Trp2063 and Trp2064 in the Factor Va C2 Domain Are Required for High-Affinity Binding to Phospholipid Membranes but Not for Assembly of the Prothrombinase Complex

Weimin Peng, Mary Ann Quinn-Allen, Suhng Wook Kim, Kenneth A. Alexander, William H. Kane

Research output: Contribution to journalArticle

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Abstract

Interactions between factor Va and membrane phosphatidylserine (PS) regulate activity of the prothrombinase complex. Two solvent-exposed hydrophobic residues located in the C2 domain, Trp2063 and Trp 2064, have been proposed to contribute to factor Va membrane interactions by insertion into the hydrophobic membrane bilayer. However, the prothrombinase activity of rHFVa W(2063, 2064)A was found to be significantly impaired only at low concentrations of PS (5 mol %). In this study, we find that 10-fold higher concentrations of mutant factor Va are required for half-maximal prothrombinase activity on membranes containing 25% PS. The ability of the mutant factor Va to interact with factor Xa on a membrane was also impaired since 4-fold higher concentrations of factor Xa were required for half-maximal prothrombinase activity. The interaction of factor Va with 25% PS membranes was also characterized using fluorescence energy transfer and surface plasmon resonance. We found that the affinity of mutant factor Va for membranes containing 25% PS was reduced at least 400-fold with a Kd > 10-7 M. The binding of mutant factor Va to 25% PS membranes was markedly enhanced in the presence of factor Xa, indicating stabilization of the factor Va-factor Xa-membrane complex. Our findings indicate that Trp 2063 and Trp2064 play a critical role in the high-affinity binding of factor Va to PS membranes. It remains to be determined whether occupancy of this PS binding site in factor Va is also required for high-affinity binding to factor Xa.

Original languageEnglish
Pages (from-to)4385-4393
Number of pages9
JournalBiochemistry
Volume43
Issue number14
DOIs
Publication statusPublished - 2004 Apr 13
Externally publishedYes

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Factor Va
Phosphatidylserines
Phospholipids
Membranes
Factor Xa
Thromboplastin
prothrombinase complex
C2 Domains
Surface Plasmon Resonance
Energy Transfer
Surface plasmon resonance
Energy transfer
Stabilization
Fluorescence
Binding Sites

ASJC Scopus subject areas

  • Biochemistry

Cite this

Trp2063 and Trp2064 in the Factor Va C2 Domain Are Required for High-Affinity Binding to Phospholipid Membranes but Not for Assembly of the Prothrombinase Complex. / Peng, Weimin; Quinn-Allen, Mary Ann; Kim, Suhng Wook; Alexander, Kenneth A.; Kane, William H.

In: Biochemistry, Vol. 43, No. 14, 13.04.2004, p. 4385-4393.

Research output: Contribution to journalArticle

Peng, Weimin ; Quinn-Allen, Mary Ann ; Kim, Suhng Wook ; Alexander, Kenneth A. ; Kane, William H. / Trp2063 and Trp2064 in the Factor Va C2 Domain Are Required for High-Affinity Binding to Phospholipid Membranes but Not for Assembly of the Prothrombinase Complex. In: Biochemistry. 2004 ; Vol. 43, No. 14. pp. 4385-4393.
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abstract = "Interactions between factor Va and membrane phosphatidylserine (PS) regulate activity of the prothrombinase complex. Two solvent-exposed hydrophobic residues located in the C2 domain, Trp2063 and Trp 2064, have been proposed to contribute to factor Va membrane interactions by insertion into the hydrophobic membrane bilayer. However, the prothrombinase activity of rHFVa W(2063, 2064)A was found to be significantly impaired only at low concentrations of PS (5 mol {\%}). In this study, we find that 10-fold higher concentrations of mutant factor Va are required for half-maximal prothrombinase activity on membranes containing 25{\%} PS. The ability of the mutant factor Va to interact with factor Xa on a membrane was also impaired since 4-fold higher concentrations of factor Xa were required for half-maximal prothrombinase activity. The interaction of factor Va with 25{\%} PS membranes was also characterized using fluorescence energy transfer and surface plasmon resonance. We found that the affinity of mutant factor Va for membranes containing 25{\%} PS was reduced at least 400-fold with a Kd > 10-7 M. The binding of mutant factor Va to 25{\%} PS membranes was markedly enhanced in the presence of factor Xa, indicating stabilization of the factor Va-factor Xa-membrane complex. Our findings indicate that Trp 2063 and Trp2064 play a critical role in the high-affinity binding of factor Va to PS membranes. It remains to be determined whether occupancy of this PS binding site in factor Va is also required for high-affinity binding to factor Xa.",
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