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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

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  • 1 Departments of Small Animal Clinical Sciences, Faculty of Life Sciences, University of Copenhagen, 1870 Frederiksberg C, Denmark
  • | 2 Biopharmaceuticals Research Unit, Novo Nordisk A/S, Novo Nordisk Park, 2760 Maaloev, Denmark
  • | 3 Biopharmaceuticals Research Unit, Novo Nordisk A/S, Novo Nordisk Park, 2760 Maaloev, Denmark
  • | 4 Biopharmaceuticals Research Unit, Novo Nordisk A/S, Novo Nordisk Park, 2760 Maaloev, Denmark
  • | 5 Department of Pathology and Laboratory Medicine, School of Medicine, University of North Carolina, Chapel Hill, NC 27599.
  • | 6 Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, 1870 Frederiksberg C, Denmark
  • | 7 Departments of Small Animal Clinical Sciences, Faculty of Life Sciences, University of Copenhagen, 1870 Frederiksberg C, Denmark

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.

Sample—Platelets from 4 dogs with hemophilia A, 4 dogs with hemophilia B, 4 dogs with von Willebrand disease, and 6 hemostatically normal dogs.

Procedures—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 and Clinical Relevance—Formation of coated platelets in dogs was similar to that in humans. Recombinant human coagulation factor VIIa bound preferentially to coated platelets from dogs.

Impact for Human Medicine—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.

Contributor Notes

Supported by a grant from the National Institutes of Health (NIH/NHLBI R24HL063098-12). Dr. Knudsen was supported by the University of Copenhagen Faculty of Life Sciences; the Danish Ministry of Science, Technology and Innovation; and Novo Nordisk A/S.

The authors thank Lone Odborg, Robin Raymer, John Nowicki, and Victoria Hamlett for technical assistance and Drs. Bo Wiinberg, Mads Kjelgaard-Hansen, Hanne Gamst-Andersen, and Christopher Scull for technical advice.

Address correspondence to Dr. Knudsen (tmkn@novonordisk.com).