PLATELET HIGH MOLECULAR WEIGHT KININOGEN

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。

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.