Evidence for an ester bond between thrombin and heparin cofactor.

Heparin cofactor, a thrombin inhibitor, is purified from human plasma by affinity chromatography on heparin-agarose. The nature of the binding between thrombin and the inhibitor is studied by treatment of the complex with 6 M guanidinium chloride, hydroxylamine, and dilute alkali. The complex is not dissociated during gel chromatography in 6 M guanidinium chloride. This result supports an earlier proposal that formation of the complex includes the formation of a covalent bond. Treatment of dodecyl sulfate-denatured complex with hydroxylamine results in dissociation of the complex to yield free thrombin and heparin cofactor. Hydroxylamine does not dissociate the complex unless it is denatured. The complex is also dissociated in dilute sodium hydroxide (pH 12) solutions. These results indicate that the covalent bond between thrombin and the inhibitor is a carboxylic ester.