title: Recombinant factor VIIa analog NN1731 (V158D/E296V/M298Q-FVIIa) enhances fibrin formation, structure and stability in lipidated hemophilic plasma.
abstract: BACKGROUND
  The bypassing agent recombinant factor VIIa (rFVIIa) is efficacious in treating bleeding in hemophilia patients with inhibitors. Efforts have focused on the rational engineering of rFVIIa variants with increased hemostatic potential. One rFVIIa analog (V158D/E296V/M298Q-FVIIa, NN1731) improves thrombin generation and clotting in purified systems, whole blood from hemophilic patients and factor VIII-deficient mice.
  
  
  METHODS
  We used calibrated automated thrombography and plasma clotting assays to compare effects of bypassing agents (rFVIIa, NN1731) on hemophilic clot formation, structure, and ability to resist fibrinolysis.
  
  
  RESULTS
  Both rFVIIa and NN1731 shortened the clotting onset and increased the maximum rate of fibrin formation and fibrin network density in hemophilic plasma clots. In the presence of tissue plasminogen activator, both rFVIIa and NN1731 shortened the time to peak turbidity (TTPeak(tPA)) and increased the area under the clot formation curve (AUC(tPA)). Phospholipids increased both rFVIIa and NN1731 activity in a lipid concentration-dependent manner. Estimated geometric mean concentrations of rFVIIa and NN1731 producing similar onset, rate, TTPeak(tPA), and AUC(tPA) as seen with 100% factors VIII and IX were: 24.5, 74.3, 29.7, and 37.1 nM rFVIIa, and 8.6, 31.2, 9.0, and 11.3 nM NN1731, respectively. In each case, the NN1731 concentration was significantly lower than rFVIIa.
  
  
  CONCLUSIONS
  These findings suggest that like rFVIIa, NN1731 improves the formation, structure, and stability of hemophilic clots. Higher lipid concentrations may facilitate assessment of both rFVIIa and NN1731 activity. NN1731 appears likely to support rapid clot formation in tissues with high endogenous fibrinolytic activity.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599–7525, USA.
authors.forename: Laura D
authors.initials: LD
authors.lastname: Gray
authors.forename: Michael A
authors.initials: MA
authors.lastname: Hussey
authors.forename: Brittany M
authors.initials: BM
authors.lastname: Larson
authors.forename: Kellie R
authors.initials: KR
authors.lastname: Machlus
authors.forename: Robert A
authors.initials: RA
authors.lastname: Campbell
authors.forename: Gary
authors.initials: G
authors.lastname: Koch
authors.forename: Mirella
authors.initials: M
authors.lastname: Ezban
authors.forename: Ulla
authors.initials: U
authors.lastname: Hedner
authors.forename: Alisa S
authors.initials: AS
authors.lastname: Wolberg
mesh_terms: D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects*; D005167:Factor VII / Q000494:pharmacology*; D015942:Factor VIIa / Q000494:pharmacology; D005337:Fibrin / Q000378:metabolism*; D005342:Fibrinolysis / Q000187:drug effects; D006467:Hemophilia A / Q000097:blood* / Q000188:drug therapy*; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D010743:Phospholipids / Q000494:pharmacology; D010949:Plasma / Q000187:drug effects*; D011994:Recombinant Proteins / Q000494:pharmacology; D013917:Thrombin / Q000096:biosynthesis
chemical_list: C519901:NN1731; D010743:Phospholipids; D011994:Recombinant Proteins; D005167:Factor VII; D005337:Fibrin; C103587:recombinant FVIIa; D015942:Factor VIIa; D013917:Thrombin