title: 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.
authors.affiliation: Therapeutic Protein Product Research Department, The Chemo-Sero-Therapeutic Research Institute, KAKETSUKEN, 1-6-1 Okubo, Kumamoto-shi, Kumamoto 860-8568, Japan. nakatomi-ya@kaketsuken.or.jp
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Nakatomi
authors.forename: Manami
authors.initials: M
authors.lastname: Tsuji
authors.forename: Soutaro
authors.initials: S
authors.lastname: Gokudan
authors.forename: Takako
authors.initials: T
authors.lastname: Hanada-Dateki
authors.forename: Teruhisa
authors.initials: T
authors.lastname: Nakashima
authors.forename: Hiroki
authors.initials: H
authors.lastname: Miyazaki
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Hamamoto
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Nakagaki
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Tomokiyo
mesh_terms: D000991:Antithrombins / Q000737:chemistry* / Q000494:pharmacology; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D005170:Factor X / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D013329:Structure-Activity Relationship
chemical_list: D000991:Antithrombins; D004792:Enzyme Precursors; D015842:Serine Proteinase Inhibitors; D012694:Serine; D005170:Factor X; D006493:Heparin; D001120:Arginine