Inhibition of the Thrombin-Thrombomodulin by a Covalent Antithrombin-Heparin.

Endothelial thrombin·thrombomodulin (IIa-TM) complex is capable of activating protein C anticoagulant. In vivo, TM can exist in two forms: one with a chondroitin sulfate (CS) (acidic) and one without (non-acidic). TM binding has been shown to reduce the rate of IIa inhibition by antithrombin (AT) in the presence of unfractionated heparin (UFH), especially when the TM possesses a glycosaminoglycan chain. We have recently developed a covalent antithrombin-heparin (ATH) with high potency for inhibition of IIa bound to surfaces. In the present study we examined the role of the CS chain in the inhibition of IIa-TM complex by the novel anticoagulant, ATH, in comparison to AT + UFH. For this study, discontinuous second order rate constant (k₂) inhibition assays of IIa-TM were performed at 37°C. IIa was incubated with TM for 30 min and inhibited using either 20nM AT + varying concentrations (0–6000nM) of UFH or a constant concentration of 10nM ATH. Residual IIa activity was measured at 405nm after stopping the reaction using a solution of polybrene and chromogenic substrate (S-2238). Second order rate constants (mean +/− SEM; n≥5) were calculated from plots of IIa-TM activity vs. time and differences determined using two-sample t-tests. Comparison of the second order rate constants for inhibition of IIa, IIa-TM(−CS), or IIa-TM(+CS) by AT + UFH (2.015 × 10⁸ +/− 7.079 × 10⁶ M⁻¹min⁻¹, 1.633 × 10⁸ +/− 8.922 × 10⁶ M⁻¹min⁻¹, and 5.248 × 10⁷ +/− 5.693 × 10⁶ M⁻¹min⁻¹, respectively) showed that the presence of TM reduced the rate of inhibition (with respect to IIa alone; p=0.012, p<0.001, respectively). Moreover, the presence of a CS on the TM inhibited IIa neutralization to a greater degree than when the CS is absent (p<0.001). Furthermore, ATH was a much faster inhibitor of IIa, IIa-TM(−CS), and TM(+CS) (1.486 × 10⁹ +/− 1.193 × 10⁸ M⁻¹min⁻¹, 1.370 × 10⁹ +/− 1.246 × 10⁸ M⁻¹min⁻¹, and 8.869 × 10⁸ +/− 1.246 × 10⁸ M⁻¹min⁻¹, respectively) compared to AT + UFH (p<0.001). CS removal by chondroitin ABC lyase (ABCase) showed that the rate of IIa-TM(+CS) inhibition significantly increased for both AT + UFH and ATH, while no significant change due to ABCase treatment was found for the rate of inhibition of IIa-TM(−CS) by either AT + UFH or ATH. We speculate that during IIa-TM(+CS) inhibition by ATH, the covalently linked heparin moiety may be able to repel (rotate) the CS chain in order for ATH to interact with the IIa-TM complex. In the case of AT + UFH, lack of permanent UFH linkage to AT may allow the CS to displace the UFH chain from binding both AT and IIa. Steric hindrance by CS chains remains a significant factor since the k₂ values were lower for the AT + UFH or ATH reactions with IIa-TM(+CS) compared to those with IIa-TM(−CS). Given the rapid reaction rate with free or bound IIa, ATH significantly limits availability of IIa for coagulant or anticoagulant functions.