REACTION OF THROMBIN AND COLLAGEN WITH PLATELETS

凝血酶和胶原蛋白与血小板的反应

• 批准号: 3335184
• 负责人: THOMAS C DETWILER
• 金额: $ 18.94万
• 依托单位: SUNY DOWNSTATE MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-04-01 至 1990-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3335184
• 关键词: adenylate cyclase; affinity chromatography; affinity labeling; cell free system; chemical association; chymotrypsin; covalent bond; crosslink; cyclic GMP; human tissue; immunochemistry; membrane activity; monoclonal antibody; platelet activating factor; platelets; proteolysis; thrombin; tissue /cell culture

The mechanism(s) of thrombin-platelet interaction(s) will be studied with three long range objectives: i) to characterize and establish the function of the thrombin-induced inhibition of platelet adenylate cyclase (AC), ii) to use inhibition of AC as an assay of a thrombin receptor in soluble preparations, and iii) to isolate thrombin-binding proteins and to identify components of the putative thrombin "receptor". Comparisons of thrombin-induced activation of control vs chymotrypsin-treated platelets and of activation of platelets by Alpha-thrombin vs Gamma-thrombin reveal that when the ability of thrombin to activate platelets quickly is impaired (pretreatment with chymotrypsin; activation by Gamma-thrombin), the ability to inhibit AC is blocked. We will test the hypotheses that i) inhibition of AC is necessary for normal platelet activation by thrombin and other agonists, and ii) thrombin regulates AC activity by either a receptor or an effector that is distinct from that for platelet activation, with only the AC system highly sensitive to proteolysis. Thrombin-induced inhibition of AC will be analyzed in membrane and soluble preparations, and the effects of proteases on regulation of cyclase by thrombin and other agonists will be correlated with the effects of protease pretreatment on the polypeptide composition of the cyclase preparation. The solubilized system will be used as an assay for receptor activity to establish an apparent mass for the receptor, to study dissociation and stability of the receptor and for following the course of purification. With similar experiments, the effect of collagen on the regulation of AC will be analyzed. To identify platelet thrombin-binding proteins, photoactivatable crosslinking reagents will be used in two ways: i) as a means for enrichment of the binding proteins, and ii) as a means for specifically attaching a radioactive label for screening for monoclonal antibodies against the binding proteins. For isolation of the binding proteins, a cleavable crosslinking reagent will be used. The derivatized thrombin will be crosslinked to the surface of labeled platelets, the complex will be solubilized and adsorbed to an immobilized antibody against thrombin, and the platelet protein will be released by cleavage of the crosslinking reagent. The product will be used as an immunogen for the preparation of monoclonal antibodies, using the immunogen and crosslinked thrombin-platelet complexes for clone selection. Antibody affinity columns will be used to isolate the binding proteins in high yield.

将研究凝血酶 - 植入术相互作用的机理（S） 三个远程目标：i）表征和建立功能 凝血酶诱导的血小板腺苷酸环化酶（AC）的抑制作用 使用AC作为可溶性凝血酶受体的测定 制剂，iii）分离凝血酶结合蛋白并识别 推定凝血酶“受体”的成分。 凝血酶诱导的对照VS激活的比较 胰凝乳蛋白酶处理的血小板和血小板激活 alpha-thrombin vs gamma-thrombin揭示了凝血酶的能力 迅速激活血小板会受到损害（用甲over蛋白预处理； 通过γ-凝血酶激活），抑制AC的能力被阻断。 我们 将检验假设i）抑制AC是正常的必需 凝血酶和其他激动剂的血小板激活，ii）凝血酶 通过不同的受体或效应器来调节AC活性 从中为血小板激活，仅AC系统高度敏感 蛋白水解。 凝血酶诱导的AC抑制作用将在 膜和可溶性制剂以及蛋白酶的影响 凝血酶和其他激动剂对环酶的调节将是相关的 蛋白酶预处理对多肽组成的影响 循环酶制备。 溶解系统将用作测定 使受体活性为受体建立明显的质量， 研究受体的解离和稳定性，并遵循 净化过程。 通过类似的实验，胶原蛋白的作用 关于AC的调节，将进行分析。 要识别血小板结合蛋白，可光活化 交联试剂将以两种方式使用：i）作为一种手段 富集结合蛋白，ii）作为特定的手段 连接放射性标签以筛选单克隆抗体 针对结合蛋白。 为了隔离结合蛋白，A 将使用可切合的交联试剂。 衍生化的凝血酶将 交联至标记为血小板的表面，该复合物将为 溶解并吸附到针对凝血酶的固定抗体，然后 血小板蛋白将通过交联的裂解释放 试剂。 该产品将用作制备的免疫原 单克隆抗体，使用免疫原并交联 用于克隆选择的凝血酶 - 地板配合物。 抗体亲和力柱 将使用高产量分离结合蛋白。