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SummaryObjectives: To characterize the cellular composition (platelets, erythrocytes, and leukocytes) and confirm reproducibility of platelet enrichment, as well as determine the platelet activation status in the final product of a commercial platelet-rich plasma kit using canine blood.Methods: Venous blood from 20 sedated client-owned dogs was used to prepare platelet-rich plasma (PRP) from a commercial kit. Complete blood counts were performed to determine erythrocyte, leukocyte, and platelet numbers in both whole blood (WB) and resultant PRP. The WB and PRP samples from jugular (fast collection) and cephalic (slow collection) venipuncture were also compared. P-selectin externalization was measured in WB and PRP samples from 15 of 20 dogs.Results: This commercial kit produced an average percent recovery in platelets of 64.7 ± 17.4; erythrocytes of 3.7 ± 0.8, and leukocytes of 31.6 ± 10.0. Neutrophil, monocyte, and lymphocyte percent recovery was 19.6 ± 7.2, 44.89 ± 19.8, and 57.5 ± 10.6, respectively. The recovery of platelets from jugular venipuncture (59.7 ± 13.6%) was lower than from cephalic recovery (68.8 ± 19.1%). The mean percent P-Selectin externalization for WB, PRP, and PRP with thrombin was 25.5 ± 30.9, 4.5 ± 6.4, and 90.6 ± 4.4 respectively.Clinical significance: Cellular reproducibility of this kit was confirmed and platelets were concentrated within autologous serum. Additionally, measurements of P-selectin externalization showed that platelets are inactive in PRP unless stimulated to degranulate.

SummaryThe molecular defects causing severe factor IX deficiency were identified in two distinct canine breed-variants. Both defects were associated with an absence of plasma factor IX coagulant activity and antigen. A large deletion mutation was found in 1 breed variant, spanning the entire 5’ region of the factor IX gene extending to exon 6. An approximately 5 kb insertion disrupted exon 8 of the second breed-variant. This insertion was associated with alternative splicing between a donor site 5’ and acceptor site 3’ to the normal exon 8 splice junction, with introduction of a new stop codon. The resultant transcript lacked most of the factor IX catalytic domain and 3’ untranslated region. Molecular analyses of canine hemophilia B define an experimental model for study of inhibitor formation and gene therapy strategies, and provide insight into spontaneous mutation mechanisms in the factor IX gene and on the X chromosome of mammalian species.

SummaryCanine plasma factor IX was purified to homogeneity by a combination of barium citrate precipitation and three-step column chromatographies of DEAE sepharose, heparin agarose and a monoclonal antifactor IX antibody-linked agarose. Canine factor IX has an apparent molecular size of 61 kDa, which is slightly smaller than that of human factor IX, as determined by denatured polyacrylamide gel electrophoresis. Its amino acid composition, amino-terminal and carboxyterminal amino acid sequences agreed well with those predicted from the reported cDNA. Unlike purified human factor IX, canine factor IX preparation often showed a discrete smaller molecular species (∼50 kDa) which was generated by a specific proteolytic cleavage between Arg310 and Val311. When purified canine factor IX was utilized as a standard for enzyme linked immunosorbent assay, the concentration of canine factor IX in the pooled normal dog plasma was determined to be 5.3 Μg/ml with 11.2% carbohydrate content (or 4.7 Μg/ml for its polypeptide chain moiety). Concentration of plasma factor IX antigen was measured in six severely affected, unrelated hemophilia B dogs. Four had factor IX antigen of less than 1% of the normal, and two had undetectable levels. The latter two had gross molecular abnormalities in their factor IX genes. Three obligate carrier females had variable but proportionately reduced factor IX antigen and factor IX coagulant activity levels. These results provide a quantitative method for measuring canine factor IX antigen which is a prerequisite for studying hemostasis and development of gene transfer approaches in the canine model of hemophilia B.

SummaryBleeding times are reported in many studies using canine models, with a variety of techniques employed to adapt these tests for dogs. We evaluated a canine model of template bleeding time, the buccal mucosa bleeding time (BMBT), by examining the test’s sensitivity and specificity for defects of primary hemostasis. We examined thirty-five dogs having defined defects of either primary hemostasis (Types I, II, III von Willebrand’s disease, thrombasthenia, thrombopathia) or secondary hemostasis (hemophilia A and B, Factor VII deficiency). Comparisons of BMBT and cuticle bleeding time were made in a subset of these dogs.All dogs having primary hemostatic disorders had long BMBT, and all factor deficient dogs had BMBT within normal range. The BMBT in canine models appears to be a specific and sensitive test of primary hemostasis; suitable for evaluating factors affecting template bleeding time and potential efficacy and thrombogenicity of treatment regimens.