The overall goal of this project is the determination of molecular mechanisms of the physical process of fibrinolysis. Although much is known about the biochemistry of fibrinolysis, much less is known about the changes in fibrin clot structure as fibers are lysed and the appearance of the pieces removed by digestion. Our preliminary results have shown that fibrinolysis of fibrin clots proceeds by transverse cutting of fibers, rather than digestion of fibers from the outside-in, and removal of large pieces held together by non-covalent forces. Confocal microscopy has revealed complex changes that take place at the lysis front. The first specific aim tests the hypothesis that fibrinolysis proceeds by a series of actions at the lysis front including transverse cutting of fibers, but these processes are affected by the conditions of lysis, including the nature of the enzyme/activator and the mode of its introduction, and the presence of other proteins, such as Lp(a), PAI-1, and TAFI. One of the principal approaches will be to follow digestion in real time using confocal microscopy in both reflectance and fluorescence modes. We also will examine the digested clot surface by scanning electron microscopy. The cleaved pieces removed from the clots will be characterized by transmission electron microscopy of negatively contrasted or rotary shadowed specimens. The process of intrinsic fibrinolysis, in which clots are formed in the presence of plasminogen and tPA so that they are digested internally, will be studied by confocal microscopy and by following turbidity and rigidity of the clots as a function of time. The second specific aim tests the hypothesis that the rate and nature of fibrinolysis is dependent on clot structure, but is not simply a function of fiber diameter. The overall approach will be to make clots with different structures, characterize them quantitatively and then to measure the rates of fibrinolysis using several different methods and to measure the binding of plasminogen and tPA and the activation of plasminogen at the lysis front. The third specific aim tests the hypothesis that the influence of platelet aggregation on fibrin structure and properties has a major impact on events at the lysis front. The fiber meshwork is much denser in the vicinity of platelet aggregates and the fibers are more oriented around the platelets. In addition, plasminogen activator inhibitor-1 is released from platelets and binds to fibrin in the vicinity of platelet aggregates, retarding lysis in these areas. Although these phenomena are well known, the physical processes involved and the local effects have not been studied. The physical process of lysis of platelet-rich clots will also be studied in the presence of abciximab, an inhibitor of interactions between fibrin(ogen) and the platelet integrin, alphaIIbbeta3, the integrin that binds fibrinogen in platelet aggregation. The results of these studies will help us to understand molecular mechanisms of fibrinolysis, which may have clinical implications for the treatment and prevention of thrombotic disorders.
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