Characterisation of the molecular basis of protein S anticoagulant function

Abstract

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.