The factor V C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis

Mahasen Saleh; Weimin Peng; Mary Ann Quinn-Allen; Sandra Macedo-Ribeiro; Pablo Fuentes-Prior; Wolfram Bode; William H. Kane

doi:10.1160/TH03-04-0222

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2004; 91(01): 16-27
DOI: 10.1160/TH03-04-0222

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

The factor V C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis

Authors

Mahasen Saleh^*

¹Division of Hematology, Departments of Medicine and Pathology, Duke University Medical Center, Durham, North Carolina, USA

³Current address: King Faisal Hospital and Research Center, Riyadh, Saudi Arabia
Weimin Peng^*

¹Division of Hematology, Departments of Medicine and Pathology, Duke University Medical Center, Durham, North Carolina, USA
Mary Ann Quinn-Allen

¹Division of Hematology, Departments of Medicine and Pathology, Duke University Medical Center, Durham, North Carolina, USA
Sandra Macedo-Ribeiro

²Max-Planck-Institut fur Biochemie, Abteilung Strukturforschung, Martinsried, Germany

⁴Current address: Centro de Neurociências de Coimbra, Coimbra, Portugal
Pablo Fuentes-Prior

²Max-Planck-Institut fur Biochemie, Abteilung Strukturforschung, Martinsried, Germany
Wolfram Bode

²Max-Planck-Institut fur Biochemie, Abteilung Strukturforschung, Martinsried, Germany
William H. Kane

¹Division of Hematology, Departments of Medicine and Pathology, Duke University Medical Center, Durham, North Carolina, USA

Abstract

Summary

The contribution of the factor Va C1 domain (fVa-C1) to assembly of the prothrombinase complex has not been previously investigated. The homologous fVa-C2 domain contains a binding site for phosphatidylserine (PS) that includes the indole moieties of Trp²⁰⁶³/Trp²⁰⁶⁴ at the apex of spike-1. In order to investigate the structure and function of fVa-C1 a molecular model was constructed based on the structure of fVa-C2. The aromatic and hydrophobic side chains of Tyr¹⁹⁵⁶/Leu¹⁹⁵⁷ in fVaC1 are located at the predicted apex of spike-3. Exposed charged and hydrophobic residues in fVa-C1 were changed to alanine in clusters of 1-3 mutations per construct. The resultant 20 mutants were expressed in COS cells and screened for binding to immobilized PS and prothrombinase activity on phospholipid vesicles containing either 25% or 5% PS. Two mutants, (Y1956,L1957)A, and (R2023,R2027)A showed both decreased binding to immobilized PS and a selective decrease in prothrombinase activity on membranes containing 5% PS. The interaction of purified (Y1956,L1957)A with phospholipid vesicles was studied using fluorescence resonance energy transfer and prothrombinase assays. The affinity of (Y1956,L1957)A binding to 25% PS membranes was reduced 12-fold compared to rHFVa. Prothrombin activation in the presence of (Y1956,L1957)A was markedly impaired on phospholipid vesicles containing 10% or less PS. We conclude that solvent exposed hydrophobic and aromatic amino acids in both fVa-C1 and fVa-C2 contribute to the interaction of factor V with PS membranes.

Keywords

Factor VIII - factor V - phospholipids / procoagulant - site-directed mutagenesis

Full Text

References

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