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.