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Definition of a factor Va binding site in factor Xa.
A E Rudolph1, R Porche-Sorbet, J P Miletich
1Departments of Pathology and Medicine, Division of Laboratory Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA. amy.e.rudolph@monsanto.com
The Journal of Biological Chemistry
|November 23, 2000
Summary
Five mutations in activated factor X (fXa) affect cofactor binding. Residues Arg-347, Lys-351, and Lys-414 are critical for factor Va (fVa) binding, impacting enzyme activity and inhibition.
Area of Science:
- Biochemistry
- Molecular Biology
- Hemostasis
Background:
- Activated factor X (fXa) is a key enzyme in the coagulation cascade.
- Cofactor binding, specifically to factor Va (fVa), is essential for fXa's catalytic efficiency.
- Previous studies identified residue 347 as important for fVa binding.
Purpose of the Study:
- To identify additional residues involved in factor Va (fVa) binding to activated factor X (fXa).
- To characterize the functional consequences of mutations in these residues on fXa activity and cofactor interaction.
Main Methods:
- Site-directed mutagenesis was used to generate five mutant fX species.
- Enzyme kinetics, prothrombin activation assays, and competition binding assays were employed.
- Functional characterization was performed in the absence and presence of phospholipid vesicles and activated platelets.
Main Results:
- Mutations at Arg-347, Lys-351, and Lys-414 significantly reduced the apparent affinity for fVa.
- Mutations at Arg-306 and Glu-310 had less impact on fVa binding but affected prothrombin activation.
- All mutant enzymes showed reduced inhibition rates by antithrombin III in the presence of heparin.
- Enzyme function was preserved on activated platelets, indicating the importance of the surface-bound fVa.
Conclusions:
- Arg-347, Lys-351, and Lys-414 constitute a core binding site for fVa on fXa.
- Arg-306 and Glu-310 play a secondary role in fVa interaction or substrate binding.
- These findings elucidate the structural basis of fXa-fVa complex formation and its regulation.