Bivalent Binding to γA/γ′-Fibrin Engages Both Exosites of Thrombin and Protects It from Inhibition by the Antithrombin-Heparin Complex

Abstract

Thrombin exosite 1 binds the predominant ␥ A /␥ A -fibrin form with low affinity. A subpopulation of fibrin molecules, ␥ A /␥-fibrin, has an extended COOH terminus ␥-chain that binds exosite 2 of thrombin. Bivalent binding to ␥ A /␥-fibrin increases the affinity of thrombin 10-fold, as determined by surface plasmon resonance. Because of its higher affinity, thrombin dissociates 7-fold more slowly from ␥ A /␥-fibrin clots than from ␥ A /␥ A -fibrin clots. After 24 h of washing, however, both ␥ A /␥-and ␥ A /␥ A -fibrin clots generate fibrinopeptide A when incubated with fibrinogen, indicating the retention of active thrombin. Previous studies demonstrated that heparin heightens the affinity of thrombin for fibrin by simultaneously binding to fibrin and exosite 2 on thrombin to generate a ternary heparinthrombin-fibrin complex that protects thrombin from inhibition by antithrombin and heparin cofactor II. In contrast, dermatan sulfate does not promote ternary complex formation because it does not bind to fibrin. Heparin-catalyzed rates of thrombin inhibition by antithrombin were 5-fold slower in ␥ A /␥-fibrin clots than they were in ␥ A /␥ A -fibrin clots. This difference reflects bivalent binding of thrombin to ␥ A /␥-fibrin because (a) it is abolished by addition of a ␥-chaindirected antibody that blocks exosite 2-mediated binding of thrombin to the ␥-chain and (b) the dermatan sulfate-catalyzed rate of thrombin inhibition by heparin cofactor II also is lower with ␥ A /␥-fibrin than with ␥ A /␥ A -fibrin clots. Thus, bivalent binding of thrombin to ␥ A /␥-fibrin protects thrombin from inhibition, raising the possibility that ␥ A /␥-fibrin serves as a reservoir of active thrombin that renders thrombi thrombogenic.

The pleiotropic role of the fibrinogen γ′ chain in hemostasis

“…Thrombin bound to fibrin was shown to remain functionally active. 32 Despite the higher affinity to ␥A/␥Ј fibrin, the release of FpA from ␥A/␥Ј fibrinogen by thrombin was similar compared with ␥A/␥A fibrin, 32 a finding supported by our data. 33 In addition, Fredenburgh et al 32 showed that binding of thrombin to the ␥Ј chain offers thrombin protection from inhibition by heparin in complex with heparin cofactor II or antithrombin, confirming a previous report by Pospisil et al 34 As exosite II provides the site of interaction for both heparin and the ␥Ј chain, it is probable that competitive inhibition for exosite II plays a role in the mechanism underpinning this.…”

“…32 Despite the higher affinity to ␥A/␥Ј fibrin, the release of FpA from ␥A/␥Ј fibrinogen by thrombin was similar compared with ␥A/␥A fibrin, 32 a finding supported by our data. 33 In addition, Fredenburgh et al 32 showed that binding of thrombin to the ␥Ј chain offers thrombin protection from inhibition by heparin in complex with heparin cofactor II or antithrombin, confirming a previous report by Pospisil et al 34 As exosite II provides the site of interaction for both heparin and the ␥Ј chain, it is probable that competitive inhibition for exosite II plays a role in the mechanism underpinning this. 21,35,36 So although on one hand the ␥Ј chain is responsible for antithrombin I activity and reduction of intrinsic activation, FpA release on the other hand is normal and thrombin is afforded protection against serpin inactivation in the presence of glycosaminoglycans.…”

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

“…Fibrinogen and fibrinogen fragment X were prepared and characterized as described (13)(14)(15). Human antithrombin was from Affinity Biologicals, Inc. (Ancaster, ON, Canada).…”

Fibrinogen-elongated γ Chain Inhibits Thrombin-induced Platelet Response, Hindering the Interaction with Different Receptors

“…Platelets are major players of arterial thrombus formation, as also demonstrated by the clinical efficacy of anti-platelet agents in cardiovascular prevention. The fibrinogen ␥Ј chain, through its ability to bind to thrombin, might enhance the amount of clot-bound thrombin, known to be active in the presence of the heparin-antithrombin complex, and thus scarcely inactivated by traditional anticoagulants (heparins, indirect Factor Xa inhibitors) (3). Thus, clot-bound, active thrombin may represent a storage pool of the enzyme, facilitating arterial thrombus formation and growth.…”

“…fibrin, binds with a considerable specificity thrombin, so that in the early studies fibrin was termed antithrombin I (2). Thrombin has a divalent interaction with two classes of binding sites on fibrin, one of low affinity in the E domain and the other of high affinity in the D domain of fibrin(ogen) molecules (3). Binding of thrombin to fibrinogen involves sequences of both A␣ and B␤ chain, which contain recognition sites in the fibrinogen E domain.…”