Inhibition of the prothrombinase complex on phospholipid vesicles, activated platelets, and red blood cells by a covalently-linked antithrombin-heparin complex

Abstract

<p>Prothrombinase is composed of a proteinase, factor Xa (Xa), its cofactor Va (Va), Ca²⁺ and a zymogen, prothrombin (II), assembled on a phospholipid surface. During coagulation, prothrombinase accelerates II to thrombin conversion; but during anticoagulation, it protects the proteinase from inhibition by antithrombin (AT) ± unfractionated heparin (UFH). Although the degree of Xa protection by prothrombinase varies according to the reports in literature, moderate to significant protective effects have been consistently reported by most investigators. To overcome the limitations of UFH, our laboratory has developed a covalent complex of AT and UFH (ATH) with superior anticoagulant responses. To further understand the mechanisms of enhanced anticoagulant activity of ATH, we proceeded to study inhibition of the prothrombinase complex<em> </em>on synthetic vesicles, activated platelets and red blood cells (RBCs). Using discontinuous inhibition assays, we determined the rate of inhibition of prothrombinase-complexed Xa compared to control Xa. With synthetic vesicles, Xa was protected from inhibition by AT+UFH when in prothrombinase, while only a mild protective effect was observed with ATH. Omission of various components of the prothrombinase led to a reduction in Xa protection for AT+UFH. However, an increased Xa protection against ATH was observed when II was omitted from the prothrombinase. In comparison to the synthetic vesicle system, activated platelets showed a similar trend for protection of Xa in reactions involving prothrombinase ± components, while no protection of Xa was observed for ATH reactions. Alternatively, RBCs showed differences relative to vesicles in that increased protection of Xa occurred with omission of II and Va for AT+UFH, whereas omission of Va increased protection against ATH inhibition. In addition, ATH had improved inhibition of thrombin generation, fibrin formation and plasma coagulation compared to AT+UFH. Studies of fluorescently labelled Xa and inhibitors detailed binding interactions with prothrombinase subunits. Overall, the results suggest that a covalent linkage between AT and heparin improves inactivation of prothrombinase complexed-Xa leading to down-regulation of prothrombinase function.</p>