CHARACTERIZATION AND ISOLATION OF PLATELET ADP RECEPTORS

血小板 ADP 受体的表征和分离

• 批准号: 2219257
• 负责人: GRAHAM A JAMIESON
• 金额: $ 25.71万
• 依托单位: AMERICAN NATIONAL RED CROSS
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1998-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2219257
• 关键词: adenosine diphosphate; adenylate cyclase; affinity chromatography; affinity labeling; antireceptor antibody; binding proteins; biological signal transduction; cell biology; cell population study; chemical binding; chemical structure function; chromatography; hormone receptor; human tissue; immunochemistry; laboratory mouse; laboratory rabbit; membrane activity; molecular biology; monoclonal antibody; nucleotide analog; platelet activating factor; platelet aggregation; protein structure; proteins; radiotracer; surface antigens; vascular endothelium

The mechanism by which ADP activates platelets is not understood. A major question is whether it acts via a single type of receptor or through two receptors, one causing platelet activation and the other inhibition of stimulated adenyl cyclase: a corollary to the two receptor hypothesis is that C2-substituted ADP analogues should act only a: the receptor modulating adenyl cyclase. The proposed studies are a major commitment by this laboratory intended to resolve these questions as well as to define the ADP receptor, or receptors, of the platelet surface in terms of number, size, specificity and structural configuration and function. In preliminary studies, formaldehyde-fixed platelets were used to avoid complications due to metabolism and secretion and high (Kd 0.35 uM) and low (Kd 7.9 uM) affinity binding sites for ADP and C2- substituted ADPs have been identified. A new C2-substituted ADP photoaffinity probe has been synthesized that specifically labels three binding sites with molecular weights of 188,000, 93,000 and 50,000 in isolated platelet membranes. In order to identify the ADP receptor, or receptors, of intact platelets the following specific aims are proposed: (i) characterize the binding sites for ADP and 2-methylthioADP in situ by equilibrium binding and radiation inactivation to determine their number and affinity and utilize radiation inactivation to differentiate them on the basis of functional sizes; (ii) utilize the C2-substituted photoaffinity label and a new affinity probe directed against a possible active thiol at the receptor site to identify putative receptors; (iii) isolate the products of photoaffinity and affinity labeling and their parent polypeptides by a variety of techniques including affinity chromatography on C2-ADP-Sepharose; (iv) measure the ability of these polypeptides, and antibodies prepared against them, to block ADP-induced platelet activation and to reverse the inhibition of stimulated adenyl cyclase; (v) use a fluid phase assay to differentiate receptors accessible to C2-ADP analogues and those accessible only to ADP; (vi) characterize the receptor, or receptors, so defined by structural analysis, including sequencing through the ADP-binding domains and by evaluation of functional activities; (vii) evaluate endothelial cells and other cells and tissues for proteins related to the ADP receptor(s) of platelets.

ADP激活血小板的机制尚不清楚。 一个主要的问题是，它是否通过单一类型的受体起作用 或者通过两个受体，一个引起血小板活化，另一个 刺激的腺苷环化酶的其他抑制：一个推论 两种受体假说认为C2取代的ADP类似物应该 仅起作用a：调节腺苷酸环化酶的受体。建议数 研究是该实验室的一项主要承诺，旨在 解决这些问题，并定义ADP受体，或 受体，在数量，大小， 专一性和结构形态和功能。在……里面 初步研究，甲醛固定的血小板用于 避免因代谢分泌和高(KD)引起的并发症 0.35um)和低(kD 7.9um)ADP和C2-亲和力结合部位 已经确定了替代的ADP。一种新的C2取代的ADP 合成了一种光亲和探针，可以特异性地标记 三个结合位点，分子量分别为188,000，93,000和 在分离的血小板膜中有50,000。为了识别 ADP受体，或完整血小板的以下受体 提出了具体目标：(1)确定结合部位的特征 ADP与2-甲硫基ADP平衡结合原位 辐射灭活以确定它们的数量和亲和力 利用辐射灭活来区分它们 功能尺寸；(Ii)利用C2取代的光亲和 标记和一种新的针对可能的活性的亲和探针 受体部位的硫醇，以识别可能的受体； 分离光亲和亲和标记产物 它们的母体多肽通过各种技术进行处理，包括 C2-ADP-琼脂糖亲和层析；(4)测定 这些多肽的能力以及针对这些多肽制备的抗体 阻断ADP诱导的血小板活化并逆转血管紧张素转换酶 抑制受刺激的腺苷环化酶；(V)使用液体相 区分C2-ADP类似物和受体的方法 只有ADP才能访问的那些；(Vi)表征受体，或 受体，通过结构分析，包括测序来定义 通过ADP结合域和通过功能评估 活动；(7)评价内皮细胞和其他细胞和 组织中寻找与血小板腺苷二磷酸受体(S)相关的蛋白质。