INITIATION OF COAGULATION AND FIBRINOLYSIS IN MAN

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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.