The roles of cofactors protein S and factor V in the TFPI anticoagulant pathway

Tissue factor pathway inhibitor (TFPI) is a principal inhibitor of the initiation of coagulation through inhibition of factor (F) Xa and tissue factor (TF)/FVIIa. While TFPI on its own is a poor inhibitor of FXa, protein S acts as cofactor for TFPI through interaction with TFPI Kunitz 3 domain, greatly enhancing FXa inhibition (~10-fold). The aim of my thesis was to further characterise the molecular basis underlying the TFPI cofactor function of protein S by identifying the complementary functional interaction site on protein S required for TFPI enhancement. I have screened over 40 protein S variants comprising substitutions spanning the whole protein S molecule and have identified the C-terminal sex hormone-binding globulin (SHBG)-like domain to be important for the enhancement of TFPI-mediated inhibition of thrombin generation in plasma. This was demonstrated using a protein S/growth arrest-specific 6 (Gas6) chimera, in which the SHBG-like domain was replaced with the corresponding domain in Gas6. Further examination of this chimera using FXa inhibition assays together with direct binding studies confirmed that the protein S SHBG-like domain is essential for the interaction with TFPI and the enhancement of TFPI-mediated inhibition of FXa. TFPI has also been reported to bind to FV in circulation. In my thesis I have aimed to evaluate the effects of FV interaction with TFPI upon its inhibition of FXa. While FV did not enhance TFPI on its own, it further enhanced the inhibition of FXa by TFPI in the presence of protein S (~32-fold by comparison to TFPI alone), suggesting that FV acts together with protein S as a synergistic cofactor for TFPI. Co-immunoprecipitation experiments revealed a direct interaction between TFPI and FV which was augmented by the presence of protein S. I propose that FV, in cooperation with protein S localise TFPI to the activated membrane surface in a position favourable for the efficient inhibition of FXa. Collectively, my findings shed light on the molecular mechanisms of TFPI enhancement by its cofactors protein S and FV.Open Acces