Studies of Recombinant Activated Human Factor VII Binding to Human Umbilical Vein Endothelial Cells Show Shared Binding Sites with Factor X and Protein C.
Authors: Steinoe Anne , Sorensen Brit B. , Stennicke Henning R. , Rao L. Vijaya Mohan 
Published: April 2020

The endothelial cell-covered vasculature is constantly interacting with the various components of the blood. The exposure to the endothelial cell surface of any compound carried in the bloodstream is thus enormous, making the endothelium a crucial player in the mechanistic action of any drug given intravenously. Coagulopathic conditions arise when inflammatory messengers (e.g. TNF-a) induce the expression of certain endothelial surface receptors like the well known receptor for factor VII (FVII), tissue factor (TF). The interaction between recombinant activated FVII (rFVIIa) and TF on cell surfaces, including stimulated endothelial cells, has been studied extensively. However, little is known about the interaction of rFVIIa with native endothelium that lines the vasculature under normal conditions. Therefore, the binding of rFVIIa to unstimulated human umbilical vein endothelial cells (HUVEC) was investigated in the present study, using iodinated rFVIIa (125I-rFVIIa). ~ 45 fmol rFVIIa/100.000 cells in a time-dependent manner. Competition studies showed that the binding of rFVIIa could be reduced by 50 % with a 100-fold molar excess of unlabeled rFVIIa, suggesting both specific and non-specific binding sites on the surface of HUVEC. The binding could not be inhibited by an anti-TF antibody that blocks rFVIIa binding to TF, which indicates that the binding of rFVIIa to the cell surface of HUVEC is TF-independent. A 100-fold molar excess of gla-domain proteins FIX, prothrombin, protein S and protein Z had no effect on the binding of 125I-rFVIIa to unstimulated HUVEC at 4 °C, indicating a gla-domain independent binding site for rFVIIa on uninduced endothelial cells. However, the other two gla-proteins FX and protein C inhibited 125I-rFVIIa binding to HUVEC by 30 % in a 100-fold molar excess, suggesting that rFVIIa to some extent share binding site(s) on the HUVEC cell-surface with FX and protein C. Furthermore, unlabeled rFVIIa in 5-fold molar excess significantly reduced the binding of 125I-rFVIIa to HUVEC whereas FX and protein C in 5-fold molar excess had no significant effect on 125I-rFVIIa binding to HUVEC. This might indicate that the affinity of the putative binding site(s) on the HUVEC cell-surface is lower for FX and Protein C than for FVIIa. These results point to the presence of mutual specific binding site(s) on unstimulated endothelial cells with different affinity for the three proteins. The ability to compete with rFVIIa for binding to the cell surface was unchanged upon activation of FIX or FX. Thus, FIXa did not inhibit binding, whereas FXa reduced the amount of bound rFVIIa by 30 % as seen with FX.

In conclusion, these data indicate the presence of both specific and non-specific binding sites for FVIIa on the surface of HUVEC. Neither binding site(s) appears to be gla-domain dependent since FIX, prothrombin, protein S and protein Z were unable to compete for rFVIIa-binding. There seems to be a common binding site for rFVIIa, FX and protein C.