CONTENT AND ASSEMBLAGE OF PLATELET GRANULE PROTEINS

血小板在调节多种生物学过程中起着关键作用。 包括细胞增殖、细胞黏附、免疫调节、 血栓形成和纤溶；这些事件中的许多都直接受到影响 由血小板α颗粒分泌的蛋白质所致。中环 这一提议的假设是，至少有两种这样的蛋白质，即 凝血酶敏感蛋白和血小板因子4参与这种调节 通过它们与特定配体的相互作用和， 此外，这种相互作用是独特和具体的功能 多肽结构域，在给定的配体和血小板蛋白中都有。 具体地说，所需的凝血酶原蛋白结合域 将鉴定Aα-Aβ-纤维蛋白原链。合成肽 每个结构域的类似物都将被合成和评估 它们调节血小板分泌依赖相的能力 聚合。利用印迹结合分析，凝血酶反应蛋白的区域 还将鉴定与纤维蛋白原结合的必需肽 领域(S)表征、合成和评估他们的能力 调节凝血酶原-纤维蛋白原相互作用和血小板聚集。 凝血酶反应蛋白的COOH末端18 kDa结构域已在 允许直接评估其结合能力的量 到未激活和激活的血小板。这些研究将进行 利用荧光标记的18 kDa结构域以及多克隆和 单抗探针。凝血酶敏感蛋白与特异性结合的研究进展 纤溶酶原的Kringle区域，特别是Kringle 5，将是 下定决心。多肽类似物对这一区域的能力将是 探讨它们对纤溶酶原激活物动力学的调节作用 对纤溶酶生成的影响。纤溶酶生成方案还将 被评估时富含组氨酸的糖蛋白和凝血酶反应蛋白 来自该蛋白质的结合结构域区包括在 富含凝血酶原蛋白-组氨酸的三分子络合物活化 糖蛋白-纤溶酶原。关于相互作用的进一步研究 凝血酶原蛋白和纤维连接蛋白的29 kDa结构域也将 已执行。将利用抗凝血球蛋白的单抗 为了定义凝血酶反应蛋白分子中的哪些区域是 由特定的蛋白质配基“装饰”。这些研究应该提供一个 更好地了解凝血酶反应蛋白在血小板中的作用 聚集，纤维蛋白形成和纤溶，并允许发展 指阻止或减弱这些过程的合成肽。

Platelets play a critical role in mediating a variety of biological processes, including cell proliferation, cell adhesion, immunoregulation, thrombosis and fibrinolysis; many of these events are directly influenced by proteins secreted from the platelet alpha-granule. The central hypothesis of this proposal is that at least two such proteins, namely thrombospondin and platelet factor 4, participate in such regulatory mechanisms through their interaction with specific ligands and, furthermore, that this interaction is the function of unique and specific peptide domains, both within a given ligand and the platelet proteins. Specifically, the requisite thrombospondin binding domains within the Aalpha- an Bbeta-fibrinogen chains will be identified. Synthetic peptide analogues to each of these domains will be synthesized and evaluated for their ability to modulate the secretion-dependent phase of platelet aggregation. Utilizing a blot-binding assay, the region of thrombospondin which binds fibrinogen will also be identified, the requisite peptide domain(s) characterized, synthesized, and assessed for their ability to regulate thrombospondin-fibrinogen interaction and platelet aggregation. The COOH-terminal 18 kDa domain of thrombospondin has been purified in quantities which will permit a direct assessment of its ability to bind to non-activated and activated platelets. These studies will be conducted using fluorescent-labeled 18 kDa domain as well as polyclonal and monoclonal antibody probes. The binding of thrombospondin to specific kringle regions of plasminogen, particularly kringle 5, will be determined. The ability of peptide analogues to this region will be probed for their potency in regulating plasminogen activators' kinetics effect on plasmin generation. The plasmin generation protocols will also be assessed when histidine rich glycoprotein, and the thrombospondin binding domain regions from that protein, are included in the trimolecular complex activation of thrombospondin-histidine rich glycoprotein-plasminogen. Further studies on the interaction of thrombospondin and the 29 kDa domain of fibronectin will also be performed. Monoclonal antibodies against thrombospondin will be utilized to define which regions within the thrombospondin molecule are "decorated" by specific protein ligands. These studies should provide a better understanding of the role of thrombospondin in platelet aggregation, fibrin formation and fibrinolysis and permit the development of synthetic peptides which prevent or attenuate these processes.