Abstract
The nonspecific binding of heparin to plasma proteins compromises its anticoagulant activity by reducing the amount of heparin available to bind antithrombin. In addition, interaction of heparin with fibrin promotes formation of a ternary heparin-thrombin-fibrin complex that protects fibrin-bound thrombin from inhibition by the heparin-antithrombin complex. Previous studies have shown that heparin binds the E domain of fibrinogen. The current investigation examines the role of Zn(2+) in this interaction because Zn(2+) is released locally by platelets and both heparin and fibrinogen bind the cation, resulting in greater protection from inhibition by antithrombin. Zn(2+) promotes heparin binding to fibrinogen, as determined by chromatography, fluorescence, and surface plasmon resonance. Compared with intact fibrinogen, there is reduced heparin binding to fragment X, a clottable plasmin degradation product of fibrinogen. A monoclonal antibody directed against a portion of the fibrinogen αC domain removed by plasmin attenuates binding of heparin to fibrinogen and a peptide analog of this region binds heparin in a Zn(2+)-dependent fashion. These results indicate that the αC domain of fibrinogen harbors a Zn(2+)-dependent heparin binding site. As a consequence, heparin-catalyzed inhibition of factor Xa by antithrombin is compromised by fibrinogen to a greater extent when Zn(2+) is present. These results reveal the mechanism by which Zn(2+) augments the capacity of fibrinogen to impair the anticoagulant activity of heparin.
Highlights
The interaction of heparin with fibrinogen compromises its anticoagulant activity
In the presence of 2 mM CaCl2, the bulk of fibrinogen was eluted at 200 mM NaCl, and a minor fraction was eluted at 400 mM NaCl (Fig. 1A)
These findings suggest that fibrinogen binds heparin with higher affinity in the presence of Zn2ϩ
Summary
The interaction of heparin with fibrinogen compromises its anticoagulant activity. Results: Zn2ϩ promotes heparin binding to His-544 –His-545 on the fibrinogen ␣-chain. Heparin-catalyzed inhibition of factor Xa by antithrombin is compromised by fibrinogen to a greater extent when Zn2؉ is present These results reveal the mechanism by which Zn2؉ augments the capacity of fibrinogen to impair the anticoagulant activity of heparin. For inhibition of factor Xa, the conformational change in antithrombin that accompanies heparin binding is essential Because this conformational change is without catalytic effect for thrombin, heparin serves as a template onto which both the protease and inhibitor bind, thereby promoting bimolecular interaction. Thrombin bound to heparin and fibrin within this ternary complex retains its catalytic activity and is protected from inhibition [6, 7] This phenomenon likely contributes to the prothrombotic nature of thrombi and to the rethrombosis that can occur despite heparin therapy [2]. The current study reveals a novel Zn2ϩ-dependent heparin binding site localized to the ␣C domains of fibrinogen that is distinct from the previously reported heparin binding site on the -chain
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