The Role of Arg46 and Arg47 of Antithrombin in Heparin Binding

Abstract

Heparin greatly accelerates the reaction between antithrombin and its target proteinases, thrombin and factor Xa, by virtue of a specific pentasaccharide sequence of heparin binding to antithrombin. The binding occurs in two steps, an initial weak interaction inducing a conformational change of antithrombin that increases the affinity for heparin and activates the inhibitor. Arg46 and Arg47 of antithrombin have been implicated in heparin binding by studies of natural and recombinant variants and by the crystal structure of a pentasaccharide-antithrombin complex. We have mutated these two residues to Ala or His to determine their role in the heparin-binding mechanism. The dissociation constants for the binding of both full-length heparin and pentasaccharide to the R46A and R47H variants were increased 3-4-fold and 20-30-fold, respectively, at pH 7.4. Arg46 thus contributes only little to the binding, whereas Arg47 is of appreciable importance. The ionic strength dependence of the dissociation constant for pentasaccharide binding to the R47H variant showed that the decrease in affinity was due to the loss of both one charge interaction and nonionic interactions. Rapid-kinetics studies further revealed that the affinity loss was caused by both a somewhat lower forward rate constant and a greater reverse rate constant of the conformational change step, while the affinity of the initial binding step was unaffected. Arg47 is thus not involved in the initial weak binding of heparin to antithrombin but is important for the heparin-induced conformational change. These results are in agreement with a previously proposed model, in which an initial low-affinity binding of the nonreducing-end trisaccharide of the heparin pentasaccharide induces the antithrombin conformational change. This change positions Arg47 and other residues for optimal interaction with the reducing-end disaccharide, thereby locking the inhibitor in the activated state.

Lysine 114 of Antithrombin Is of Crucial Importance for the Affinity and Kinetics of Heparin Pentasaccharide Binding

“…Stoichiometries and Affinities of Heparin Binding-Stoichiometries and dissociation equilibrium constants for the binding of the different heparin forms to the antithrombin variants were measured by titrations monitored by the enhancement of intrinsic protein fluorescence accompanying the interaction, as described previously (19,20,22,25). Stoichiometries of full-length heparin binding to the N135A and N135Q variants were measured at I 0.15, pH 7.4, with antithrombin concentrations of 0.1-0.3 M. Stoichiometries of pentasaccharide or full-length heparin binding to the K114A/N135A, K114M/N135A, and K114M/ N135Q variants were determined at I 0.025, pH 6.0, and 1-2 M antithrombin.…”

“…Kinetics of Heparin Binding-The kinetics of binding of pentasaccharide or full-length heparin to the N135A and K114A/N135A antithrombin variants were analyzed under pseudo-first order conditions by monitoring the increase in protein fluorescence in an SX-17MV stopped-flow instrument (Applied Biophysics, Leatherhead, United Kingdom) as in earlier work (11,19,20,22). Experiments with both saccharides were done at I 0.075, although at pH 6.0 for the pentasaccharide and at pH 7.4 for full-length heparin.…”

“…Substitution of Arg 47 , Lys 125 , and Arg 129 led to 20 -30-, 30 -150-, and 400 -2500-fold, respectively, losses of affinity for pentasaccharide and full-length heparin (15,19,20), reflecting a larger contribution of Arg 129 than of the other two residues to the binding. Kinetic studies showed that both Arg 47 and Arg 129 are involved in the second step of heparin binding (19,20), predominantly by decreasing the reverse rate constant of this step and thus aiding in keeping antithrombin in its activated form. In contrast, they only moderately contribute to increasing the rate of induction of the conformational change.…”

“…The importance of Arg 47 and Arg 129 for the binding is consistent with the reduced heparin affinities of natural antithrombin variants in which these residues are altered (13,14). The roles of the four residues have been further investigated by mutations in recombinant variants of the in-hibitor (15)(16)(17)(18)(19)(20). Substitution of Arg 47 , Lys 125 , and Arg 129 led to 20 -30-, 30 -150-, and 400 -2500-fold, respectively, losses of affinity for pentasaccharide and full-length heparin (15,19,20), reflecting a larger contribution of Arg 129 than of the other two residues to the binding.…”

“…The roles of the four residues have been further investigated by mutations in recombinant variants of the in-hibitor (15)(16)(17)(18)(19)(20). Substitution of Arg 47 , Lys 125 , and Arg 129 led to 20 -30-, 30 -150-, and 400 -2500-fold, respectively, losses of affinity for pentasaccharide and full-length heparin (15,19,20), reflecting a larger contribution of Arg 129 than of the other two residues to the binding. Kinetic studies showed that both Arg 47 and Arg 129 are involved in the second step of heparin binding (19,20), predominantly by decreasing the reverse rate constant of this step and thus aiding in keeping antithrombin in its activated form.…”