REACTION OF THROMBIN AND COLLAGEN WITH PLATELETS

凝血酶-血小板相互作用的机制将通过以下方法进行研究： 三个长期目标：i）确定和确定职能 凝血酶诱导的血小板腺苷酸环化酶（AC）抑制，ii） 使用AC的抑制作为可溶性凝血酶受体的测定 iii）分离凝血酶结合蛋白并鉴定 凝血酶“受体”的组成部分。 凝血酶诱导的对照与 胰凝乳蛋白酶处理的血小板和血小板活化 α-凝血酶与γ-凝血酶的对比表明，当凝血酶的能力 快速激活血小板的能力受损（用胰凝乳蛋白酶预处理; 通过γ-凝血酶活化），抑制AC的能力被阻断。 我们 将检验以下假设：i）AC的抑制对于正常的 通过凝血酶和其它激动剂的血小板活化，和ii）凝血酶 通过不同的受体或效应物调节AC活性 与血小板活化相比，只有AC系统高度敏感 蛋白质水解 凝血酶诱导的AC抑制将在 膜和可溶性制剂，以及蛋白酶对 凝血酶和其他激动剂对环化酶的调节将与 研究了蛋白酶预处理对大豆蛋白多肽组成的影响 环化酶制剂。 增溶系统将用作试验 为了确定受体的表观质量， 研究受体的解离和稳定性， 净化过程。 通过类似的实验， 对AC的调节进行分析。 为了鉴定血小板凝血酶结合蛋白， 交联试剂将以两种方式使用：i）作为 结合蛋白的富集，和ii）作为特异性地 连接放射性标记用于筛选单克隆抗体 对抗结合蛋白。 对于结合蛋白的分离， 将使用可裂解的交联剂。 衍生的凝血酶将 交联到标记血小板的表面，复合物将 溶解并吸附到固定化的抗凝血酶抗体上，和 血小板蛋白将通过交联的裂解而释放 试剂. 该产品将用作免疫原，用于制备 单克隆抗体，使用免疫原和交联 凝血酶-血小板复合物用于克隆选择。 抗体亲和柱 将用于以高产率分离结合蛋白。

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.