Mechanism of inhibition of the prothrombinase complex by a covalent antithrombin-heparin complex

Abstract

Factor-Xa assembly into the prothrombinase complex decreases its availability for inhibition by antithrombin + unfractionated heparin (AT + UFH). We have developed a novel covalent antithrombin-heparin complex (ATH), with enhanced anticoagulant actions compared with AT + UFH. The present study was performed to extend understanding of the anticoagulant mechanisms of ATH by determining its inhibition of Xa within the critical prothrombinase. Discontinuous inhibition assays were performed to determine final k(2) values for inhibition of Xa. Fluorescent microscopy was conducted to evaluate inhibitor-prothrombinase interactions. The k(2) for inhibition of prothrombinase versus free Xa by AT + UFH was lower, whereas for ATH were much higher. Relative to intact prothrombinase, rates for Xa inhibition by AT + UFH in complexes devoid of prothrombin/vesicles/factor-Va were higher. For ATH, exclusion of prothrombin decreased k(2), removal of vesicles increased k(2) and exclusion of factor-Va gave no effect. While UFH may displace Xa from prothrombinase, Xa is detained within prothrombinase during ATH reactions. We confirm prothrombinase hinders inhibitory action of AT + UFH, whereas ATH is less affected with prothrombin being a key component in the complex responsible for the opposing effects. Overall, the results suggest that covalent linkage between AT-heparin assists access and neutralization of complexed Xa, with concomitant inhibition of prothrombinase function compared with conventional non-conjugated heparin.

Inhibition of the prothrombinase complex on red blood cells by heparin and covalent antithrombin-heparin complex

“…We also observed a significant reduction in Xa inhibition when factor Xa was incorporated in the prothrombinase complex for AT þ UFH reactions. This is consistent with previous findings using vesicle or platelet surfaces (18,19,23). The decrease in Xa inhibition is often referred to as the protective effect, where certain components of the complex protect the catalytically active enzyme from inhibition by anticoagulants (38).…”

“…Many studies on prothrombinase inhibition by AT þ heparin, or heparin derivatives such as low molecular weight heparin (LMWH) and pentasaccharide (fondaparinux), have been performed using platelets and vesicles (18,19,23). In these previous studies, it was noticed that UFH yielded the highest inhibition rates, followed by LMWH then fondaparinux.…”

“…k 2 values for inhibition of Xa AE RBC-prothrombinase complex by AT þ UFH versus ATH were determined by performing discontinuous second-order rate constant assays under pseudo first-order conditions, as described earlier (23). All rate experiments were performed at physiological temperature (37 C) and a minimum inhibitor:enzyme ratio of 10:1 was maintained for all reactions.…”

“…All rate experiments were performed at physiological temperature (37 C) and a minimum inhibitor:enzyme ratio of 10:1 was maintained for all reactions. In addition, we have previously determined that maximal inhibition of Xa by AT occurs in the presence of 10-fold mole ratio of heparin to AT, or 30 nM AT þ 300 nM UFH (23). For rate experiments involving the inhibition of free-Xa by AT þ UFH and ATH, the reaction protocol was as follows: First, 10 ml aliquots of TSP buffer (0.02 M Tris HCl þ 0.15 M NaCl þ 0.6% polyethylene glycol, pH 7.4) was added to six separate wells of a 96-well flat bottom microtiter plate (Fisher, Nepean, ON, Canada), followed by the addition of 10 ml aliquots containing 24 nM Xa, 32 mM CaCl 2 and 320 mM Pefabloc-TH in TSP buffer to all the wells and incubated for 3 min.…”

“…Thus, to overcome these limitations, Chan et al have developed a covalent antithrombinheparin complex (ATH) with high anticoagulant activity compared with heparin (21,22). Recently, we have investigated comparison of non-covalent ATþUFH versus ATH to inhibit Xa in prothrombinase composed of synthetic vesicles (23) or platelets (24) and have noticed greater inhibition of Xa by the ATH. Much of the work with prothrombinase and use of anticoagulants has been performed on the native platelet system or synthetic vesicles (25,26).…”