Binding of high affinity heparin to antithrombin III. Stopped flow kinetic studies of the binding interaction.

The kinetics of high affinity heparin binding to human antithrombin III has been studied by stopped flow fluorimetry, using the 40% antithrombin fluorescence enhancement resulting from this interaction. At mu 0.15, pH 7.4, and 25 degrees C, the observed pseudo-first order rate constant varies hyperbolically with heparin concentration with a limiting rate constant of 440 +/- 90 s-1, demonstrating that heparin binding is a two-step process involving a conformational change in antithrombin III. An identical dependence is produced when antithrombin is varied, consistent with a symmetrical mechanism in which heparin binding induces a conformational change in antithrombin rather than perturbing an equilibrium between two conformational states of the protein. The rate constant for dissociation of the antithrombin-heparin complex is 1.1-1.5 s-1 at mu 0.15, as determined from the ordinate intercept at low heparin concentrations or by dissociation of the antithrombin-heparin complex with iodide. Observation of a single pseudo-first order binding rates over a 400-fold heparin concentration range with no detectable lags is compatible with the initial binding step being in rapid equilibrium with a KD of 4.3 +/- 1.3 X 10(-5) M at mu 0.15. Variation in ionic strength primarily affects the KD for the initial binding step with little effect on the conformational change rate constants, implying that binding involves ionic interactions. Calculation of the overall dissociation equilibrium constant from these rate parameters agrees with the directly determined value of 7.2 +/- 1.9 X 10(-8) M at mu 0.15. A major function of the conformational change is, thus, to increase the affinity of heparin for antithrombin III greater than 300-fold. The implications of these findings for the mechanism of the heparin-catalyzed inhibition of coagulation proteases by antithrombin III are discussed.