INITIATION OF COAGULATION AND FIBRINOLYSIS IN MAN

我们的长期目标是确定启动的机制， 内源性凝血-激肽系统在人血浆中发生， 开发用于评估疾病中该级联反应激活的测定方法， states. 我们希望区分酶的表达是否 未切割的酶原中的活性在起始期间可能是显著的;这 这个问题将通过动力学分析的反应，使用 裂解产物的特异性抑制剂和通过化学修饰， 特别是关注精氨酸残基。 复合物的类型 将使用交联试剂来限定在表面上形成的 Hageman因子（HF）的可能聚集以及与已知的 含有前激肽释放酶或因子Xi和HMW-激肽原的复合物。 我们将 还确定C1 INH是否控制HF自动激活的速率， 测试引发表面阻碍抑制的假设 通过C1 INH结合、活化HF。 用于定量的复合物的测定 血浆中活化的HF和C1 INH将使用单克隆抗体进一步精制。 将测定抗血清和产生的活化HF的种类。 辅助因子HMW激肽原的研究将集中在以下方面 问题：1）它是可激活的辅因子，即，一个或一个的分裂 更多的键会增加其内在辅因子活性或影响其能力 与其他血浆蛋白竞争结合表面; 2）什么是 当它被不同的酶消化时键断裂的顺序， 产生的凝血剂链的大小; 3）单克隆抗体 将产生针对最小种类的凝血剂链的抗体， 表征表面相互作用所需链部分，或 结合底物前激肽释放酶和HMW-激肽原; 4） 切割的分子可用于设计仅检测切割的 在血浆中形成。 最后，我们将研究HF依赖和 血浆的独立纤维蛋白溶解途径，并确定血浆是否 尿激酶原在这两种情况下都起作用。 补体系统在 血浆纤维蛋白溶解将被确定，新的纤溶酶原激活剂将被确定。 寻求，任何纤溶酶原激活剂或新的纤溶酶将被 纯化和表征。

Our long term objectives are to determine the mechanism by which initiation of the intrinsic coagulation-kinin system occurs in human plasma and to develop assays by which to assess activation of this cascade in disease states. We hope to distinguish whether the expression of enzymatic activity in uncleaved zymogens might be significant during initiation; this issue will be approached by kinetic analysis of the reactions using specific inhibitors of the cleaved products and by chemical modifications, particularly focusing upon arginine residues. The types of complexes formed upon surfaces will be defined using cross linking reagents including possible aggregation of Hageman factor (HF) and interaction with known complexes containing prekallikrein or factor XI and HMW-kininogen. We will also determine whether C1INH controls the rate of HF autoactivation and test the hypothesis that initiating surfaces impede the inhibition of bound, activated HF by C1INH. The assay for quantitating complexes of activated HF and C1INH in plasma will be further refined using monoclonal antisera and the species of activated HF produced will be determined. Studies of the cofactor HMW kininogen will focus upon the following issues: 1) Is it an activatable cofactor, i.e., does cleavage of one or more bonds increase its intrinsic cofactor activity or affect its ability to compete with other plasma proteins for binding to surfaces; 2) What is the order of bond cleavages when it is digested by different enzymes and what is the size of the coagulant chain that is produced; 3) Monoclonal antibodies to the smallest species of coagulant chain will be produced to characterize portions of that chain requisite for surface interaction or binding the substrates prekallikrein and HMW-kininogen; 4) Antibodies to the cleaved molecule can be used to devise an assay detecting only cleaved forms in plasma. Finally, we will investigate the HF-dependent and independent fibrinolytic pathways of plasma and determine whether a plasma prourokinase plays a role in either. The role of the complement system on plasma fibrinolysis will be determined, new plasminogen activators will be sought, and any plasminogen activators or new fibrinolytic enzymes will be purified and characterized.