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The evaluation of complex-dependent alterations in human factor VIIa.
J H Lawson1, S Butenas, K G Mann
1Department of Biochemistry, University of Vermont, Burlington 05405.
The Journal of Biological Chemistry
|March 5, 1992
Summary
A new fluorescent substrate enables direct measurement of factor VIIa enzymatic activity. Complexing factor VIIa with tissue factor enhances its catalytic efficiency over 100-fold, primarily by increasing kcat.
Area of Science:
- Biochemistry
- Enzymology
- Hemostasis
Background:
- Factor VIIa is a key glycoprotein in hemostasis.
- Tissue factor (TF) is an integral membrane glycoprotein.
- The enzymatic complex of Factor VIIa and TF is crucial for blood clotting.
Purpose of the Study:
- To develop a fluorescent substrate for measuring Factor VIIa enzymatic activity.
- To investigate the effect of TF on Factor VIIa catalytic efficiency.
- To determine the binding kinetics of Factor VIIa to TF.
Main Methods:
- Development of a novel fluorescent substrate (6-(Mes-D-Leu-Gly-Arg)amino-1-naphthalenediethylsulfamide).
- Direct measurement of Factor VIIa enzymatic activity in the presence and absence of TF and phospholipids.
- Kinetic analysis (kcat/Km) of substrate hydrolysis by Factor VIIa and its complex with TF.
Main Results:
- The substrate allows detection of Factor VIIa at concentrations below 10(-9) M.
- Binding to TF increases Factor VIIa catalytic efficiency by over 100-fold, mainly via increased kcat.
- TF binding enhances Factor VIIa activity independently of the supporting surface.
- Apparent dissociation constant for Factor VIIa-TF binding is 1.1–2.1 nM with 1:1 stoichiometry.
Conclusions:
- TF binding induces a conformational change in Factor VIIa's catalytic site, enhancing substrate hydrolysis.
- The developed substrate is sensitive and useful for studying Factor VIIa kinetics and TF interactions.
- No substrate hydrolysis was observed for single-chain Factor VII, indicating specific activation requirements.