A novel variant of
antithrombin, the major
serpin inhibitor of coagulation
proteases, has been identified in a patient with early onset
thrombosis and abnormal plasma
antithrombin activity. Sequencing of the
antithrombin genes of the patient revealed that one of the two alleles was abnormal due to an in-frame deletion of the
codon for the P1
arginine residue. The abnormal
antithrombin was separated from the normal inhibitor by complexing the latter with
thrombin followed by
heparin-agarose affinity chromatography. The purified variant,
antithrombin London, was completely inactive as a
thrombin or
factor Xa inhibitor even after
heparin activation. Surprisingly, the variant bound
heparin with a K(D) reflecting an approximately 10-fold greater affinity than the normal inhibitor. Stopped-flow kinetic analysis showed that this was almost entirely due to a more favorable conformational activation of the variant than the normal inhibitor, as reflected by a decreased rate constant for reversal of the activation. Consistent with its higher than normal
heparin affinity, the inactive
antithrombin variant was a potent competitive antagonist of the
heparin-catalyzed reaction of normal
antithrombin with
thrombin but did not affect the uncatalyzed reaction. These results suggest that deletion of the
antithrombin P1 residue partially activates the
serpin by inducing strain in the reactive center loop, which destabilizes the native loop-buried state and favors the activated loop-exposed state with high
heparin affinity. The unusually severe
thrombosis associated with the heterozygous mutation may be explained by the ability of
antithrombin London to bind endogenous
heparan sulfate or
heparin molecules with high affinity and to thereby block activation of the normal inhibitor.