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Characterization of the molecular defect in factor VR506Q
M Kalafatis1, R M Bertina, M D Rand
1Department of Biochemistry, University of Vermont, College of Medicine, Burlington 05405-0068.
The Journal of Biological Chemistry
|February 24, 1995
Summary
A mutation in factor V (Arg506-->Gln) causes poor anticoagulant response to activated protein C (APC). Factor V from patients with this mutation shows normal procoagulant activity but reduced APC inactivation, highlighting the role of Arg506 cleavage in factor Va inactivation.
Area of Science:
- Biochemistry
- Hematology
- Molecular Biology
Background:
- Activated protein C (APC) resistance is linked to a specific mutation in factor V (Arg506-->Gln).
- Factor V is a crucial cofactor in the coagulation cascade, and its regulation by APC is vital for hemostasis.
Purpose of the Study:
- To investigate the mechanism of APC resistance in individuals homozygous for the factor V Arg506-->Gln mutation.
- To compare the cleavage and inactivation of factor Va from these patients with normal factor Va by APC.
Main Methods:
- Purification of factor V from homozygous patients and normal individuals.
- Assays for procoagulant activity (clotting time, alpha-thrombin generation).
- Analysis of factor Va cleavage patterns and inactivation kinetics by APC using SDS-PAGE and functional assays.
Main Results:
- Factor V from patients exhibited normal procoagulant activity but significantly reduced inactivation by APC compared to normal factor V.
- Cleavage at Arg506 in normal factor Va is essential for optimal exposure of cleavage sites at Arg306 and Arg679, facilitating rapid inactivation.
- Despite the mutation, cleavage at Arg306 and Arg679 by APC still occurred, leading to inactivation, albeit at a slower rate initially.
Conclusions:
- Cleavage at Arg506 is critical for the efficient and rapid inactivation of membrane-bound factor Va by APC.
- APC-mediated cleavage at Arg306 on membrane-bound factor V occurs at comparable rates in both normal and APC-resistant individuals.
- The study elucidates the molecular basis of APC resistance, emphasizing the importance of the Arg506 cleavage site for effective anticoagulant function.