The response of the fibrinolytic system to hypophysectomy in the rat was studied. 4 and 6 weeks after hypophysectomy the tissue plasminogen activator activity (PAA) was significantly increased in the intima of the aorta, slightly but significantly increased in the renal medulla, and significantly decreased in the vessels of the testis. No change in the PAA of the caudal vena cava, heart, lung and renal cortex was noted. Also, the tissue plasmin inhibition remained unchanged. No effect on the PAA in plasma euglobulin fractions was observed, whereas F VIII was slightly but not significantly decreased 6 weeks after hypophysectomy.
Monoclonal antibodies against four different epitopes of plasminogen (PG) were obtained and characterized for their binding towards PG using immunoblotting of native, degraded, elastase cleaved PG and alpha-2-antiplasmin complexed plasmin. One antibody (MPW1PG) recognized exclusively Glu-forms of PG and plasmin, a second antibody (MPW2PG) was bound to the kringle 1-3 fragment, one antibody (MPW7PG) recognized the kringle 4 fragment and the fourth antibody (MPW4PG) miniplasminogen. All antibodies exhibited affinities towards PG in the same order of magnitude. From these antibodies only MPW4PG inhibited plasmin action (competitive inhibitor, Ki = 4nM) and after complex formation of plasmin with alpha-2-antiplasmin the complex was not recognized. All other antibodies exhibited binding also to the respective alpha-2-antiplasmin complexed forms. The three antibodies directed against the different epitopes of the heavy chain of plasmin stimulated activation of Glu-PG by urokinase (UK), thereby mimicking the effect of lysine (KM unchanged, kcat increased) but not activation of Lys-PG. When single chain tPA was used instead of UK and PG activation was studied in the absence and presence of CNBr fragments of fibrinogen none of the three antibodies (MPW1PG, MPW2PG, MPW7PG) interfered significantly with the stimulating effect of fibrin, but the lag phase, until maximal fibrin stimulated plasmin formation occured, was reduced. In the absence of fibrin all three antibodies increased the rate of Glu-PG activation by tPA also increasing kcat values while addition of lysine rather inhibited PG activation by tPA and completely abolished the fibrin effect. These results are consistent with the plasmin induced decrease in lag time of the fibrin effect found for these antibodies. From these data it can be concluded that binding of an antibody to one of the three described domains in the heavy chain induces conformational changes in PG leading to an increased rate of activation by both activators. Interestingly, binding of these antibodies did not interfere with the fibrin effect and is different from the effect of lysine.
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