Zinc²⁺ Promotes Heparin Binding to Fibrin and Subsequent Formation of Ternary Heparin-Thrombin-Fibrin Complexes.

Binding of heparin to fibrinogen and fibrin can compromise its anticoagulant function in two ways. First, binding to fibrinogen renders less heparin available to interact with antithrombin. Second, fibrin-bound heparin can sequester thrombin and protect it from inhibition by antithrombin. Protection results from the formation of a ternary heparin-thrombin-fibrin complex mediated by binary heparin-thrombin, heparin-fibrin, and thrombin-fibrin interactions. Thrombin within the ternary complex is protected from inhibition by antithrombin because exosite 2 is occupied by fibrin-bound heparin, which impairs binding of antithrombin-bound heparin. Because heparin binding to fibrin compromises the anticoagulant functions of heparin, it was of interest to further investigate this interaction. Previous studies have not considered the role of Zn²⁺, a divalent cation that binds both fibrinogen and heparin and promotes their interactions with numerous proteins. The effect of Zn²⁺ on binding of heparin to fibrin(ogen) and the subsequent effect on formation of the ternary heparin-thrombin-fibrin complex were investigated. Clots prepared with fibrinogen, ¹²⁵I-heparin, 2 mM CaCl₂, and increasing concentrations of Zn²⁺ were compacted by centrifugation and aliquots of the supernatant were removed to quantify ¹²⁵I-heparin bound to fibrin. Titration of Zn²⁺ showed a saturable 3-fold increase in ¹²⁵I-heparin bound to fibrin, with maximal binding observed at physiological Zn²⁺ concentration of 12 μM. When ¹²⁵I-heparin was titrated with fibrinogen and the samples were clotted in the presence of 12 μM Zn²⁺, ¹²⁵I-heparin bound to fibrin with 5-fold higher affinity in the presence of Zn²⁺ than its absence (K_d values of 0.3 and 1.5 μM, respectively). Comparable results were obtained regardless of whether clots were formed with thrombin or batroxobin, demonstrating that the increased affinity of heparin for fibrin was not the result of formation of thrombin-fibrin-heparin complexes. Investigating ternary complex formation, Zn²⁺ promoted a saturable 60% increase in thrombin binding to fibrin in the presence of heparin and the Zn²⁺ dose response mirrored that of the heparin-fibrin interaction. In the absence of heparin, Zn²⁺ had little effect on thrombin binding to fibrin. To further examine the effect of Zn²⁺ on heparin-fibrinogen interaction, surface plasmon resonance studies were performed. Varying concentrations of fibrinogen were applied to a biotinylated medium molecular weight heparin (6700 kDa) that was bound to a streptavidin sensor chip. Fibrinogen bound immobilized heparin with a K_d of 379 nM in the presence of EDTA. In the presence 2 mM CaCl₂ or 12 μM Zn²⁺, the K_d values were 10 nM and 8 nM, respectively. However, the mass of heparin bound was ten-fold higher in the presence of Zn²⁺ than it was with CaCl₂ or EDTA. These data reveal that Zn²⁺ promotes the interaction of heparin with fibrinogen and that this effect is maintained when fibrinogen is converted to fibrin. Because the increased heparin that binds to fibrin in the presence of Zn²⁺ has greater capacity to bind thrombin, this phenomenon may augment the protection of thrombin from inhibition by antithrombin. Therefore, the extent to which fibrin-bound thrombin is protected from inhibition is likely to be underestimated in studies that do not include physiological concentrations of Zn²⁺. This gives further emphasis for the need to develop heparin-derived anticoagulants that resist formation of the protective ternary complex.