Importance of individual residues in hydrophobic patch PLVIVGL (1481-1487) in FV-Short for synergistic TFPIα-cofactor activity with protein S, an alanine-scanning study. AlphaFold-mediated prediction of FV-Short/TFPIα/protein S tri-molecular complex structure.

Abstract

BackgroundIn the splice variant Factor V-Short, 702 residues are deleted from the B domain, resulting in exposure of an acid region (AR2; 1493-1537) that binds TFPIα. FV-Short and protein S serve as synergistic TFPIα cofactors in inhibition of FXa. In the preAR2 region, a hydrophobic patch PLVIVGL (1481-1487) is crucial for synergistic TFPIα-cofactor activity and assembly of FV-Short, TFPIα and protein S. ObjectiveTo elucidate importance of individual residues in the PLVIVGL patch for synergism between FV-Short and protein S as TFPIα cofactors. MethodsAn alanine-scanning of the hydrophobic patch was performed in which seven FV-Short variants were created. The synergistic TFPIα-cofactor activity was analyzed by FXa-inhibition and a microtiter-based assay tested binding between the proteins. AlphaFold 3 was used to predict protein-protein interactions between FV-Short, protein S and TFPIα. ResultsFive of the seven variants (V1483A, I1484A, V1485A, G1486A, L1487A) demonstrated decreased synergistic TFPIα-cofactor activity, in particular G1486A and L1487A were severely affected. Neither wild-type FV-Short nor any of the mutants bound protein S in the absence of TFPIα. In the presence of TFPIα, wild-type FV-Short, P1481A, L1482A and V1485A bound protein S, whereas V1483A, I1484A, G1486A, and L1487A did not. AlphaFold predicted an interaction between the hydrophobic patch in FV-Short and a hydrophobic patch in protein S involving residues 268-276 and 422-426. ConclusionIndividual residues (V1483, I1484, G1486 and L1487) in the hydrophobic patch are demonstrated to be important for the synergistic TFPIα-cofactor activity and for the assembly of a trimolecular FXa-inhibitory complex.