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J. Biol. Chem., Vol. 283, Issue 5, 2470-2477, February 1, 2008

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Bivalent Binding to _A/'-Fibrin Engages Both Exosites of Thrombin and Protects It from Inhibition by the Antithrombin-Heparin Complex^*

From the Henderson Research Centre and McMaster University, Hamilton, Ontario L8V 1C3, Canada

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 heparin-thrombin-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 '-chain-directed 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.

Received for publication, September 13, 2007 , and in revised form, November 29, 2007.

^* This work was supported in part by Canadian Institutes of Health Research Grants MOP 3992 and CTP 79846, Heart and Stroke Foundation of Ontario Grants T4729 and T4730, and funds from the Ontario Research and Development Challenge Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a Career Investigator Award from the Heart and Stroke Foundation of Ontario, Heart and Stroke Foundation of Ontario/J. Fraser Mustard Endowed Chair in Cardiovascular Research, and Canada Research Chair (Tier 1) in Thrombosis. To whom correspondence should be addressed: 711 Concession St., Hamilton, Ontario L8V 1C3, Canada. Tel.: 905-574-8770; Fax: 905-575-2646; E-mail: jweitz{at}thrombosis.hhscr.org.

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