OVERALL OBJECTIVES: These are (1) to obtain sufficient information about the fibrinolytic cascade to summarize and describe it by a computer model, and (2) to determine the role of the enzyme TAFIa in down regulating the fibrinolytic cascade and connecting the fibrinolytic and coagulation cascades. BACKGROUND AND SIGNIFICANCE: The fibrinolytic cascade removes inappropriately deposited fibrin and assures an unimpeded flow of blood in the vascular system. Although many of the features of the cascade have been elucidated, many quantitative details are lacking. In addition, a new potential connection between the coagulation and fibrinolytic cascades is suggested by the discovery of TAFI (Thrombin Activatable Fibrinolytic Inhibitor). These studies should provide new information on the basic properties of the fibrinolytic cascade, its connection to coagulation, and its regulation. These studies also should provide new rationale for improving thrombolytic therapy.
SPECIFIC AIMS : (1) to determine the antifibrinolytic mechanism of TAFIa and its potential to modulate coagulation. (2) to study fibrin-dependent plasminogen activation with fluorescently labelled (S741C) plasminogen. (3) to investigate the enzymology of plasmin-catalyzed fibrin degradation. (4) to study the role of platelets in plasminogen activation and fibrinolysis. (5) to continue to develop the Lys-Speed computer model of the fibrinolytic cascade. METHODS OF PROCEDURE: The effects of TAFIa will be studied by exposing purified components to TAFIa and determining their loss of COOH Arg or Lys and quantifying their changes in functional properties. Plasminogen activation in fibrin will be measured by the fluorescence change that occurs when the plasminogen derivative is cleaved. Fibrin breakdown will be studied by perfusing fibrin with plasmin, capturing the solubilized material in a plasmin inhibitor, and analyzing released material with respect to concentration, size, protomer, and chain composition. Washed platelets and purified components will be used to determine the role of platelets in modulating plasminogen and TAFI activation, tPA inhibition, and fibrin breakdown. The computer model will be developed by incorporating new parameter values and mechanistic equations obtained from work carried out to satisfy the first four specific aims.
Showing the most recent 10 out of 247 publications
