OC 36.2 - Hydrophobic patch (PLVIVG 1481-1486) in FV-Short crucial for its synergistic TFPI-cofactor activity with protein S and the formation of a FXa-inhibitory complex comprising FV-Short, protein S and TFPI
Monday, July 11, 2022
11:00 AM – 11:15 AM
Location: ExCel Center, ICC Capital Suite Room 10&11
MLS Lund University, Dept Translational Medicine Malmö, Skane Lan, Sweden
Background: The natural factor V (FV) splice isoform FV-Short (FV756-1458) functions in synergy with protein S as a TFPIα-cofactor in inhibition of FXa. TFPIα binds to an exposed acid region (AR2; 1493-1537) in the B domain and protein S binds to the FV-Short/TFPIα-complex. 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 between FV-Short, protein S and TFPIα.
Aims: To identify which part of preAR2 is required for the synergistic TFPIα-cofactor activity between FV-Short and protein S and the assembly of the FXa-inhibitory complex.
Methods: New FV-Short truncation variants FV709-1476, FV712-1478, FV712-1481, FV712-1484, FV712-1487, and FV712-1490 were created and tested in the FXa-inhibition assay to identify the site required for the synergistic TFPIα cofactor activity between protein S and FV-Short. A microtiter-based assay analyzed the binding between FV-Short variants, protein S and TFPIα.
Results: Three of the FV-Short variants (FV709-1476, FV712-1478, 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. Although TFPIα bound to all FV-Short variants in the absence of protein S, strongest binding was observed with FV712-1478 and FV712-1481. In the absence of TFPIα no direct binding of protein S was observed to any of the FV-Short variants. However, in presence of TFPIα, efficient cooperative binding was demonstrated between protein S, TFPIα and FV709-1476, FV712-1478, or FV712-1481. In contrast, even in the presence of TFPIα, no binding of protein S was observed when FV712-1484, FV712-1487, or FV712-1490 were tested.
Conclusion(s): 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 cooperative assembly of a trimolecular FXa-inhibitory complex between FV-Short, protein S and TFPIα.