Reaction of Fibrin-Bound Thrombin with a Covalent Antithrombin-Heparin Complex Results in an Anticoagulant Surface.

Abstract Thrombin bound to fibrin is protected from inhibition by mixtures of antithrombin (AT) and heparin (H). Resistance of thrombin to reaction with AT + H when fibrin is present results from the formation of a ternary fibrin:thrombin:H complex. However, recent investigations using an antithrombin-heparin conjugate (ATH) showed that the covalent ATH complex rapidly inhibits fibrin-bound thrombin, with the thrombin-ATH product remaining attached to the fibrin. One intriguing possibility from these findings was that the heparin chain of ATH (or thrombin-ATH) on the fibrin surface might still be available to interact with fluid-phase molecules. We studied the anticoagulant activity of fibrin-bound ATH by determining the ability to catalyze the inhibition of added thrombin + AT. Excess fibrin monomer (polymerization inhibited by GPRP) was mixed with thrombin to form soluble complexes. Fibrin:thrombin was then incubated in the absence or presence of AT, H, AT + H or ATH, at various concentrations relative to the thrombin. Following treatment with anticoagulant, a vast excess of exogenous thrombin + AT was added and the ability of fibrin:thrombin-bound heparin to catalyze inhibition of the exogenous thrombin was assessed by measuring the loss of thrombin activity (chromogenic substrate assay). In the absence of heparin, reaction of exogenous thrombin + AT was essentially unaffected by the presence of soluble fibrin:thrombin. When fibrin:thrombin complex was mixed with AT + H (concentrations equimolar to thrombin), an insignificant ability to catalyze inhibition of excess thrombin by AT was observed (remaining thrombin activity = 49 +/− 4 vs 43 +/− 3 mOD/min for reaction with AT + H vs without AT + H, respectively). However, when similar concentrations of ATH were combined with fibrin:thrombin, a marked catalytic activity was seen (remaining thrombin = 34 +/− 3 mOD/min). Furthermore, the significant catalytic effect of ATH could be demonstrated over a wide range of concentrations relative to fibrin:thrombin. At these same concentrations, AT + H had little effect on exogenous thrombin + AT reactions. We conclude that although AT + H is unable to effectively inhibit fibrin-bound thrombin, covalently linked ATH not only overcomes this defect but can convert the fibrin:thrombin into a new site for anticoagulant activity to catalyze thrombin inhibition by exogenous AT. If clot-based antithrombin activity is demonstrated in vivo, we speculate that ATH may be useful for transforming thrombi from procoagulant to anticoagulant entities.