Zinc Enhances the Protection of Fibrin-Bound Thrombin from Antithro Inhibition.

Introduction: Unfractionated heparin (UFH) binds to thrombin (IIa), which bridges IIa to antithrombin (AT) and promotes inhibition. UFH also binds fibrin (Fn) and, by bridging IIa onto Fn, promotes the formation of a ternary UFH-IIa-Fn complex that protects IIa from AT inactivation. Within this ternary complex, UFH binds to exosite 2 on IIa, whereas Fn binds to exosite 1. γ′-fibrinogen (γ′-Fg), which comprises about 10% of circulating Fg, is a variant with an extended γ-chain. Because IIa binds to the γ′ chain via exosite 2, IIa binds γ′-Fn with higher affinity than it does to the bulk γ_A-Fn. Zinc (Zn) binds both Fn and UFH. Accordingly, we examined the possibility that Zn promotes the formation of the UFH-IIa-Fn complex, thereby increasing the protection of IIa from AT inhibition.

Methods: The affinity of ¹²⁵I-labelled UFH for γ _A-Fn was obtained by measuring unbound UFH in the supernatant after γ _A-fg was clotted with IIa in the absence or presence of Zn. Similarly, the affinity of IIa for γ^A- or γ′-Fn was determined by quantifying the chromogenic activity of unbound IIa in the supernatants of clots prepared from γ^A- or γ′-Fg with or without Zn and UFH. Rate constants of IIa inhibition by AT were determined under pseudo-first order conditions in the absence or presence of soluble fibrin monomer (Fm) prepared from γ^A- or γ′-Fg.

Results: UFH binds γ^A-fibrin with 4-fold higher affinity in the presence of 12.5 μM Zn than in its absence (Kd values of 0.4 and 1.5 μM, respectively). Therefore, Zn enhances the affinity of UFH for γ^A-Fn. Likewise, the affinity of IIa for γ^A-Fn was 3-fold higher in the presence of UFH than in its absence (Kd values of 0.8 and 2.6 μM, respectively). Zn produced an additional 4-fold increase in affinity in the presence of UFH (Kd value of 0.2 μM), but had no effect on IIa binding in its absence. These data suggest that, by heightening the affinity of UFH for γ^A-Fn, Zn enhances the formation of a UFH-IIa complex with γ^A-Fn. In contrast, IIa binds γ′-Fn with high affinity (Kd 0.1 μM), and this interaction was unaffected by UFH or Zn. The heparin-catalyzed rate of IIa inhibition by AT was 2 ± 0.2 × 10⁸ M⁻¹ min⁻¹. Using a concentration of γ^A-Fm that reduced this rate by 25%, the addition of Zn produced an additional 30% reduction in the rate. Contrarily, an equivalent concentration of γ′-Fm reduced the rate by 60%, and there was no further reduction with the addition of Zn.

Conclusion: Physiological concentrations of Zn enhance the formation of the UFH-IIa-Fn complex with γ^A-Fn, thereby increasing the protection of Fn-bound IIa from inhibition by AT. Zn has no effect on ternary complex formation with γ′-Fn, which already binds IIa with high affinity and affords IIa with greater protection than γ^A-Fn. These findings suggest that the extent of protection of Fn-bound IIa from inhibition by the AT-UFH complex is greater than previously suspected. Because Fn-bound IIa can trigger thrombus growth, its resistance to inhibition helps to explain the limitations of UFH in patients with arterial thrombosis.