Time course of PAI-1 cleavage by thrombin and of thrombin/PAI-1 complex formation. Thrombin (600 nmol/L) and PAI-1 (3,600 nmol/L) were incubated at 37°C as described under the Experimental Procedures. At the indicated time points aliquots were withdrawn and subjected to 10% (wt/vol) SDS-polyacrylamide gel electrophoresis, stained with Coomassie Brilliant Blue (A). The indicated reaction products were quantified by densitometry and the amounts obtained were expressed as percentage of maximum intensity observed for each individual band (B). The lines represent the outcome of nonlinear regression analysis, according to a single-exponential reaction mechanism. The rate of disappearance of the thrombin band yields a second-order rate constant for inhibition of 9.8 × 10² L mol⁻¹ s⁻¹. (A) “A” represents PAI-1; “B” represents thrombin. Times were as indicated (min). (B) (○), thrombin; (▪), cleaved PAI-1; (▴), PAI-1/thrombin complex.