Stable complex formation between serine protease inhibitor and zymogen: coagulation factor X cleaves the Arg393–Ser394 bond in a reactive centre loop of antithrombin in the presence of heparin

Abstract

Antithrombin (AT) inhibits several blood coagulation proteases, including activated factor X (FXa), by forming stable complexes with these proteases. Herein, we demonstrate that AT forms a stable complex with zymogen factor X (FX). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion chromatography analyses showed that AT and FX formed an SDS-stable complex, which is distinct in apparent molecular mass from an FXa-AT complex, in the presence of heparin. Amino-terminal sequence analysis of the complex following SDS-PAGE under reducing conditions provided clear evidence that AT forms this complex with the heavy chain of FX, because two sequences, HGSPVDI (residues 1-7 of AT) and SVAQATS (residues 1-7 of the heavy chain of FX), were identified. Furthermore, sequence SLNPNRV, which corresponds to residues 394-400 of AT, was identified in the non-reduced FX-AT complex, indicating that FX cleaved the Arg393-Ser394 bond in a reactive centre loop of AT. Unfractionated heparin induced FX-AT complex formation more effectively than low-molecular weight heparin or AT-binding pentasaccharide, and appeared to promote complex formation mainly via a template effect. These data suggest that AT is capable of forming a stable complex with zymogen FX by acting as an inhibitor in the presence of heparin.

Crystal structure of the prothrombinase complex from the venom of Pseudonaja textilis

“…In addition, a recent report has demonstrated the formation of a human antithrombin-fX complex, suggesting that human fX is also catalytically competent to some extent. 48 The similar affinities between the components of the activated and zymogen forms of pseutarin C also support a single conserved mode of binding. We therefore consider it reasonable to conclude that the interface between fV and fX revealed in our crystal structure is the same as that formed with the active components of pseutarin C. Due to the low resolution of the structure, some details of the interaction are not observed, and the flexibility inherent in certain regions of the zymogen fX also influenced what could be observed in the structure.…”

Identification and characterization of two SERPINC1 mutations causing congenital antithrombin deficiency

“…During coagulation inhibition, RCL first identifies the substrate-binding site of the target protease, breaks its own Arg425-Ser426 peptide bond (P1-P1′), inserts the hinge of the broken RCL into the notch region of the A-β fold, and then covalently binds to Ser150 in the thrombin active center to form a stable AT-thrombin complex such that thrombin activity is lost or weakened. When the pentasaccharide sequence in the heparin molecule binds to AT, the hinge of the RCL is removed from the A-β fold, leaving the RCL completely exposed, which binds more thrombin and rapidly amplifies the anticoagulant activity of AT [51][52][53][54].…”

The mechanism by which enoxaparin sodium–high-viscosity bone cement reduces thrombosis by regulating CD40 expression in endothelial cells

“…CD40 is a member of the tumour necrosis factor receptor superfamily. The effect of enoxaparin sodium is mainly dependent on by anticoagulant factor Xa [ 25 ], which is a serine protease [ 26 ]. Serine protease exists in the digestive enzyme and complement system [ 27 , 28 ].…”