Role of the γ-carboxyglutamic acid domain of activated factor X in the presence of calcium during inhibition by antithrombin–heparin

doi:10.1111/j.1538-7836.2004.00796.x

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Wiley InterScience

Journal of Thrombosis and Haemostasis

Volume 2 Issue 7, Pages 1127 - 1134

Published Online: 23 Jun 2004

The official journal of the International Society on Thrombosis and Haemostasis

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ORIGINAL ARTICLE

Role of the γ-carboxyglutamic acid domain of activated factor X in the presence of calcium during inhibition by antithrombin–heparin

H. C. Whinna*, E. B. Lesesky*, D. M. Monroe‡, K. A. High§, P. J. Larson§ and F. C. Church*†‡

Departments of *Pathology and Laboratory Medicine, †Pharmacology and ‡Medicine, Carolina Cardiovascular Biology Center, The University of North Carolina School of Medicine, Chapel Hill, NC, USA; and §Division of Hematology, The Children's Hospital of Philadelphia, Abramson Research Center, Philadelphia, PA, USA

Correspondence to Herbert C. Whinna, Division of Hematology-Oncology/Medicine, Campus Box 7035, 932 Mary Ellen Jones Bldg., University of North Carolina at Chapel Hill, Chapel Hill NC 27599-7035, USA.
Fax: +1 919 966 7639; e-mail: whinna@med.unc.edu

P.J.L. current address: Bayer HealthCare, Biological Products, PO Box 13887, Research Commons, 79 TW Alexander Dr., Research Triangle Park, NC 27709, USA.

H.C.W. and E.B.L. contributed equally to this study.

KEYWORDS

Factor Xa • antithrombin • serpin • calcium heparin

ABSTRACT

Summary.

Background: Factor (F)Xa has 11 γ-carboxylated glutamic acid (Gla) residues that are involved in calcium-dependent membrane binding. The serpin antithrombin (AT) is an important physiological regulator of FXa activity in an inhibition reaction that is enhanced by heparin. Recently, Rezaie (Rezaie AR. J Biol Chem 1998; 273: 16824–7) showed that calcium further enhanced the heparin-catalyzed AT inhibition of FXa by promoting 'ternary complex' formation, and these results showed a role for the γ-carboxyl-glutamate (Gla)-domain of FXa.

Objectives: In this study, we used recombinant FXa mutants to assess the role of individual Gla residues in augmenting or antagonizing the AT–heparin inhibition reaction in the presence of calcium.

Results and conclusions: In the absence of heparin, AT inhibition of plasma and the recombinant FXas were essentially equivalent. Similar to plasma-derived FXa, calcium increased about 3-fold the inhibition rate of wild-type recombinant FXa by AT–heparin over that in the presence of EDTA. Interestingly, three different effects were found with the recombinant FXa Gla-mutants for AT–heparin inhibition: (i) Gla→Asp 14 and 29 were enhanced without calcium; (ii) Gla→Asp 16 and 26 were not enhanced by calcium; and (iii) Gla→Asp 19 was essentially the same as wild-type recombinant FXa. These results support a theory that mutating individual Gla residues in FXa alters the calcium-induced conformational changes in the Gla region and affects the antithrombin–heparin inhibition reaction.

Received 16 June 2003, accepted 2 March 2004

DIGITAL OBJECT IDENTIFIER (DOI)

10.1111/j.1538-7836.2004.00796.x About DOI