Aspirin therapy has been associated with significant clinical antithrombotic effects in certain arterial thromboembolic disorders, particularly for the treatment of transient ischemic attacks and unstable angina, and for the secondary prevention of myocardial infarction. It is generally thought that its antithrombotic mechanism involves an irreversible inactivation of the platelet enzyme cyclooxygenase through o- acetylation of active site serine. However, results obtained in experimental models of thromboembolism and data on non-platelet-related effects of aspirin suggest that its antithrombotic effect may also involve mechanism(s) other than cyclooxygenase inhibition. Our preliminary studies on the fibrinolytic effect of aspirin suggest that its acetylation of fibrinogen to form epsilon-N-acetyl-lysyl residues is associated with the mechanism underlying this effect. The overall objective of this proposal is concerned with the detailed elucidation of he biological consequences of the acetylation of fibrinogen as it relates to the fibrinolytic effects of aspirin. The six major specific aims of this proposal are: 1) To determine the in vivo dose-response relationship for the fibrinolytic effect of aspirin. These studies will be carried out in human subjects, using six different daily doses of aspirin, ranging from 80 to 2,600 mg, administered for two weeks. 2) To elucidate the mechanism(s) of action underlying the fibrinolytic effect of aspirin. This involve a series of in vitro studies on normal and acetylated fibrinogens aimed at obtaining a biological mechanistic explanation for the fibrinolytic effect of aspirin, including studies on fibrin polymerization and cross-link formation, and on interactions between fibrin(ogen) and fibrinolytic system proteins. 3) To define the effects of acetylation of fibrinogen on the three=dimensional structure of fibrinogen and the fibrin assembly process. This will involve electron microscopy studies aimed at determining structures, diameters, and branching of fibrin protofibrils and fibers. 4) To demonstrate the in vivo formation of epsilon-N-acetyl-lysyl residues on fibrinogen. this will be done by obtaining fibrinogen from aspirin-treated subjects and measuring the epsilon-N-acetyl-lysyl to lysyl residue ratio of fibrinogen by a specific and sensitive assay. 5) To characterize the effects of acetylation of fibrinogen on its in vivo turnover kinetics. These studies will be performed in rabbits, and will involve the characterization of the turnover kinetics parameters of iodinated fibrinogen receptor-fibrinogen interaction. These studies will be performed using isolated platelets and different fibrinogen components. The long-term goals of this project are to further our understanding of the mechanism(s) underlying aspirin's antithrombotic effect and to elucidate biological mechanisms for pharmacological interventions of fibrin aggregation, assembly, cross- linking and lysis. It is anticipated that the results of the proposed studies will also aid in the optimal clinical use of aspirin.
