Journal Article

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

Ivan Stevic, Leslie R. Berry and Anthony K.C. Chan

in The Journal of Biochemistry

Published on behalf of The Japanese Biochemical Society

Volume 152, issue 2, pages 139-148
Published in print August 2012 | ISSN: 0021-924X
Published online April 2012 | e-ISSN: 1756-2651 | DOI: http://dx.doi.org/10.1093/jb/mvs039
Mechanism of inhibition of the prothrombinase complex by a covalent antithrombin–heparin complex

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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 k2 values for inhibition of Xa. Fluorescent microscopy was conducted to evaluate inhibitor–prothrombinase interactions. The k2 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 k2, removal of vesicles increased k2 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.

Keywords: antithrombin; covalent antithrombin–heparin complex; heparin; prothrombinase complex

Journal Article.  7285 words.  Illustrated.

Subjects: Biochemistry

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