Abstract
Antithrombin (AT) is a serine protease inhibitor that regulates the activity of coagulation
proteases of both intrinsic and extrinsic pathways. We identified an AT-deficient
patient with a heterozygous Thr90Ser (T90S) mutation who experiences recurrent venous
thrombosis. To understand the molecular basis of the clotting defect, we expressed
AT-T90S in mammalian cells, purified it to homogeneity, and characterized its properties
in established kinetics, binding, and coagulation assays. The possible effect of mutation
on the AT structure was also evaluated by molecular modeling. Results demonstrate
the inhibitory activity of AT-T90S toward thrombin and factor Xa has been impaired
three- to fivefold in both the absence and presence of heparin. The affinity of heparin
for AT-T90S has been decreased by four- to fivefold. Kinetic analysis revealed the
stoichiometry of AT-T90S inhibition of both thrombin and factor Xa has been elevated
by three- to fourfold in both the absence and presence of heparin, suggesting that
the reactivity of coagulation proteases with AT-T90S has been elevated in the substrate
pathway. The anticoagulant activity of AT-T90S has been significantly impaired as
analyzed in the AT-deficient plasma supplemented with AT-T90S. The anti-inflammatory
effect of AT-T90S was also decreased. Structural analysis predicts the shorter side-chain
of Ser in AT-T90S has a destabilizing effect on the structure of AT and/or the AT-protease
complex, possibly increasing the size of an internal cavity and altering a hydrogen-bonding
network that modulates conformations of the allosterically linked heparin-binding
site and reactive center loop of the serpin. This mutational effect increases the
reactivity of AT-T90S with coagulation proteases in the substrate pathway.
Keywords
antithrombin - thrombosis - thrombin - factor Xa - stoichiometry
