Detection of a new heparin-dependent inhibitor of thrombin in human plasma

We have demonstrated that human plasma contains a heparin-dependent inhibitor of thrombin that is distinguishable from antithrombin III (AT III). When a 1:50 dilution of plasma was incubated with ≥0.01 U/ml heparin and 1 U/ml ¹²⁵I-thrombin, the labeled thrombin B-chains became incorporated into the two complexes of M(r)-96,000 and M(r)-85,000 that were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and β-mercaptoethanol. Neither complex was detectable at heparin concentrations <0.01 U/ml. When a limiting amount of ¹²⁵I-thrombin was present, the proportion of radioactivity incorporated into each of the two complexes varied with the heparin concentration. Thus, the M(r)-85,000 complex predominated at 0.01-5 U/ml heparin, whereas the M(r)-96,000 complex predominated at 5-100 U/ml heparin. The M(r)-85,000 complex reacted with antibodies to human AT III and comigrated with the purified thrombin-AT III complex. The M(r)-96,000 complex did not react with antibodies to AT III or to α1-antitrypsin, and it was detected in normal quantities after incubating ¹²⁵I-thrombin with plasma immunodepleted of AT III, α2-antiplasmin, α2-macroglobulin, C1 inactivator, α1-antichymotrypsin, or inter-α-trypsin inhibitor. The protein that combines with thrombin to form the M(r)-96,000 complex was estimated to be present at a minimum concentration of 90±26 μg/ml (mean ±SD) in normal plasma. We conclude that the protein is not identical to any of the known plasma protease inhibitors and that at relatively high heparin concentrations in vitro it reacts with thrombin more rapidly than does AT III.