PLATELET HIGH MOLECULAR WEIGHT KININOGEN

本提案的目的是确定各种机制的特点， 血小板高分子量激肽原（HMWK）可以是细胞外的 接触相酶原激活的调节剂，一种可能的细胞内 血小板钙蛋白酶抑制剂和内皮细胞的间接调节剂 功能 本研究的具体目的如下：（1） 存在于活化血小板外膜上的血小板HMWK将 通过125 I-单克隆抗HMWK Fab结合进行定量和表征 （2）血小板HMWK将在未受刺激和 活化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 将进行与血小板的结合以确定HMWK的部分 与血小板结合的分子，并确定 当血小板被激活时，125 I-HMWK-血小板结合改变;（4） 将进行协调因子Xi/Xia-HMWK血小板结合， 确定HMWK是否作为其血小板受体;（5）分子 血小板HMWK的结构将通过免疫印迹表征以确定 如果它不同于血浆HMWK或如果它被血小板裂解 （6）血小板钙蛋白酶对纯化HMWK的影响 进行研究，以确定血小板钙蛋白酶是否激活HMWK并产生 改变的HMWK具有与血小板中观察到的HMWK相似的结构， 确定HMWk本身是否可以是血小板钙蛋白酶的抑制剂;（7） 从血小板HMWK衍生的缓激肽的可用性和功能将是 确定其是否直接从血小板分泌， 以确定是否有足够的数量可以从 血小板HMWK刺激内皮细胞分泌前列环素。 这些研究旨在表征血小板表面作为一个位点， 对于通过HMWK在细胞膜上的表达进行的接触相酶原活化， 血小板外膜;证明HMWK被激活， 血小板钙蛋白酶抑制剂;并确定缓激肽从 分泌的血小板HMWK足以刺激内皮细胞 前列环素分泌 这些目标符合 假设血小板HMWK是一种介质和调节剂， Hageman因子蛋白的表面活化防御反应 参与启动凝血的途径， 炎症反应和局部损伤部位血压控制。

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.