title: Development of a flow cytometric assay for detection of coated platelets in dogs and evaluation of binding of coated platelets to recombinant human coagulation factor VIIa.
abstract: OBJECTIVE
  To develop an antibody-based flow cytometric assay to detect coated platelets in dogs and to characterize the interaction of recombinant human coagulation factor VIIa with activated platelets from dogs with hemophilia A.
  
  
  METHODS
  Platelets from 4 dogs with hemophilia A, 4 dogs with hemophilia B, 4 dogs with von Willebrand disease, and 6 hemostatically normal dogs.
  
  
  METHODS
  Freshly isolated platelets were activated with thrombin, convulxin, or a thrombin-convulxin combination. Resulting platelet phenotypes were resolved on the basis of P-selectin and fibrinogen expression, and binding of recombinant human coagulation factor VIIa to these distinct platelet subpopulations was measured by use of a flow cytometric assay.
  
  
  RESULTS
  Coated platelets were identified on the basis of expression of α-granule fibrinogen and were generated in response to stimulation with the thrombin-convulxin combination but not to stimulation with either agonist alone. Approximately 70% of the platelets from dogs with hemophilia A, hemophilia B, and von Willebrand disease and from the control dogs had the coated platelet phenotype. Recombinant human coagulation factor VIIa bound preferentially to coated platelets with a mean ± SD binding equilibrium constant of 2.6 ± 0.5μM.
  
  
  CONCLUSIONS
  Formation of coated platelets in dogs was similar to that in humans. Recombinant human coagulation factor VIIa bound preferentially to coated platelets from dogs.
  
  
  CONCLUSIONS
  A similar mechanism of action for recombinant human coagulation factor VIIa may exist in dogs and humans. The potential for use of dogs in the study of bleeding disorders in humans was strengthened.
authors.affiliation: Department of Small Animal Clinical Sciences, Faculty of Life Sciences, University of Copenhagen, 1870 Frederiksberg C, Denmark. tmkn@novonordisk.com
authors.forename: Tom
authors.initials: T
authors.lastname: Knudsen
authors.forename: Marianne
authors.initials: M
authors.lastname: Kjalke
authors.forename: Mikael
authors.initials: M
authors.lastname: Tranholm
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Nichols
authors.forename: Asger L
authors.initials: AL
authors.lastname: Jensen
authors.forename: Annemarie T
authors.initials: AT
authors.lastname: Kristensen
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000378:metabolism; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D003435:Crotalid Venoms / Q000378:metabolism; D004195:Disease Models, Animal; D004283:Dog Diseases / Q000378:metabolism / Q000503:physiopathology; D004285:Dogs; D015942:Factor VIIa / Q000378:metabolism*; D005340:Fibrinogen / Q000032:analysis / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods / Q000662:veterinary*; D006467:Hemophilia A / Q000378:metabolism / Q000503:physiopathology / Q000662:veterinary; D002836:Hemophilia B / Q000378:metabolism / Q000503:physiopathology / Q000662:veterinary; D006801:Humans; D037181:Lectins, C-Type / Q000378:metabolism; D019007:P-Selectin / Q000032:analysis / Q000378:metabolism; D015539:Platelet Activation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D014842:von Willebrand Diseases / Q000378:metabolism / Q000503:physiopathology / Q000662:veterinary
chemical_list: D000906:Antibodies; D003435:Crotalid Venoms; D037181:Lectins, C-Type; D019007:P-Selectin; D011994:Recombinant Proteins; C028890:convulxin; D005340:Fibrinogen; C103587:recombinant FVIIa; D015942:Factor VIIa; D013917:Thrombin