CHARACTERIZATION AND ISOLATION OF PLATELET ADP RECEPTORS

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 或通过两个受体，一个引起血小板活化， 其他抑制受刺激的腺苷酸环化酶：一个推论， 两个受体假说是C2-取代的ADP类似物应该 仅作用A：调节腺苷酸环化酶的受体。 拟议 研究是该实验室的主要承诺， 解决这些问题以及确定ADP受体，或 受体，血小板表面的数量，大小， 特异性和结构构型及功能。 在 初步研究中，使用甲醛固定的血小板， 避免因代谢和分泌引起的并发症， 0.35μ M）和低亲和力（Kd 7.9 μ M）的ADP和C2- 已经鉴定了替代的ADP。 一种新的C2-取代ADP 已经合成了一种光亲和探针， 分子量分别为188，000、93，000和 5万个在分离的血小板膜中。 为了识别 完整血小板的一种或多种ADP受体： 提出了具体的目标：（一）表征结合位点， ADP和2-甲硫基ADP原位平衡结合， 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， （ii）利用C2-取代的光亲和性 标记和针对可能活性物的新亲和探针 在受体位点处的巯基以鉴定推定的受体;（iii） 分离光亲和和亲和标记的产物， 它们的亲本多肽通过多种技术，包括 在C2-ADP-琼脂糖上进行亲和色谱;（iv）测量 这些多肽的能力，以及针对 他们，阻止ADP诱导的血小板活化，并逆转 抑制受刺激腺苷酸环化酶;（v）使用流体相 用于区分C2-ADP类似物可及受体的试验， （vi）表征受体，或 受体，通过结构分析（包括测序）定义 通过ADP结合结构域和功能评估 （vii）评估内皮细胞和其他细胞，以及 组织中与血小板ADP受体相关的蛋白质。

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.