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Thromb Res. 2014 Feb;133(2):293-8. doi: 10.1016/j.thromres.2013.11.021. Epub 2013 Dec 1.

Development of a new laboratory test to evaluate antithrombin resistance in plasma.

Author information

  • 1Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya 461-8673, Japan.
  • 2Department of Transfusion Medicine, Nagoya University Hospital, Nagoya 466-8550, Japan.
  • 3National Hospital Organization-Nagoya Medical Center, Nagoya 460-0001, Japan.
  • 4Department 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.

Abstract

INTRODUCTION:

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.

MATERIALS AND 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.

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

PMID:
24325876
DOI:
10.1016/j.thromres.2013.11.021
[PubMed - indexed for MEDLINE]
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