Elimination of P1 arginine 393 interaction with underlying glutamic acid 255 partially activates antithrombin III for thrombin inhibition but not factor Xa inhibition

Abstract

The mechanism for heparin activation of antithrombin III has been postulated to involve disruption of interactions between its reactive loop P1 residue and Glu255 on the underlying protein surface. To test this hypothesis, the potential P1-constraining Arg393-Glu255 hydrogen bond and ionic interactions were eliminated by converting Glu255 to alanine. E255A and wild-type ATIIIs have identical reactive loop sequences (including the P1 and P14 residues), but differ in that Glu255-mediated, P1-constraining interactions with the underlying surface cannot form in the mutant. Relative to its wild-type parent, E255A had a 5-fold higher affinity for heparin and pentasaccharide. In the absence of cofactor, E255A exhibited a 5-fold activation of thrombin inhibition but no activation of factor Xa inhibition. Pentasaccharide addition elicited no further activation of thrombin inhibition but increased the factor Xa inhibition rate 100-fold. E255A heparin-dependent thrombin and factor Xa inhibition rates were 1000- and 2-fold faster, respectively, than pentasaccharide-catalyzed rates. Although "approximation" is the predominant factor in heparin activation of ATIII thrombin inhibition, and removal of the P1 constraint plays a distinct but minor role, the primary determinant for activation of factor Xa inhibition is the pentasaccharide-induced conformational change, with approximation making a further minor contribution, and removal of the P1 constraint playing no role at all.