title: Activity and regulation of factor VIIa analogs with increased potency at the endothelial cell surface.
abstract: BACKGROUND
  Variants of recombinant factor VIIa (rFVIIa) with increased intrinsic activity have been developed to improve efficacy in the treatment of bleeding disorders in the future. The increased potency of FVIIa variants was demonstrated in limited in vitro and in vivo studies. However, further characterization of FVIIa variants is needed to evaluate their potential clinical use.
  
  
  METHODS
  In the present study, we investigated the interactions of two FVIIa variants, FVIIa(Q) and FVIIa(DVQ), with plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT), and vascular endothelium. TF-FVIIa activity or its inhibition was measured directly in an amidolytic activity assay or for its ability to activate factor X.
  
  
  RESULTS
  Both TFPI and AT/heparin inhibited the FVIIa variants more rapidly than the wild-type (WT) FVIIa in the absence of tissue factor (TF). In the presence of TF, TFPI, TFPI-Xa, and AT/heparin inhibited FVIIa and FVIIa variants at similar rates. Although the WT FVIIa failed to generate significant amounts of FXa on unperturbed endothelial cells, FVIIa variants, particularly FVIIa(DVQ), generated a substantial amount of FXa on unperturbed endothelium. Annexin V fully attenuated the FVIIa-mediated activation of FX on unperturbed endothelial cells. On stimulated human umbilical vein endothelial cells, FVIIa and FVIIa variants activated FX at similar rates, and annexin V blocked the activation only partly. AT/heparin and TFPI-Xa inhibited the activity of FVIIa and FVIIa variants bound to endothelial cell TF in a similar fashion. Interestingly, despite significant differences observed in FXa generation on unperturbed endothelium exposed to FVIIa and FVIIa analogs, no differences were found in thrombin generation when cells were exposed to FVIIa or FVIIa analogs under plasma mimicking conditions.
  
  
  CONCLUSIONS
  Overall, the present data suggest that although FVIIa variants generate substantial amounts of FXa, they do not generate excessive thrombin on the surface of endothelium.
authors.affiliation: Biomedical Research Division, The University of Texas Health Center at Tyler, Tyler, TX 75708, USA.
authors.forename: S
authors.initials: S
authors.lastname: Ghosh
authors.forename: M
authors.initials: M
authors.lastname: Ezban
authors.forename: E
authors.initials: E
authors.lastname: Persson
authors.forename: U
authors.initials: U
authors.lastname: Pendurthi
authors.forename: U
authors.initials: U
authors.lastname: Hedner
authors.forename: L V M
authors.initials: LV
authors.lastname: Rao
mesh_terms: D017304:Annexin A5 / Q000494:pharmacology; D000990:Antithrombin III / Q000494:pharmacology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D005167:Factor VII / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D015942:Factor VIIa; D015951:Factor Xa / Q000096:biosynthesis; D006801:Humans; D008074:Lipoproteins / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000096:biosynthesis; D013925:Thromboplastin / Q000494:pharmacology
chemical_list: D017304:Annexin A5; D008074:Lipoproteins; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor; D000990:Antithrombin III; D005167:Factor VII; D013925:Thromboplastin; C103587:recombinant FVIIa; D015942:Factor VIIa; D013917:Thrombin; D015951:Factor Xa