Elevated function of blood clotting factor VIIa mutants that have enhanced affinity for membranes: Behavior in a diffusion-limited reaction

Gary L. Nelsestuen, Matthew Stone, Michael B. Martinez, Stephen B. Harvey, Donald Foster, Walter Kisiel

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Blood clotting factor VIIa is involved in the first step of the blood coagulation cascade, as a membrane-associated enzyme in complex with tissue factor (TF). Factor VIIa is also an important therapeutic agent for hemophilia where its function may include TF-independent as well as TF-dependent mechanisms. This study compared the activity of wild type factor VIIa (WT-VIIa) with that of a mutant with elevated affinity for membrane (P10Q/Q32E, QE-VIIa). Phospholipid and cell-based assays showed the mutant to have up to 40-fold higher function than WT-VIIa in both TF-dependent and TF-independent reactions. Tissue factor-dependent reactions displayed the maximum enhancement when binding had reached equilibrium in competition with another TF-binding protein. In liposome-based assays, the association rate of WT-VIIa with TF occurred at a physical maximum and could not be improved by site-directed mutagenesis. A practical consequence was identical function of WT-VIIa and QE-VIIa in assays that depended entirely on assembly kinetics. Thus, factor VIIa mutants provided unique reagents for probing the mechanism of factor VIIa action. They may also offer superior agents for therapy.

Original languageEnglish (US)
Pages (from-to)39825-39831
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number43
DOIs
StatePublished - Oct 26 2001

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Factor VIIa
Blood Coagulation Factors
Thromboplastin
Blood Coagulation
Blood
Membranes
Assays
Mutagenesis
Hemophilia A
Site-Directed Mutagenesis
Coagulation
Liposomes
Phospholipids
Carrier Proteins
Association reactions
Kinetics
Enzymes
Therapeutics

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Elevated function of blood clotting factor VIIa mutants that have enhanced affinity for membranes : Behavior in a diffusion-limited reaction. / Nelsestuen, Gary L.; Stone, Matthew; Martinez, Michael B.; Harvey, Stephen B.; Foster, Donald; Kisiel, Walter.

In: Journal of Biological Chemistry, Vol. 276, No. 43, 26.10.2001, p. 39825-39831.

Research output: Contribution to journalArticle

Nelsestuen, Gary L. ; Stone, Matthew ; Martinez, Michael B. ; Harvey, Stephen B. ; Foster, Donald ; Kisiel, Walter. / Elevated function of blood clotting factor VIIa mutants that have enhanced affinity for membranes : Behavior in a diffusion-limited reaction. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 43. pp. 39825-39831.
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