HEMOSTATIC EFFECTS OF FIBRINOLYTIC THERAPY

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Fibrinolytic (thrombolytic) therapy has been used in treating arterial and venous thrombosis. Administration of thrombolytic agents, such as streptokinase, urokinase or tissue-type plasminogen activator (t-PA), is standard treatment for acute myocardial infarction patients. Bach agent results in cleavage of plasminogen to plasmin. Plasmin degrades the fibrin strands and dissolves thrombi. In addition, plasmin degrades platelet receptors (GP Ib) and plasma proteins that play a pivotal role in hemostasis, eg. fibrinogen, von Willebrand factor (vWF). Both the dispersion of platelet aggregates, through the lysis of fibrin, and the plasmin-induced systemic effects may partly account for inhibition of mural thrombus formation and maintenance of the vessel patency after successful recanalization. Non intervention-related hemorrhage may be also associated with the plasmin-induced hemostatic defects. Molecular mechanisms employed for platelet adhesion/aggregation are different under flow vs. static conditions: vWF initiates platelet adhesion to subendothelium under the high flow conditions typically encountered in arteries. Fibrinogen is important at low flow rates, close to stasis. The aim of this project is to investigate the hemostatic consequences of fibrinolytic therapy under in vitro conditions that mimic blood flow in a vessel. An experimental model is designed that allows both: (a) real-time imaging of fluorescent platelet thrombus formation from whole blood under defined rheologic conditions, with added agents, at localized injury sites of cultured human umbilical vein endothelial cell (HUVEC) monolayers, and (b) measurement of the extent of degradation of plasma proteins and platelet receptors. Fibrinogen degradation products and vWF multimers will be quantified by immunoblotting, and platelet receptors by flow cytometry. t-PA variants, which are more fibrin-specific or resistant to inactivation by the plasminogen activator inhibitor-1, will be tested for their interference with the thrombotic process and systemic side effects. The ability of endothelial cell (EC)-secreted t-PA to regulate thrombus formation and systemic defects will be evaluated by: (a) inhibiting EC t- PA synthesis by antisense oligonucleotides, and (b) infecting ECs with a recombinant t-PA adenovirus vector. Each agent or method of fibrinolysis with participating biochemical reactions will be computer-simulated using finite element methods to model blood flow over a thrombotic site. Concentration profiles and peak values of plasmin and fibrinogen will be correlated with thrombolytic agent and wall shear rate. The results of these studies will further our comprehension of the interconnection between thrombotic and thrombolytic processes in a dynamic flow environment. Moreover, the information obtained may form the foundation for the design of better molecules or methods of delivery that will either inhibit thrombosis or support thrombolysis with greater thrombus specificity and with less adverse effects on hemostasis.