Probing the molecular basis of factor Xa specificity by mutagenesis of theserpin, antithrombin

The molecular basis of the substrate and inhibitor specificity of factor Xa , the serine proteinase of the prothrombinase complex, was investigated by constructing two mutants of human antithrombin (HAT) in which the reactive site loop of the serpin from the P4-P4' site was replaced with the correspo nding residues of the two factor Xa cleavage sites in prothrombin (HAT/Prot h-1 and HAT/Proth-2). These mutants together with prethrombin-2, the smalle st zymogen form of thrombin containing only the second factor Xa cleavage s ite, were expressed in mammalian cells, purified to homogeneity and charact erized in kinetic reactions with factor Xa in both the absence and presence of cofactors; factor Va, high affinity heparin and pentasaccharide fragmen t of heparin. HAT/Proth-1 inactivated factor Xa similar to3-4-fold better t han HAT/Proth-2 in either the absence or presence of heparin cofactors. In the absence of a cofactor, factor Xa reacted with the HAT/Proth-2 and preth rombin-2 with similar second-order rate constants (similar to2-3x10(2) M(-1 )s(-1)). Pentasaccharide catalyzed the inactivation rate of factor Xa by th e HAT mutants 300-500-fold. A similar 10(4)-10(5)-fold enhancement in the r eactivity of factor Xa with prethrombin-2 and the HAT mutants was observed in the presence of the cofactors Va and heparin, respectively. Factor Va di d not influence the reactivity of factor Xa with either one of the HAT muta nts. These results suggest that (1) in the absence of a cofactor, the P4-P4 ' residues of HAT and prethrombin-2 primarily determine the specificity rea ctions with factor Xa, (2) factor Va binding to factor Xa is not associated with allosteric changes in the catalytic pocket of enzyme that would invol ve interactions with the P4-P4' binding sites, and (3) similar to allosteri c activation of HAT by heparin, a role for factor Va in the prothrombinase complex may involve rearrangement of the residues surrounding the scissile bond of the substrate to facilitate its optimal docking into the catalytic pocket of factor Xa. (C) 2001 Elsevier Science BN. All rights reserved.