Protein S has an established role in the protein C anticoagulant pathway as a
cofactor for anticoagulant protein C (APC) and has also recently been shown to
serve as a cofactor enhancing the anticoagulant activity of tissue factor pathway
inhibitor (TFPI). Despite its physiological role and clinical importance, the
molecular bases of its functions are not fully understood. The aim of my thesis
was to clarify the molecular mechanisms involved in the protein S interaction with
APC and TFPI. More than 30 point or composite protein S variants were
constructed and analysed during this project. These variants spanned the Gla,
thrombin sensitive region (TSR), epidermal growth-factor1 (EGF1) and EGF2
domains of protein S. Protein S was expressed in mammalian cells and was
purified by chromatography, as required. Protein S was characterised by size,
cleavage, multimerisation, γ-carboxylation of the Gla domain, binding to
phospholipids and to domain specific monoclonal antibodies. Variants were
evaluated for their APC and TFPI cofactor activities both by calibrated automated
thrombography and in purified FVa inactivation or FXa inhibition assays,
respectively. The protein S variant, protein S D95A, with substitution in EGF1 was
found to be largely devoid of functional APC cofactor activity and I believe that
this residue plays an important role in protein S anticoagulant function. It was γ-
carboxylated and bound phospholipids and domain specific monoclonal
antibodies with an apparent dissociation constant similar to that of wild type
protein S. Importantly, protein S D95A enhanced the anticoagulant activity of
TFPI, suggesting that distinct residues in protein S mediate its APC and TFPI
cofactor activity. Two composite mutants in the protein S EGF1 domain had
partially reduced TFPI cofactor activity in plasma. However, none of the more
than 30 variants spanning the Gla-TSR-EGF1-EGF2 domain of protein S
completely disrupted the protein S cofactor activity towards TFPI. Collectively,
these results shed light on the molecular basis of protein S cofactor function and
suggest distinct residues in protein S are involved in the binding to APC and to
TFPI.