PLATELET HIGH MOLECULAR WEIGHT KININOGEN

血小板高分子量激肽原

• 批准号: 3349535
• 负责人: ALVIN H SCHMAIER
• 金额: $ 14.09万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 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/3349535
• 关键词: bradykinin; calpain; cell membrane; crosslink; genetic library; human subject; kallikreins; membrane proteins; monoclonal antibody; platelets; protein purification; protein transport; receptor; 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.

这项提议涉及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 和低分子激肽原(LK)]是缓激肽(BK)所必需的。 传送到细胞。为了描述激肽原如何将BK传递到 细胞这项提议的目标是确定表位的特征 与血小板膜结合的HK和LK测定血小板 激肽原受体(S)。这项建议的具体目标是 实现这些目标的顺序如下：(1)我们将确定 与血小板结合的激肽原上的表位。纯化结构域 每一种激肽原都会被用来间接和直接作用于血小板 结合实验确定这些蛋白质上的区域(S) 在膜结合中。抑制激肽原的单抗 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 通过确定激肽原上的表位确定激肽原的细胞结合域 这些抗体识别的蛋白质和通过表征多肽 由lambdagt11重组DNA激肽原表达文库分泌 被这些抗体识别。合成肽，对应于 推导出的激肽原结合域，将被产生以确定 它们抑制亲本分子与血小板结合的能力，并可以 直接与血小板表面结合。(2)将进行调查 进行鉴定和鉴定的血小板受体(S)为 激肽原。将进行研究以确定一种物理化学物质 激肽原在未刺激的血小板和HEL细胞上的受体 配基印迹和化学交联剂。一种天然产物的提纯 未刺激的血小板和HEL激肽原的生理化学受体 细胞将通过免疫亲和层析或配基亲和层析进行。 将进行调查，以确定(S) 激活的血小板膜上的血小板钙化酶 如果它的表达允许它成为一个额外的受体 凝血酶激活的血小板上的激肽原。刻画人物形象 激肽原与细胞结合的表位及其存在与否 血小板和HEL激肽原的物理化学受体(S) 细胞应揭示调节BK的重要机制 对细胞的可用性。因为这两种激肽原都是 缓激肽，知道激肽原是如何在细胞膜上表达的 有助于理解缓激肽是如何被调节到组织的。 卡托普利和依那普利对缓激肽向细胞递送的调节作用 对于高血压的管理和治疗已经变得非常重要 对于心力衰竭，拟议的研究可能提供新的治疗方法 缓激肽递送到细胞的拮抗剂的研究进展 调节这种多肽的可利用性的治疗潜力。