FUNCTIONAL-PROPERTIES OF A RECOMBINANT CHIMERIC PROTEIN WITH COMBINEDTHROMBIN INHIBITORY AND PLASMINOGEN-ACTIVATING POTENTIAL

A chimeric protein (rscu-PA-40-kDa/Hir), consisting of the C-terminal amino acids 53-65 of hirudin (Hir), fused via a 14-amino-acid linker s equence to the C-terminal of a 40-kDa fragment (Ser47-Leu411) of recom binant (r) single-chain (sc) urokinase-type plasminogen activator (rsc u-PA), was produced by expression of the corresponding chimeric cDNA i n Escherichia coli cells. The thrombin inhibitory potential of purifie d rscu-PA-40-kDa/Hir was confirmed by complete inhibition of the coagu lant activity of thrombin at 20-30-fold molar excess of the chimera, a nd by the resistance of rscu-PA-40-kDa/Hir to proteolytic cleavage by thrombin. rscu-PA-40-kDa/Hir prolonged the thrombin time of normal hum an plasma in a dose-dependent way (reduction of the apparent thrombin concentration to 50% with 95 nM chimeric protein as compared to 4.7 nM hirudin), and inhibited thrombin-mediated platelet aggregation (reduc tion of the apparent thrombin concentration to 50% with 40 nM chimeric protein). The chimera had a specific activity on fibrin films of 5700 0 IU/mg as compared to 95000 IU/mg for rscu-PA. The urokinase-like ami dolytic activity of the single-chain protein was only 220 IU/mg but in creased to 169000 IU/mg after treatment with plasmin, which resulted i n quantitative conversion to a two-chain (tc) derivative (rtcu-PA-40-k Da/Hir). Corresponding values for rscu-PA were 270 and 226000 IU/mg. T he catalytic efficiencies for plasmin-mediated conversion to two-chain molecules were comparable for rscu-PA-40-kDa/Hir and rscu-PA (0.63 an d 0.65 mu M(-1). s(-1), respectively). The plasminogen-activating pote ntial of the single-chain chimera was comparable to that of rscu-PA; t he catalytic efficiencies for plasminogen activation by their two-chai n counterparts were also similar (0.55 and 0.73 mu M(-1). s(-1), respe ctively). In 2 h, 50% lysis of I-125-fibrin-labeled clots prepared fro m platelet-poor human plasma and immersed in normal plasma was obtaine d with 1.3 mu g/ml rscu-PA-40-kDa/Hir and with 0.67 mu g/ml rscu-PA, w ith corresponding residual fibrinogen levels of 74% and 87%, respectiv ely. In the absence of fibrin, 50% fibrinogenolysis in 2 h in normal h uman plasma required 2.1 mu g/ml rscu-PA, but 7.9 mu g/ml rscu-PA-40-k Da/Hir. Thus, the chimera rscu-PA-40-kDB/Hir has maintained the specif ic fibrinolytic and plasminogen acti vating activity of rscu-PA as wel l as its fibrinolytic potency in plasma: whereas it displayed a simila r or somewhat better fibrin specificity. In addition, the fibrinolytic ally active concentration in a plasma medium is severalfold lower than the concentration required for thrombin inhibition, which may limit s ystemic anticoagulant activity. Therefore, further evaluation of the t hrombolytic and antithrombotic potential of such chimeric molecules se ems to be war-ranted.