CHARACTERIZATION OF PLATELET FIBRINOGEN RECEPTORS

这项研究的一个主要目标是阐明分子机制，通过 哪种纤维蛋白原与其血小板表面受体相互作用 导致血小板聚集。几条强有力的调查路线 表面糖蛋白(GPIIIa)在血管内皮细胞的功能中的作用 纤维蛋白原受体。我们的研究揭示了66,000个MR蛋白质 它存在于经胰凝乳酶和链霉蛋白酶处理的表面 血小板。这种蛋白质，由多克隆抗体沉淀而成，可阻断 纤维蛋白原诱导的血小板聚集和125I-纤维蛋白原结合 在胰凝乳酶或链霉蛋白酶对GPIIIa进行有限的蛋白质分解时形成。这个 来源于GPIIIa的66,000 mR蛋白在细胞表面的形成 蛋白水解性血小板与纤维蛋白原诱导的相关性 血小板聚集和125I-纤维蛋白原与这些血小板的结合。我们 提出一个假设，即MR=66,000是糖蛋白IIIa的一部分 存在于经蛋白质分解处理的血小板表面，它可能 纤维蛋白原结合和纤维蛋白原诱导的血小板聚集中的作用 完整血小板中糖蛋白IIIa的66,000个道尔顿区域 可能代表糖蛋白IIIa的纤维蛋白原结合域。这个 这里提出的研究的目的是识别和表征 GPIIIa的纤维蛋白原结合结构域(S)及其66,000个MR 蛋白质片段。我们的具体目标是：1)进一步发展和 针对纯化人的多克隆抗体的鉴定 与纤维蛋白原受体相关的血小板膜蛋白； 抗日本血吸虫66,000个MR衍生物GPIIIa的单抗研制 GPIIIa，以及存在于胰凝乳酶表面的蛋白分解片段- 和链霉蛋白酶处理的血小板；3)多克隆和单克隆的使用 抗体在血小板纤维蛋白原受体纯化中的应用 膜，特别强调GPIIIa和66,000 MR衍生物 GPIIIa；4)研究这些膜糖蛋白与 在细胞水平和纯化系统中的纤维蛋白原；以及5)使用 抗GPIIIa和蛋白水解酶抗体库的建立 GPIIIa产品在人体暴露机制研究中的应用 血小板聚集过程中的纤维蛋白原受体。我的长期目标是 测定纤维蛋白原受体的化学结构并对其进行研究。 ADP暴露和激活纤维蛋白原受体的机制， 肾上腺素和凝血酶在血小板聚集过程中。这些研究将 有助于阐明血栓形成和止血的基本机制。

A major objective of this research is to elucidate molecular mechanisms by which fibrinogen interacts with its receptor on the platelet surface resulting in platelet aggregation. Several lines of investigation strongly implicate the roles of the surface glycoprotein (GPIIIa) in the function of the fibrinogen receptor. Our studies have revealed a 66,000 Mr protein that is present on the surface of chymotrypsin- and pronase-treated platelets. This protein, precipitated by polyclonal antibodies which block fibrinogen-induced platelet aggregation and 125I-fibrinogen binding, is formed during limited proteolysis of GPIIIa by chymotrypsin or pronase. The formation of this 66,000 Mr protein derived from GPIIIa on the surface of proteolytically-treated platelets correlated with fibrinogen-induced platelet aggregation and 125I-fibrinogen binding to these platelets. We propose a hypothesis that the Mr = 66,000 is a part of glycoprotein IIIa present on the surface of proteolytically-treated platelets and it may function in fibrinogen binding and fibrinogen-induced platelet aggregation, and that the 66,000-dalton region of glycoprotein IIIa in intact platelets may represent the fibrinogen-binding domain of glycoprotein IIIa. The purpose of the research proposed here is to identify and characterize the fibrinogen-binding domains(s) of GPIIIa with emphasis on its 66,000 Mr protein fragment. Our specific aims are: 1) further development and characterization of polyclonal antibodies directed against purified human platelet membrane proteins associated with the fibrinogen receptor; 2) development of monoclonal antibodies to GPIIIa, the 66,000 Mr derivative of GPIIIa, and proteolytic fragments present on the surface of chymotrypsin- and pronase-treated platelets; 3) use of polyclonal and monoclonal antibodies in the purification of fibrinogen receptors from platelet membranes with special emphasis on GPIIIa and the 66,000 Mr derivative of GPIIIa; 4) study the interaction of these membrane glycoproteins with fibrinogen at the cellular level and in the purified system; and 5) use of a library of antibodies developed against GPIIIa and proteolytic digest products of GPIIIa in studies on the mechanism of exposure of the fibrinogen receptor during platelet aggregation. My long term goals are to determine the chemicl structure of the fibrinogen receptor and to study the mechanism of fibrinogen receptor exposure and activation by ADP, epinephrine, and thrombin during platelet aggregation. These studies will serve to elucidate basic mechanisms involved in thrombosis and hemostasis.