PLATELET HIGH MOLECULAR WEIGHT KININOGEN

血小板高分子量激肽原

• 批准号: 3349537
• 负责人: ALVIN H SCHMAIER
• 金额: $ 6.08万
• 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 1990-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3349537
• 关键词: alpha globulin; binding proteins; bradykinin; calcium binding protein; coagulation factor XI; coagulation factor XII; cofactor; enzyme mechanism; exopeptidase; human subject; immunochemistry; kallikreins; laboratory mouse; membrane proteins; molecular weight; monoclonal antibody; platelets; prostacyclins; protease inhibitor; radiotracer; receptor; vascular endothelium; western blottings; zymogens

The objectives of this proposal are to characterize the mechanisms whereby platelet high molecular weight kininogen (HMWK) can be an extracellular regulator of contact phase zymogen activation, a possible intracellular inhibitor of platelet calpains, and an indirect regulator of endothelial function. The specific aims of this investigation are as follows: (1) Platelet HMWK present on the external membrane of activated platelets will be quantified and characterized by 125I-monoclonal anti HMWK Fab binding studies; (2) Platelet HMWK will be characterized in unstimulated and activated platelets as to its location and distribution by immunocytochemical staining using colloidal gold; (3) Purified HMWK subunit binding to platelets will be done to determine the portion of the HMWK molecule that binds to platelets and to determine if the affinity of 125I-HMWK-platelet binding is altered when platelets are activated; (4) Coordinate factor XI/XIa-HMWK platelet binding will be performed to determine if HMWK serves as their platelet receptor; (5) The molecular structure of platelet HMWK will be characterized by immunoblot to determine if it is different from plasma HMWK or if it is cleaved by platelet calpain; (6) The effect of purified platelet calpain on purified HMWK will be studied to determine if platelet calpain activates HMWK and produces an altered HMWK with a structure similar to the HMWK seen in platelets, and to determine if HMWk itself can be an inhibitor of platelet calpain; (7) The availability and function of bradykinin derived from platelet HMWK will be ascertained to determine whether it is directly secreted from platelets and to determine whether a sufficient amount can be made available from platelet HMWK to stimulate endothelial cell secretion of prostacyclin. These studies are intended to characterize the platelet surface as a site for contact phase zymogen activation through the expression of HMWK on the platelet external membrane; to demonstrate that HMWK is activated by and is an inhibitor of platelet calpain; and to determine that bradykinin from secreted platelet HMWK is sufficient to stimulate endothelial cell prostacyclin secretion. These objectives are consistent with the hypothesis that platelet HMWK is a mediator and regulator of the surface-activated defense reactions of the proteins of the Hageman factor pathways which participate in the initiation of coagulation, the inflammatory response, and local injury-site blood pressure control.

这项提案的目标是说明下列机制的特点 血小板高分子激肽原(Hmwk)可以是一种细胞外信号。 接触相酶原激活的调节因子，可能是细胞内的一种 血小板钙蛋白酶抑制因子和内皮间接调节因子 功能。本次调查的具体目的如下：(一) 存在于激活的血小板外膜上的血小板HMWK将 用125I-抗hmwk Fab单抗结合进行定量和鉴定 研究；(2)血小板hmwk的特征是无刺激和 激活的血小板的位置和分布 胶体金免疫细胞化学染色；(3)纯化的hmwk亚基 将与血小板结合以确定hmwk的部分 与血小板结合的分子，并确定其亲和力是否 当血小板被激活时，125I-hmwk-血小板结合发生改变； 协调因子XI/XIA-HMWK将进行血小板结合以 确定hmwk是否作为他们的血小板受体；(5)分子 用免疫印迹法对血小板hmwk的结构进行表征 如果它与血浆hmwk不同，或者它被血小板切割 (6)纯化的血小板钙蛋白酶对纯化的HMWK的影响 被研究以确定血小板钙蛋白酶是否激活hmwk并产生 改变了hmwk，其结构类似于在血小板中看到的hmwk，并 确定hmwk本身是否可以抑制血小板钙蛋白酶；(7) 血小板hmwk来源的缓激肽的可用性和功能将是 以确定它是否是直接从血小板和 确定是否可以从以下来源获得足够的数量 血小板hmwk刺激内皮细胞分泌前列环素。 这些研究旨在将血小板表面定性为一个部位。 通过hmwk在细胞表面的表达激活接触相酶原 血小板外膜；以证明hmwk被激活 一种血小板钙蛋白酶的抑制剂；并从 分泌的血小板hmwk足以刺激内皮细胞 前列环素分泌。这些目标与 假设血小板HMWK是血管紧张素转换酶的介体和调节因子 Hageman因子蛋白的表面活性防御反应 参与凝血启动的途径，即 炎症反应，以及局部损伤部位的血压控制。