments to determine the region(s) on these proteins involved in membrane binding. Monoclonal antibodies that inhibit kininogens' binding to platelets will be used to isolate kininogens' cell binding domains by determining the epitopes on the proteins will be used to isolate kininogens' cell binding domains by determining the epitopes on the proteins that these antibodies recognize and by characterizing peptides secreted from a lambdagt11 recombinant DNA kininogen expression library recognized by these antibodies. Synthetic peptides, corresponding to the deduced binding domains on the kininogens, will be produced to determine if they inhibit the parent molecules' ability to bind to platelets and can directly bind to the platelet surface. (2) Investigations will be performed to identify and characterize the platelet receptor(s) for the kininogens. Studies will be performed to identify a physiochemical receptor for the kininogens on unstimulated platelets and HEL cells by ligand blotting and chemical crosslinking agents. Purification of a physiochemical receptor for kininogen from unstimulated platelets and HEL cells will be performed by immuno- or ligand-affinity chromatography. Investigations will be performed to determine the mechanisms(s) by which platelet calpain becomes externalized on the activated platelet membrane and if its expression allows it to become an additional receptor for kininogen on thrombin-activated platelets. Characterization of the epitopes on the kininogens that bind to cells and determining the presence of a physiochemical receptor(s) for the kininogens on platelets and HEL cells should uncover mechanisms of importance for the regulation of BK availability to cells. Since both kininogens are the parent proteins for bradykinin, knowing how kininogens are expressed on cell membranes would be helpful to understand how bradykinin delivery to tissues can be modulated. Since modulation of bradykinin delivery to cells by captopril and enalapril has become very important for blood pressure management and treatment of heart failure, the proposed studies may provide for new approaches for the development of antagonists to bradykinin delivery to cells which could have therapeutic potential to modulate this peptide's availability.

这一建议的一般假设是，细胞膜 血浆激肽原[高分子量激肽原（HK）]的表达 和低分子量激肽原（LK）]是缓激肽（BK）的必需物质 递送至细胞。 为了描述激肽原如何将BK递送至 该建议的目的是表征细胞上的表位， HK和LK与血小板膜结合， 激肽原的受体。 该提案的具体目标是， 为了达到这些目标，我们将：（1）我们将确定 结合血小板的激肽原上的表位 纯化结构域 将用于间接和直接血小板输注， 结合实验，以确定这些蛋白质上的区域 在膜结合中。 抑制激肽原的单克隆抗体 与血小板的结合将用于分离激肽原的细胞结合 通过确定蛋白质上的表位来确定结构域将用于分离 激肽原的细胞结合结构域，通过确定 这些抗体识别的蛋白质， 从重组DNA激肽原表达文库中分泌 被这些抗体识别。 合成肽，相当于 将产生激肽原上的推导的结合结构域，以确定 它们抑制母体分子与血小板结合的能力， 直接与血小板表面结合。 (2)调查将 用于鉴定和表征血小板受体， 激肽原 将进行研究，以确定 未受刺激的血小板和HEL细胞上的激肽原受体， 配体印迹和化学交联剂。 纯化 未受刺激血小板和HEL激肽原理化受体 细胞将通过免疫或配体亲和层析进行。 将进行调查，以确定 血小板钙蛋白酶在活化的血小板膜上外化 如果它的表达允许它成为一个额外的受体， 激肽原对凝血酶活化的血小板的作用。 表征 结合细胞的激肽原上的表位， 血小板和HEL上激肽原的理化受体 细胞应该揭示调节BK的重要机制， 细胞的可用性。 由于这两种激肽原都是 缓激肽，知道激肽原如何在细胞膜上表达， 这有助于理解缓激肽向组织的递送如何被调节。 巯甲丙脯酸和依那普利对缓激肽向细胞转运的调节作用 对于血压管理和治疗已经变得非常重要， 心力衰竭，拟议的研究可能提供新的方法， 开发缓激肽递送至细胞的拮抗剂， 治疗潜力来调节这种肽的可用性。