Engineered recombinant factor VII Q217 variants with altered inhibitor specificities

Yu Jia Chang, Nobuko Hamaguchi, Shu Chuan Chang, Wolfram Ruf, Ming Ching Shen, Shu Wha Lin

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Recombinant factor VII with residue 217 (chymotrypsinogen numbering system) converted to alanine (VIIQ217A), glutamic acid (VIIQ217E), or glycine (VIIQ217G) was characterized. In a prothrombin time assay, VIIQ217E demonstrated 100%, VIIQ217A 15%, and VIIQ217G -1 s-1 x 107) for wild-type VIIa, 1.57 for VIIaQ217A, and 0.05 with VIIaQ217G. In comparison to wild-type VIIa, VIIaQ217E cleaved the chromogenic substrate S2765 (Z-D-Arg-Gly-Arg-pNA) with 10-fold higher k(cat). Analysis of the interactions with the inhibitors TFPI and antithrombin III demonstrated that VIIaQ217A but not VIIaQ217E or VIIaQ217G was inhibited less efficiently by TFPI either in the presence or in the absence of factor Xa. In contrast, VIIaQ217A association with antithrombin III in the presence of heparin was the fastest among the variants with a second-order rate constant of 2.31 (x 103 M-1 min-1), as compared to 0.47 and 1.47 for VIIaQ217E and wild-type VIIa, respectively. Our results demonstrate that residue Q217 is important in regulating substrate and, more importantly, inhibitor recognition by VIIa.

Original languageEnglish
Pages (from-to)10940-10948
Number of pages9
JournalBiochemistry
Volume38
Issue number34
DOIs
Publication statusPublished - Aug 24 1999
Externally publishedYes

Fingerprint

Factor VII
Antithrombin III
Glycine
benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide
Chymotrypsinogen
Chromogenic Compounds
Numbering systems
Prothrombin Time
Prothrombin
Alanine
Heparin
Glutamic Acid
Rate constants
Assays
Association reactions
Substrates
lipoprotein-associated coagulation inhibitor

ASJC Scopus subject areas

  • Biochemistry

Cite this

Chang, Y. J., Hamaguchi, N., Chang, S. C., Ruf, W., Shen, M. C., & Lin, S. W. (1999). Engineered recombinant factor VII Q217 variants with altered inhibitor specificities. Biochemistry, 38(34), 10940-10948. https://doi.org/10.1021/bi990055h

Engineered recombinant factor VII Q217 variants with altered inhibitor specificities. / Chang, Yu Jia; Hamaguchi, Nobuko; Chang, Shu Chuan; Ruf, Wolfram; Shen, Ming Ching; Lin, Shu Wha.

In: Biochemistry, Vol. 38, No. 34, 24.08.1999, p. 10940-10948.

Research output: Contribution to journalArticle

Chang, YJ, Hamaguchi, N, Chang, SC, Ruf, W, Shen, MC & Lin, SW 1999, 'Engineered recombinant factor VII Q217 variants with altered inhibitor specificities', Biochemistry, vol. 38, no. 34, pp. 10940-10948. https://doi.org/10.1021/bi990055h
Chang, Yu Jia ; Hamaguchi, Nobuko ; Chang, Shu Chuan ; Ruf, Wolfram ; Shen, Ming Ching ; Lin, Shu Wha. / Engineered recombinant factor VII Q217 variants with altered inhibitor specificities. In: Biochemistry. 1999 ; Vol. 38, No. 34. pp. 10940-10948.
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abstract = "Recombinant factor VII with residue 217 (chymotrypsinogen numbering system) converted to alanine (VIIQ217A), glutamic acid (VIIQ217E), or glycine (VIIQ217G) was characterized. In a prothrombin time assay, VIIQ217E demonstrated 100{\%}, VIIQ217A 15{\%}, and VIIQ217G -1 s-1 x 107) for wild-type VIIa, 1.57 for VIIaQ217A, and 0.05 with VIIaQ217G. In comparison to wild-type VIIa, VIIaQ217E cleaved the chromogenic substrate S2765 (Z-D-Arg-Gly-Arg-pNA) with 10-fold higher k(cat). Analysis of the interactions with the inhibitors TFPI and antithrombin III demonstrated that VIIaQ217A but not VIIaQ217E or VIIaQ217G was inhibited less efficiently by TFPI either in the presence or in the absence of factor Xa. In contrast, VIIaQ217A association with antithrombin III in the presence of heparin was the fastest among the variants with a second-order rate constant of 2.31 (x 103 M-1 min-1), as compared to 0.47 and 1.47 for VIIaQ217E and wild-type VIIa, respectively. Our results demonstrate that residue Q217 is important in regulating substrate and, more importantly, inhibitor recognition by VIIa.",
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