NOVEL RGD-BINDING MEMBRANE PROTEIN ON BLOOD PLATELETS

血小板上新型 RGD 结合膜蛋白

• 批准号: 3472444
• 负责人: Stephen CT Lam
• 金额: $ 9.73万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-05-01 至 1993-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3472444
• 关键词: affinity chromatography; binding proteins; bioassay; blocking antibody; cell adhesion; cell adhesion molecules; chemical binding; crosslink; dimer; glycoproteins; human tissue; immunochemistry; laboratory rabbit; ligands; liposomes; membrane proteins; peptides; platelet aggregation; platelets; protein purification; protein sequence; protein structure function; radiotracer; receptor; receptor binding; synthetic peptide

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.

血小板粘附于内皮下成分和血小板 聚集在止血和血栓形成中是重要的， 部分由3种粘附蛋白的结合介导， 含有Arg-Gly-Asp（RGD）序列：纤维蛋白原、纤连蛋白和 血管性血友病因子 GPIIb-IIIa是这些粘合剂的受体 蛋白质并结合含有RGD序列的肽。 此外，GPIIb-IIIa是广泛分布的粘附分子， 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， 某些其它整联蛋白也识别RGD序列。 我们 最近发现了一种血小板膜蛋白， 160 kDa（P160），与含有RGD序列的肽结合。 这一提议将检验P160形成一个 异二聚体复合物与另一种血小板膜蛋白， 构成一种新的血小板整合素。 为了确定P160是否形成 一个β亚基的异源二聚体，我们将分析多肽 与抗体反应形成的免疫沉淀物含量 纯化的P160 此外，我们还将研究 纯化的P160的性质，以建立其在非- 变性条件。 此外，我们将调查是否 P160序列与已知的整联蛋白相似， P160的氨基末端和片段的测序。 类似 将对P160的假定β亚基进行研究 如果一个被识别。 P160的RGD结合结构域将是 通过亲和层析定位和表征， 纯化的P160的蛋白水解酶，并通过化学交联 P160的RGD肽，然后进行蛋白水解切割。 以来 几种结合RGD的膜蛋白具有粘附功能， 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重构为放射性脂质体， 检测其与各种含RGD的粘附蛋白的结合 在固相分析中。 其次，我们将识别抗体， 优先阻断P160结合的RGD肽 固定化RGD肽。 这些抗体的能力， 评价抑制血小板粘附功能的肽。 这些研究将提供有关膜的基本信息 可能参与血小板机制的蛋白质 粘附或聚集。