Proposed mechanism of the protein S/TFPI/FXa complex assembly on a phospholipid surface. Our results suggest that the mechanism of protein S cofactor enhancement of TFPI involves formation of a complex between the SHBG-like domain of protein S and the Kunitz domain 3 (K3) of TFPI (particularly Glu226). Protein S helps localize TFPI onto an activated membrane surface in a position favorable for the interaction of Kunitz domain 2 (K2) with the serine protease domain (SP) of FXa. The boxes show the respective models of the protein S SHBG-like domain and the TFPI Kunitz domain 3. TFPI Glu226 and the residues substituted in protein S/Gas6 chimera I are highlighted in black. The TFPI Kunitz domain 3 and the protein S SHGB -domain models are adapted from Mine et al and Villoutreix et al, respectively.^37,42