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Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

Helena M Andersson1, Márcia J Arantes, James T B Crawley

  • 1Department of Haematology, Faculty of Medicine, Imperial College London, London, UK.

Blood
|March 24, 2010
PubMed

Insights

Protein S is crucial for blood clotting regulation. Researchers found that a specific residue, Asp95 in the EGF1 domain, is essential for Protein S to function as a cofactor for activated protein C (APC).

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Hematology

Background:

  • Protein S is a vital cofactor in the protein C anticoagulant pathway.
  • It enhances the inactivation of factors Va and VIIIa by activated protein C (APC).
  • The precise molecular mechanisms underlying Protein S's cofactor activity remain incompletely understood.

Purpose of the Study:

  • To elucidate the molecular basis of Protein S interaction with APC.
  • To identify specific residues critical for Protein S's cofactor function.

Main Methods:

  • Construction and expression of a library of human Protein S variants with substitutions in key domains (Gla, TSR, EGF1, EGF2).
  • Evaluation of APC cofactor activity using calibrated automated thrombography (CAT) with Protein S-deficient plasma.
  • Biochemical assays to assess gamma-carboxylation, phospholipid binding, and APC-mediated cleavage enhancement of factor Va variants.

Main Results:

  • A variant, Protein S D95A, located in the EGF1 domain, exhibited significantly reduced APC cofactor activity.
  • Protein S D95A retained gamma-carboxylation and phospholipid binding capabilities comparable to wild-type Protein S.
  • Purified Protein S D95A demonstrated a markedly diminished ability to enhance APC-induced cleavage of factor Va.

Conclusions:

  • Aspartic acid at residue 95 (Asp95) within the EGF1 domain is critical for the APC cofactor function of Protein S.
  • This residue likely represents a key interaction site between Protein S and APC.

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