Platelet-targeted fibrinolysis enhances clot lysis and inhibits platelet aggregation.

Abstract

BACKGROUND: Although plasminogen activator therapy has been shown to reduce mortality in patients with severe myocardial infarction, several problems fuel the search for more potent and specific thrombolytic agents. METHODS AND RESULTS: To explore the effect of plasminogen activator targeting to platelets, we covalently linked urokinase that had been modified with N-succinimidyl-3-(2-pyridyldithio)propionate to the Fab' of a monoclonal antibody (7E3) that selectively binds to platelet membrane glycoprotein (GP) IIb/IIIa. In an assay measuring (as reflected by plasmin generation) a plasminogen activator's ability to bind GP IIb/IIIa immobilized on plastic, urokinase-7E3 Fab' produced 31-fold more plasmin than did urokinase (p = 0.0001). The addition of solubilized GP IIb/IIIa blocked this enhancement of plasmin generation, indicating that binding was impaired. Plasmin generation reflecting binding to immobilized intact platelets was 2.4-fold greater for urokinase-7E3 Fab' than for unconjugated urokinase (p = 0.002). In a plasma clot lysis assay, urokinase-7E3 Fab' was at least 25-fold more potent than either urokinase alone or a mixture of urokinase and 7E3 (Fab')2 (p less than 0.009), and potency could be related to platelet concentration in the clot. Ex vivo, ADP-induced platelet aggregation was inhibited by a urokinase-7E3 IgG conjugate at a concentration of 8 nM, whereas a mixture of urokinase and 7E3 (Fab')2 in equimolar amounts required 60 nM and urokinase alone required 1 microM to achieve the same effect. CONCLUSIONS: Therefore, the targeting of urokinase to the GP IIb/IIIa platelet receptor both accelerates clot lysis (when platelets are associated with a fibrin clot) and inhibits platelet aggregation.