The binding of factor Va to phospholipid vesicles

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Chemistry

Background:

• Factor Va is a critical cofactor in the coagulation cascade.
• Understanding its structure and function is essential for hemostasis research.
• Previous studies have investigated factor Va's sulfhydryl groups and binding properties.

Purpose of the Study:

• To characterize the accessible sulfhydryl groups in factor Va.
• To investigate the binding of factor Va to phospholipid vesicles.
• To identify the domains involved in factor Va-phospholipid interactions.

Main Methods:

• Chemical modification with dithiobis-(nitrobenzoic acid) (DTNB) to quantify accessible thiols.
• Fluorescent labeling of factor Va with N-(1-pyrene)maleimide.
• Fluorescence polarization to study factor Va binding to phosphatidylcholine/phosphatidylserine (PCPS) vesicles.
• Competition assays to determine binding affinities and identify binding domains.

Main Results:

• Factor Va contains one accessible thiol group under native conditions, located in component D.
• Fluorescently labeled factor Va (Pyr-Va) retains activity and binds specifically to PS-containing vesicles.
• Binding is characterized by a dissociation constant of 2.7 x 10(-9) M, with a defined number of PS molecules per binding site.
• Component E, not D, mediates binding to vesicles, and its N-terminal region contains the lipid-binding domain.

Conclusions:

• Accessible thiols in factor Va are primarily located in component D.
• Factor Va binds to phospholipid vesicles via component E, specifically through its N-terminal lipid-binding domain.
• This binding is independent of calcium and can be competitively inhibited by unlabeled factor Va or component E.