Regulation Of The Fibrinolysis System And Molecular Studies Of The Plasminogen Activator DSPA

Thrombolytic diseases, such as acute myocardial infarction and stroke, are life-threatening diseases that affect millions of people each year. Therapeutic approaches to treat arterial thrombosis use plaminogen activators in combination with antiplatelet drugs and anticoagulants. Despite its tremendous clinical success, several major limitations of the therapy still need to be addressed. These include major bleeding, inadequate reperfusion, and thrombotic reocclusion. Thus, identification of new therapeutic targets and/or development of new generations of thrombolytic agents are the focal points in the field of fibrinolysis.The theme of my graduate study is to focus on regulation of fibrinolysis and molecular biology of a highly fibrin-selective plasminogen activator, DSPA, using genetic and biochemical approaches. My dissertation consists of four chapters: The chapter I describes the physiology and pathophysiology of the fibrinolysis system, focusing on regulation of fibrinolysis and progresses in thrombolytic agents. The chapter II describes the verification of negative regulatory roles of PAI-1 and TAFI on fibrinolysis in vivo using a newly developed rat model. The chapter III describes the biochemical characterization of DSPA, providing a molecular explanation for the fibrin-selectivity of DSPA. The chapter IV describes my initial attempt to develop a new-type of thrombolytic agent by targeting the highly-selective DSPA to freshly formed arterial thrombi.In the chapter I, we developed a batroxobin-induced rat lung fibrin deposition model. In this model, we observed a dramatic fibrin deposition in the lungs (elevated radioactivity) at 5 min following batroxobin administration, and the fibrin deposition in the lungs was clear up by endogenous fibrinolysis within 30 min (decreased radioactivity). We used this model to determine whether elevated levels of PAI-1 and TAFIa affect endogenous fibrinolysis. We either intravenously injected recombination PAI-1 or gave LPS to increase endogenous PAI-1 expression. In bothcases, the elevated level of PAI-1 prevented the reduction of radioactivity in the lungs at 30 min. Similarly, increased amounts of activated TAFI (TAFIa) through intravenous administration also prevented the fibrin clear -up in the lungs. These results provide direct evidence that an increase of PAI-1 and TAFIa in circulation impaired endogenous fmbinolysis, suggesting that by inhibiting PAI-1 or TAFI may be a new avenue for thrombolytic therapy.DSPA is a plasminogen activator originally isolated from the saliva of the vampire bat Desmodus rotundus. It consists of finger-, EGF-, kringle- and protease-domains, and exhibits a remarkable fibrin-specificity, which is several hundred-fold better than that of tPA. However, the molecular mechanism for such a highly fibrin-selectivity has not been elucidated, although previous studies suggest that the finger domain is involved in the fibrin selectivity. In the chapter II, I construct and express deletion mutant DSPA that only consists of the kringle- and pro tease-domains. I compare the enzymatic and fibrinolytic properties of DSPA and DSPK. In the absence of fibrin, the kinetic parameters of S2765 hydrolysis by DSPA and DSPK were similar. By contrast, in the presence of fibrin, the kcat/Km ratio of DSPA increases 3.9 fold, whereas the kcat/Km ratio of DSPK increases only 1.6 times. Furthermore, the fibrinolytic activities of DSPK decrease dramatically as compared those of DSPA. In a microtiter plate clot lysis assay, the potency of DSPK was decreased 48 times compare to that of DSPA. In 12S I-labled human plasma clot lysis assay, there was no thrombolytic activity was detected when DSPK was tested at concentrations up to 50 nM. The data indicate that DSPK retains a similar enzymatic activity towards substrate S2765 as with DSPA. However, DSPK lacks fibrin-stimulatory effects on its catalysis towards fibrin, which is in a striking contrast with DSPA. The lack of fibrin-stimulatory effect towards fibrin is not due to the inhibitory effect of PAI-1, beca...