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 microM) was incubated with peptide (55 microM) 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 T1/2 of PAI-1 activity in the presence of peptide was 4 +/- 3 min compared to a control T1/2 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.
Eitzman
D T
DT
Division of Cardiology, University of Michigan Medical Center, Ann Arbor 48109, USA.
Fay
W P
WP
Lawrence
D A
DA
Francis-Chmura
A M
AM
Shore
J D
JD
Olson
S T
ST
Ginsburg
D
D
D000595:Amino Acid Sequence; D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects*; D001792:Blood Platelets / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology*; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000494:pharmacology* / Q000502:physiology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000494:pharmacology; D013997:Time Factors
D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins