A hydrophobic patch (PLVIVG; 1481–1486) in the B‐domain of factor V‐short is crucial for its synergistic TFPIα‐cofactor activity with protein S and for the formation of the FXa‐inhibitory complex comprising FV‐short, TFPIα, and protein S

BACKGROUND: Factor V‐short (FV756‐1458) is a natural splice variant functioning in synergy with protein S as tissue factor pathway inhibitor alpha (TFPIα)–cofactor in inhibition of factor Xa (FXa). An exposed acid region (AR2; 1493–1537) in the B domain binds TFPIα. The preAR2 (1458–1492) is crucial for the synergistic TFPIα–cofactor activity between FV‐short and protein S and for assembly of a trimolecular FXa‐inhibitory complex among FV‐short, protein S, and TFPIα. OBJECTIVE: To identify which part of preAR2 is required for the synergistic TFPIα–cofactor activity between FV‐short and protein S. METHODS: A FXa‐inhibition assay was used to test the synergistic TFPIα cofactor activity between protein S and new FV‐short variants FV709‐1476, FV712‐1478, FV712‐1481, FV712‐1484, FV712‐1487, and FV712‐1490. A microtiter‐based assay analyzed binding among FV‐short variants, protein S, and TFPIα. RESULTS: FV709‐1476, FV712‐1478, and FV712‐1481 were fully active as synergistic TFPIα cofactors with protein S; FV712‐1484 showed intermediate activity; and FV712‐1487 and FV712‐1490 were inactive. TFPIα interacted with all variants in the absence of protein S but FV712‐1478 and FV712‐1481 bound TFPIα with highest affinity. None of the FV‐short variants bound directly to protein S in the absence of TFPIα. In the presence of TFPIα, efficient cooperative binding was demonstrated between protein S, TFPIα, and FV709‐1476, FV712‐1478, or FV712‐1481. In contrast, no cooperativity among TFPIα, protein S, and FV712‐1484, FV712‐1487, or FV712‐1490 was seen. CONCLUSION: A short hydrophobic patch in preAR2 (PLVIVG, 1481–1486) in FV‐short is crucial for the synergistic TFPIα‐cofactor activity between FV‐short and protein S and for the assembly of a trimolecular FXa‐inhibitory complex among FV‐short, protein S, and TFPIα.