Low-affinity heparin stimulates the inactivation of plasminogen activator inhibitor-1 by thrombin.
The influence of heparin on the reaction between thrombin and plasminogen activator inhibitor-1 (PAI-1) has been examined. With a 50-fold excess of PAI-1, the rate constant for the inhibition of thrombin was 458 mol/L-1s-1, which increased to 5,000 mol/L-1s-1 in the presence of 25 micrograms/mL unfractionated heparin or heparin with low affinity for antithrombin. The effect of low affinity heparin was then examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using close to equimolar concentrations of reactants. Thrombin and PAI-1 formed a stable stoichiometric complex in the absence of heparin, which did not dissociate after the addition of 25 micrograms/mL low-affinity heparin. In contrast, when low-affinity heparin was added at the beginning of the reaction, there was an initial increase in PAI-1-thrombin complex formation, but this was rapidly followed by substantial proteolytic cleavage of unreacted PAI-1 and of the thrombin-PAI-1 complex. The idea that the relative concentrations of thrombin and PAI-1, and the presence of low affinity heparin, could influence the products of the reaction was examined in detail. Quantitative zymographic analysis of tissue plasminogen activator and PAI-1 activities and chromogenic substrate assay of thrombin activity showed that low-affinity heparin stimulated the inactivation of PAI-1 by an equimolar amount of thrombin, but caused only a minimal stimulation of thrombin inhibition. It is concluded that low-affinity heparin stimulates thrombin inhibition when PAI-1 is in excess, but, unexpectedly, that low-affinity heparin enhances PAI-1 inactivation when thrombin is equimolar to PAI-1.
Patston
P A
PA
Department of Medicine, Vanderbilt University, Nashville, TN 37232-2287.
Schapira
M
M
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides; D017395:Plasminogen Activator Inhibitor 1 / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013379:Substrate Specificity; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D009842:Oligopeptides; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D006493:Heparin; D013917:Thrombin; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator