REACTION OF THROMBIN AND COLLAGEN WITH PLATELETS

凝血酶-血小板相互作用的机制(S)(S)将与 三个长期目标：一)确定和确立职能 凝血酶抑制血小板腺苷环化酶(AC)，II) 用AC抑制作为检测可溶性凝血酶受体的方法 制备，以及iii)分离凝血酶结合蛋白并鉴定 推测的凝血酶“受体”的成分。 对照组与对照组凝血酶诱导活化的比较 凝乳酶处理的血小板及其对血小板的激活作用 α-凝血酶VS-γ-凝血酶揭示当凝血酶能力 快速激活血小板的能力受损(用胰凝乳杆菌酶预处理； 被伽玛-凝血酶激活)，抑制AC的能力被阻断。我们 将检验以下假设：i)抑制AC是正常所必需的 凝血酶和其他激动剂激活血小板，以及ii)凝血酶 通过不同的受体或效应器调节AC活动 只有AC系统高度敏感 蛋白水解酶。凝血酶诱导的AC抑制将在 膜和可溶性制剂，以及蛋白水解酶对 凝血酶和其他激动剂对环化酶的调节将是相关的 蛋白水解酶预处理对红曲霉菌多肽组成的影响 环化酶制剂。增溶体系将被用作一种分析方法。 对于受体活性，以确定受体的表观质量，以 研究受体的解离和稳定性，并跟踪 净化过程。在类似的实验中，胶原蛋白的作用 对空调的调节进行了分析。 鉴定可光激活的血小板凝血酶结合蛋白 交联剂将以两种方式使用：i)作为 浓缩结合蛋白，以及ii)作为一种特定的 用于筛选单抗的放射性标记 对抗结合蛋白。为了分离结合蛋白，一种 将使用可分解的交联剂。衍生的凝血酶将 被交联到标记的血小板表面，该复合体将被 溶解并吸附到固定化的抗凝血酶抗体上，以及 交联物的裂解将释放血小板蛋白。 试剂。该产品将作为免疫原用于制备 单抗，使用免疫原和交联剂 凝血酶-血小板复合体用于克隆选择。抗体亲和柱 将用于高产率地分离结合蛋白。

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.