title: 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.
authors.affiliation: Department of Pediatrics, Thrombosis and Atherosclerosis Research Institute, McMaster University, Hamilton, ON, Canada, L8L 2X2.
authors.forename: Ivan
authors.initials: I
authors.lastname: Stevic
authors.forename: Leslie R
authors.initials: LR
authors.lastname: Berry
authors.forename: Anthony K C
authors.initials: AK
authors.lastname: Chan
mesh_terms: D000925:Anticoagulants / Q000378:metabolism / Q000494:pharmacology*; D000991:Antithrombins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D005165:Factor V / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology*; D015951:Factor Xa / Q000378:metabolism / Q000494:pharmacology; D065427:Factor Xa Inhibitors; D006493:Heparin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008856:Microscopy, Fluorescence; D011516:Prothrombin / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism
chemical_list: D000925:Anticoagulants; D000991:Antithrombins; D065427:Factor Xa Inhibitors; C022475:prothrombinase complex; D005165:Factor V; D011516:Prothrombin; D006493:Heparin; D013925:Thromboplastin; D013917:Thrombin; D015951:Factor Xa