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Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay
Published on: September 9, 2012
Factor VIII lacking the C2 domain retains cofactor activity in vitro
Hironao Wakabayashi1, Amy E Griffiths, Philip J Fay
1Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, USA.
The Journal of Biological Chemistry
|June 10, 2010
Summary
Factor VIII (FVIII) C2 domain deletion reveals significant contributions of other domains, like C1, to membrane binding and function. This finding impacts understanding of blood coagulation and FVIII-related disorders.
Area of Science:
- Biochemistry
- Hematology
- Molecular Biology
Background:
- Factor VIII (FVIII) is a critical coagulation cofactor.
- The FVIII C2 domain is traditionally implicated in phospholipid binding.
- Emerging evidence suggests other domains, such as C1, may also contribute to FVIIIa membrane anchoring.
Purpose of the Study:
- To investigate the role of the FVIII C1 domain in membrane binding and function.
- To characterize a FVIII variant lacking the C2 domain (DeltaC2).
Main Methods:
- Construction of a FVIII variant lacking the C2 domain (DeltaC2).
- Measurement of binding affinity to phospholipid vesicles using energy transfer.
- Assays for FXa generation, one-stage clotting, functional affinity for FIXa, thrombin activation rates, and FVIIIa stability.
- Thrombin generation assays to assess peak thrombin and endogenous thrombin potential.
Main Results:
- DeltaC2 exhibited a 14-fold reduced binding affinity to phospholipid vesicles.
- Despite reduced binding, DeltaC2 retained 76% FXa generation activity and 36% one-stage clotting activity compared to wild-type (WT) FVIII.
- Functional affinity for FIXa and thrombin activation rates were modestly reduced (approx. 4-fold).
- FVIIIa stability was significantly reduced (approx. 3.6-fold) in the DeltaC2 variant.
- Thrombin generation assays showed up to 60-fold reductions in peak thrombin and endogenous thrombin potential.
Conclusions:
- FVIII domains beyond C2, particularly C1, play significant roles in membrane binding and membrane-dependent functions.
- The loss of the C2 domain impacts FVIIIa stability and overall procoagulant activity.
- These findings provide new insights into the structural basis of FVIII function in hemostasis.
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