PEPTIDE-MEDIATED INACTIVATION OF RECOMBINANT AND PLATELET PLASMINOGEN-ACTIVATOR INHIBITOR-1 IN-VITRO

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator, is an important regulator of the blood fibrinolytic system, Elevated plasma levels of PAI-1 are associated with thrombosis, and high levels of PAI-1 within platelet-rich clots contribute to their resistance to lysis by t-PA, Consequently, strategies aimed at inhibition of PAI-1 may prove clinically useful, This study was designed to test the hypothesis that a 14-amino acid peptide, corresponding to the PAI-1 reactive center loop (residues 333-346), can rapidly inhibit PAI-1 function, PAI-1 (0.7 mu M) was incubated,vith peptide (55 mu M) at 37 degrees C, At timed intervals, residual PAI-1 activity was determined by addition of reaction mixture samples to t-PA and chromogenic substrate, The T-1/2 of PAI-1 activity in the presence of peptide was 4+/-3 min compared to a control T-1/2 of 98+/-18 min, The peptide also inhibited complex formation between PAI-1 and t-PA as demonstrated by SDS-PAGE analysis, However, the capacity of the peptide to inhibit PAI-1 bound to vitronectin, a plasma protein that stabilizes PAI-1 activity, was markedly attenuated, Finally, the peptide significantly enhanced in vitro lysis of platelet-rich clots and platelet-poor clots containing recombinant PAI-1, These results indicate that a 14-amino acid peptide can rapidly inactivate PAI-1 and accelerate fibrinolysis in vitro, These studies also demonstrate that PAI-1 function can be directly attenuated in a physiologic setting and suggest a novel approach for augmenting fibrinolysis in vivo.