Measurement of the heparin enhanced-antithrombin III/thrombin reaction rate in the presence of synthetic substrate.

An assay approach, designed to allow accurate measurement of the rate of thrombin inhibition by antithrombin III in the presence of heparin, is described. In essence the approach takes advantage of the fact that synthetic substrates, e.g. N-alpha-p-tosyl-L-glycyl-L-prolyl-L-arginine-p-nitroanilide, bind to the active site of thrombin, which slows the rate of reaction with antithrombin III. The rate of thrombin inhibition can be monitored continuously by change in absorbance at 400 nm or measured by end point determination. In either case, reproducible rate measurements can be made under a variety of experimental conditions not easily examined with conventional assay approaches.