The significance of fibrin binding by plasminogen activator inhibitor 1 for the mechanism of tissue-type plasminogen activator-mediated fibrinolysis.
The specific, reversible interaction between plasminogen activator inhibitor 1 (PAI-1) and intact fibrin polymers was studied using both purified components and isolated activated platelets as a source of PAI-1. A key reagent in these experiments is a PAI-1 mutant, having its P1 reactive center residue arginine replaced by methionine (PAI-1 R346M). The second-order association rate of PAI-1 R346M with tissue-type plasminogen activator is over 10,000-fold lower than that of wild-type PAI-1, whereas the ability of the variant to bind to fibrin is unaltered. Competition experiments demonstrated that PAI-1 R346M is equally effective as wild-type PAI-1 in displacing 125I-labeled PAI-1 from fibrin. Fibrinolysis, mediated by tissue-type plasminogen activator, is inhibited in a dose-dependent manner by purified PAI-1. The inhibition can be relieved in a dose-dependent manner by PAI-1 R346M, presumably due to displacement of wild-type PAI-1 by PAI-1 R346M. Perfusion studies, using platelet-rich clots, revealed that the incorporation of PAI-1 R346M dose dependently decreased the 50% clot lysis time. These data indicate that PAI-1 R346M displaces fibrin-bound, endogenous PAI-1 released from activated platelets. Implications to manipulate PAI-1 activity for the management of clinical complications, in particular reocclusion after thrombolytic therapy, are discussed.
Stringer
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HA
Department of Biochemistry, University of Amsterdam, The Netherlands.
Pannekoek
H
H
D000595:Amino Acid Sequence; D001120:Arginine; D001665:Binding Sites; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism*; D005342:Fibrinolysis; D006801:Humans; D007700:Kinetics; D008715:Methionine; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
D017395:Plasminogen Activator Inhibitor 1; D005337:Fibrin; D001120:Arginine; D008715:Methionine; D010959:Tissue Plasminogen Activator