NOVEL RGD-BINDING MEMBRANE PROTEIN ON BLOOD PLATELETS

血小板与内皮下成分和血小板的黏附 聚集在止血和血栓形成中很重要，而且 部分由3种黏附蛋白的结合所介导 含有Arg-Gly-Asp(RGD)序列：纤维蛋白原、纤维连接蛋白和 冯·威勒布兰德因素。GPIIb-IIIa是这些粘合剂的受体 蛋白质并与含有RGD序列的多肽结合。 此外，GPIIb-IIIa是广泛分布的黏附分子之一 受体超家族称为整合素，其成员包括 两个不同的α和β亚基。除了GPIIb-IIIa， 某些其他整合素也识别RGD序列。我们 最近发现了一种MR的血小板膜蛋白， 160 kDa(P160)，与含有RGD序列的多肽结合。 这一提议将检验P160形成一个 异二聚体与另一种血小板膜蛋白的复合体 构成一种新型的血小板整合素。以确定P160是否形成 一个带有β亚基的杂二聚体，我们将分析多肽 与抗体反应形成的免疫沉淀物的含量 纯化的P160。此外，我们还将研究流体力学 纯化的P160的性质以确定其在非 变性条件。此外，我们将调查是否 P160序列与已知的整合素序列相似 P160氨基末端及片段的测序。类似 研究将用P160的假定的β亚基进行 如果有人被发现的话。P160的RGD结合域将是 亲和层析法定位和表征 纯化的P160蛋白水解酶及其化学交联法 将RGD多肽转化为P160，然后进行蛋白水解性切割。自.以来 结合RGD的几种膜蛋白具有黏附功能， 我们将检验P160在血小板中起作用的假设 粘合反应。首先，这将通过以下方式实现 将纯化的P160重组为放射性脂质体 检测其与多种含RGD的黏附蛋白的结合 在固相分析中。其次，我们将鉴定抗体和 优先阻断P160与其结合的RGD多肽 固定化RGD多肽。这些抗体的能力和 将对抑制血小板黏附功能的多肽进行评估。 这些研究将提供有关膜的基本信息 可能与血小板相关机制有关的蛋白质 粘连或聚集。

Platelet adhesion to subendothelial constituents and platelet aggregation are important in hemostasis and thrombosis, and are mediated in part by the binding of 3 adhesive proteins which contain Arg-Gly-Asp (RGD) sequences: fibrinogen, fibronectin and von Willebrand factor. GPIIb-IIIa is a receptor for these adhesive proteins and binds to peptides containing the RGD sequence. Moreover, GPIIb-IIIa is a member of widely distributed adhesion receptor superfamily termed Integrins whose members are comprised of two non-identical alpha and beta subunits. Besides GPIIb-IIIa, certain other integrins also recognize the RGD sequence. We recently identified a platelet membrane protein of Mr approximately 160 kDa (P160) which binds to peptides containing the RGD sequence. This proposal will test the hypothesis that P160 forms a heterodimer complex with another platelet membrane protein to constitute a novel platelet integrin. To determine if P160 forms a heterodimer with a beta subunit, we will analyze the polypeptide content of immunoprecipitates formed with antibodies reactive with purified P160. In addition, we will examine the hydrodynamic properties of purified P160 to establish its Mr under non- denaturing conditions. Furthermore, we will investigate whether the sequence of P160 is similar to known integrins by direct sequencing of the amino-terminus and fragments of P160. Similar studies will be performed with the putative beta subunit of P160 should one be identified. The RGD-binding domain of P160 will be localized and characterized by affinity chromatography of proteolytic digests of purified P160, and by chemical crosslinking of RGD peptides to P160 followed by proteolytic cleavage. Since several membrane proteins which bind RGD serve adhesive functions, we will test the hypothesis that P160 plays a role in platelet adhesive reactions. Firstly, this will be accomplished by reconstituting purified P160 into radioactive liposomes and examining its binding to various RGD-containing adhesive proteins in a solid phase assay. Secondly, we will identify antibodies and RGD peptides which preferentially block the binding of P160 to immobilized RGD peptides. The capacity of these antibodies and peptides to inhibit platelet adhesive functions will be assessed. These studies will provide fundamental information about a membrane protein likely to be involved in mechanisms underlying platelet adhesion or aggregation.