The preAR2 region (1458-1492) in factor V-Short is crucial for the synergistic TFPIα-cofactor activity with protein S and the assembly of a trimolecular factor Xa-inhibitory complex comprising FV-Short, protein S, and TFPIα

Background Factor V-Short (FV756-1458) is a natural splice variant in which 702 residues are deleted from the B domain. It exposes an acid region (AR2; 1493-1537) that binds tissue factor pathway inhibitor alpha (TFPI alpha). Protein S also interacts with TFPI alpha and serves as TFPI alpha-cofactor in factor Xa (FXa) inhibition. FV-Short and protein S function as synergistic TFPI alpha-cofactors in inhibition of FXa. FV810-1492 is an artificial FV-Short variant that cannot synergize with protein S as TFPI alpha cofactor even though it contains AR2 and binds TFPI alpha. Objective To elucidate the mechanisms for the synergism between FV756-1458 and protein S as TFPI alpha cofactors. Methods Four FV-Short variants were created, FV756-1458 and FV712-1458 contained the preAR2 region (1458-1492), whereas FV810-1492 and FV713-1492 lacked this region. The synergistic TFPI alpha cofactor activity between FV-Short variants and protein S was analyzed by FXa-inhibition. A microtiter-based assay tested binding between FV-Short variants, protein S, and TFPI alpha. Results The two preAR2-containing FV-Short variants were active as synergistic TFPI alpha cofactors, whereas the other two were inactive. All variants bound to TFPI alpha. None of the FV-Short variants bound directly to protein S. The combination of TFPI alpha and preAR2-containing FV-Short variants bound protein S, whereas TFPI alpha together with the preAR2-minus variants did not. Protein S potentiated TFPI alpha-binding to the preAR2-containing variants and binding between TFPI alpha and protein S was stimulated only by the preAR2-containing variants. Conclusion The preAR2 region is demonstrated to be crucial for the synergistic TFPI alpha-cofactor activity between FV-Short and protein S and for the assembly of a trimolecular FXa-inhibitory complex comprising FV-Short, protein S, and TFPI alpha.