title: Development of a new laboratory test to evaluate antithrombin resistance in plasma.
abstract: BACKGROUND
  We recently reported a variant prothrombin (p.Arg596Leu: prothrombin Yukuhashi) that confers antithrombin resistance to patients with hereditary thrombosis. To detect antithrombin resistance in plasma, we devised a laboratory test analyzing the kinetics of thrombin inactivation using antithrombin.
  
  
  METHODS
  After incubation with prothrombin activator components (phospholipids, CaCl2, and snake venom), samples were treated with excess antithrombin in the presence or absence of heparin for various time periods. Subsequently, H-D-Phe-Pip-Arg-p-nitoranilide was added and changes in absorbance/min (ΔA/min) were measured at 405 nm.
  
  
  RESULTS
  After 1 min inactivation using antithrombin and heparin, the relative residual thrombin activity of recombinant mutant prothrombin (34.3% ± 2.2%) was higher than that of the wild-type (6.3% ± 1.2 %). After 30 min without heparin, the relative residual thrombin activity of recombinant mutant prothrombin (95.8% ± 0.4%) was higher than that of the wild-type (10.1% ± 1.7%), indicating that this assay could detect antithrombin resistance of the variant 596Leu prothrombin. Moreover, warfarinized plasmas from 2 heterozygous patients with prothrombin Yukuhashi mutation clearly showed higher values of the relative residual thrombin activity than those from 5 thrombosis patients lacking the mutation in the presence or absence of heparin.
  
  
  CONCLUSIONS
  We have devised a laboratory test to detect antithrombin resistance in plasma by analyzing the kinetics of thrombin inactivation using antithrombin. This assay may be useful for detecting antithrombin resistance in plasma, even in warfarinized patients.
authors: Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya 461-8673, Japan.
authors: Moe
authors: M
authors: Murata
authors: Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya 461-8673, Japan.
authors: Akira
authors: A
authors: Takagi
authors: Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya 461-8673, Japan.
authors: Atsuo
authors: A
authors: Suzuki
authors: Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya 461-8673, Japan.
authors: Eriko
authors: E
authors: Okuyama
authors: Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya 461-8673, Japan.
authors: Yuki
authors: Y
authors: Takagi
authors: Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya 461-8673, Japan.
authors: Yumi
authors: Y
authors: Ando
authors: Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya 461-8673, Japan.
authors: Io
authors: I
authors: Kato
authors: Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya 461-8673, Japan.
authors: Yuki
authors: Y
authors: Nakamura
authors: Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya 461-8673, Japan.
authors: Takashi
authors: T
authors: Murate
authors: Department of Transfusion Medicine, Nagoya University Hospital, Nagoya 466-8550, Japan.
authors: Tadashi
authors: T
authors: Matsushita
authors: National Hospital Organization-Nagoya Medical Center, Nagoya 460-0001, Japan.
authors: Hidehiko
authors: H
authors: Saito
authors: Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya 461-8673, Japan; Department of Transfusion Medicine, Nagoya University Hospital, Nagoya 466-8550, Japan. Electronic address: kojima@met.nagoya-u.ac.jp.
authors: Tetsuhito
authors: T
authors: Kojima
mesh_terms: D000925:Anticoagulants / Q000494:pharmacology; D000991:Antithrombins / Q000378:metabolism*; D001780:Blood Coagulation Tests / Q000379:methods*; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D009154:Mutation; D011516:Prothrombin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*; D013927:Thrombosis / Q000097:blood / Q000188:drug therapy; D014859:Warfarin / Q000494:pharmacology
chemical_list: D000925:Anticoagulants; D000991:Antithrombins; D011994:Recombinant Proteins; D014859:Warfarin; D011516:Prothrombin; D006493:Heparin; D013917:Thrombin
keywords: APC; AT; Antithrombin resistance; ELISA; Laboratory test; Ox; Oxyuranus scutellatus; Oxyuranus scutellatus venom; PT-INR; Prothrombin; SD; TGA; VTE; activated protein C; antithrombin; enzyme-linked immunosorbent assay; prothrombin time-international normalized ratios; standard deviation; thrombin generation assay; venous thromboembolism