Abstract
A sequence-specific heparin pentasaccharide specifically activates antithrombin (AT) to inhibit factor Xa whereas longer heparins accelerate AT inactivation of thrombin by bridging both proteins in a ternary complex. To determine whether the reactive center loop IAGR sequence of AT is responsible for the factor Xa specificity of the activated inhibitor, we mutated the P2 Gly residue of this sequence to Pro, the preferred P2 residue in thrombin substrates. Fluorescence titrations showed that heparin binding and conformational activation of G392P AT were normal. Kinetic analyses revealed that the G392P mutation enhanced the basal rate of thrombin inhibition 3-fold but reduced the rate of factor Xa inhibition 4-fold. Significantly, pentasaccharide subtantially accelerated the rate of thrombin inhibition by G392P AT (5-fold) than by wild-type AT (1.7-fold). Full-length heparin similarly produced a greater enhancement of the thrombin inhibition rate with G392P AT (2700-fold) than with wild-type AT (1300-fold), in keeping with an enhanced pentasaccharide activation rate and normal ternary complex bridging. By contrast, pentasaccharide or full-length heparins accelerated factor Xa inhibition rates 2-fold less for G392P AT than for wild-type AT. Rapid kinetic studies revealed that the G392P mutation enhanced AT recognition of thrombin in the transition state for stable complex formation and not in the ternary bridging complex. These results indicate that the IAGR sequence determines the factor Xa specificity of pentasaccharide-activated AT and that this specificity can be changed by protein engineering.