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