By Increasing the Affinity of Heparin for Fibrin, Zn²⁺ Promotes the Formation of a Ternary Heparin–Thrombin–Fibrin Complex That Protects Thrombin from Inhibition by Antithrombin

Abstract

Heparin binds fibrin and, by bridging thrombin onto fibrin, promotes the formation of a ternary heparin-thrombin-fibrin complex that protects thrombin from inhibition by antithrombin. Because thrombin binds γ(A)/γ'-fibrin, a variant with an extended γ-chain, with higher affinity than the bulk γ(A)/γ(A)-fibrin, γ(A)/γ'-fibrin affords bound thrombin more protection from inhibition by antithrombin-heparin. We examined the effect of Zn(2+) on heparin-thrombin-fibrin complex formation because Zn(2+) modulates heparin-protein interactions. Zn(2+) increased the affinity of heparin for γ(A)/γ(A)- and γ(A)/γ'-fibrin by 4.3- and 3.7-fold, respectively, but had no effect on the affinity of thrombin for either form of fibrin. In contrast, in the presence of heparin, Zn(2+) increased the affinity of thrombin for γ(A)/γ(A)-fibrin 4-fold (from a K(d) value of 0.8 to 0.2 μM) and slowed the rate of thrombin dissociation from γ(A)/γ(A)-fibrin clots. These findings suggest that Zn(2+) enhances the formation of ternary heparin-thrombin-fibrin complexes with γ(A)/γ(A)-fibrin but does not influence the already high affinity interaction of thrombin with γ(A)/γ'-fibrin. Consistent with this concept, in the presence of Zn(2+), γ(A)/γ(A)-fibrin protected thrombin from inhibition by antithrombin-heparin to a similar extent as γ(A)/γ'-fibrin. Therefore, by enhancing the binding of heparin to fibrin, physiological concentrations of Zn(2+) render fibrin-bound thrombin more protected from inhibition by antithrombin. Because fibrin-bound thrombin can trigger thrombus expansion, these findings help to explain why recurrent thrombosis can occur despite heparin treatment.

Zn2+ Mediates High Affinity Binding of Heparin to the αC Domain of Fibrinogen

“…The importance of the heparin-fibrin interaction is highlighted by the fact that higher doses of heparin are needed to achieve a therapeutic anticoagulant response in patients with venous thromboembolism than in those with acute coronary syndrome; a phenomenon that has been attributed to the larger thrombus burden in patients with venous thrombosis (47). Recently, we demonstrated that Zn 2ϩ promotes ternary complex formation, thereby augmenting the protection of fibrinbound thrombin from inhibition by antithrombin (10). The current work shows that fibrinogen also compromises heparincatalyzed inhibition of factor Xa in the presence of Zn 2ϩ .…”

“…Recently, we demonstrated that Zn 2ϩ augments ternary heparin-thrombin-fibrin complex formation and increases the protection of thrombin from inhibition by antithrombin in the presence of fibrin (10). The 4-fold increase in the affinity of heparin for fibrin in the presence of Zn 2ϩ leads to a similar elevation in the apparent affinity of thrombin for fibrin.…”

Thrombin and fibrinogen γ′ impact clot structure by marked effects on intrafibrillar structure and protofibril packing

“…6,8 The gA/g9 sequence contains a thrombin binding site, which reduces thrombin inhibition by antithrombin and heparin cofactor II. 9 On the other hand, binding to fibrinogen gA/g9 reduces the availability of thrombin in the circulation, an effect previously described as antithrombin I. 10 We previously found reduced fibrinopeptide (Fp)B but normal FpA release from gA/g9 compared with gA/gA fibrin, leading to gA/g9 fibrin networks made of thinner fibers and increased branching.…”

Reduced Plasminogen Binding and Delayed Activation Render γ′-Fibrin More Resistant to Lysis than γA-Fibrin

“…Although thrombin utilizes exosite 1 to bind to the E-domains of ␥ A -Fg and ␥Ј-Fg, the interaction of thrombin with the ␥Ј-chain is mediated by exosite 2, a negatively charged domain that lies opposite to exosite 1 and serves as the heparin binding site on thrombin. By simultaneously binding to the E-domain and the ␥Ј-chain via exosite 1 and exosite 2, respectively, thrombin forms a bivalent interaction with ␥Ј-Fg that heightens its affinity relative to the univalent interaction of thrombin with ␥ A -Fg and renders thrombin more resistant to inhibition by antithrombin and heparin cofactor II (9). The unique interaction of thrombin with ␥Ј-Fg not only attenuates the reactivity of the enzyme with its inhibitors, but also with its substrates, thereby reducing the activity and availability of thrombin.…”