Camilo Bulla

In vitro and in vivo assessment of platelet function in healthy dogs during administration of a low-dose aspirin regimen

OBJECTIVE To assess the in vitro and in vivo platelet function of healthy dogs during administration of a low-dose aspirin regimen. ANIMALS 16 dogs. PROCEDURES Dogs received aspirin (1 mg/kg, PO, q 24 h) for 7 days. Blood and urine samples were collected before (day 1; baseline) and on days 3 and 7 of the low-dose aspirin regimen. Platelet function was evaluated by use of turbidimetric and conventional impedance aggregometry, multiple-electrode impedance aggregometry, a platelet function analyzer (PFA), and determination of urine 11-dehydro-thromboxane B2 concentration. Turbidimetric aggregometry results were compared with the results obtained by the other 4 methods. Fourteen days after cessation of aspirin, platelet-rich plasma was incubated with acetylsalicylic acid and platelet function was assessed by turbidimetric aggregometry to determine whether this technique could accurately identify dogs that responded to the low-dose aspirin regimen. RESULTS Of the 16 dogs, 13 had turbidimetric and conventional impedance aggregometry results that were decreased by > 25% from baseline on days 3 and 7, and 4 and 7 dogs had PFA closure times > 300 seconds on days 3 and 7, respectively. The median urine 11-dehydro-thromboxane B2 concentration-to-creatinine concentration ratio decreased by 49% between days 1 and 7. Turbidimetric aggregometry results were correlated with conventional impedance aggregometry results. There was poor agreement between the turbidimetric aggregometry and PFA results. The multiple-electrode impedance aggregometry protocol failed to reliably detect aspirin-induced platelet dysfunction. In vitro incubation of platelet-rich plasma with acetylsalicylic acid followed by turbidimetric aggregometry did not predict whether dogs responded to the low-dose aspirin regimen. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that the response to a low-dose aspirin regimen varied among healthy dogs.

Ultra-pure platelet isolation from canine whole blood

BackgroundSeveral research applications involving platelets, such as proteomic and transcriptomic analysis, require samples with very low numbers of contaminating leukocytes, which have considerably higher RNA and protein content than platelets. We sought to develop a platelet purification protocol that would minimize contamination, involve minimal centrifugation steps, and yield highly pure platelet samples derived from low volume whole blood samples from healthy dogs.ResultsUsing an optimized OptiPrep density gradient technique, platelet recovery was 51.56% with 99.99% platelet purity and leukocyte contamination of 100 leukocytes per 108 platelets, on average. Platelet samples were subjected to additional purification with CD45-labeled Dynabeads after density barrier centrifugation resulting in a 95-fold depletion of residual leukocytes. Platelets purified using these methods remained inactivated as assessed by Annexin V and P-selectin labeling with flow cytometry.ConclusionsThe use of OptiPrep density gradient is a quick method for obtaining highly purified platelet samples from low volumes of canine whole blood with minimal contamination. Additional depletion of residual leukocytes can be achieved using CD45-labeled beads. These platelet samples can then be used for many downstream applications that require ultra-pure platelet samples such as RNA and protein analysis.

Expression of the thrombopoietin gene in tissues from healthy dogs.

Thrombopoietin (THPO) is the major cytokine that regulates megakaryopoiesis and platelet production. Several human and murine studies have demonstrated that THPO is primarily synthesized in the liver, but the kidney, spleen and bone marrow are also sites of expression. The aim of this study was to determine THPO mRNA levels in a range of canine tissues by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Samples of bone marrow (n = 5), liver (n = 10), lung (n = 10), renal cortex (n = 10), renal medulla (n = 5) and spleen (n = 10) were obtained from 10 healthy, hound-cross dogs aged 6-8 months. The highest THPO mRNA levels were found in the liver, followed by the bone marrow, spleen, lung and kidney. There was a 13-fold difference in expression between liver and kidney. The bone marrow showed high levels of THPO mRNA in the absence of disease. The liver and bone marrow are likely to be the major sites of THPO production in the dog.

Suppression of vascular endothelial growth factor expression by cannabinoids in a canine osteosarcoma cell line

Vascular endothelial growth factor (VEGF) is a key regulator in both physiologic and pathologic angiogenesis, and cannabinoids decrease VEGF release in human and murine cancer cells. The aim of this study was to assess the in vitro effects of a synthetic cannabinoid, WIN-55,212-2, on the expression of the proangiogenic factor VEGF-A in the canine osteosarcoma cell line 8. After analysis of gene expression by quantitative real-time polymerase chain reaction, the compound decreased VEGF-A expression by 35% ± 10% (P , 0.0001) as compared with the control. This synthetic cannabinoid shows promise as a potential inhibitor of angiogenesis, and further studies are warranted to investigate its in vivo effects and to explore the potential of this and related compounds as adjuvant cancer therapy in the dog.

Evidence of Selective Packaging and Different α-Granule Subtypes in Canine Platelets

Platelets are circulating megakaryocytic cytoplasmic fragments that are required to maintain vascular integrity and prevent haemorrhage. During activation, platelets release hundreds of bioactive proteins, mainly by secretion of α-granule content. These proteins include von Willebrand factor (vWF) and fibrinogen, major adhesive proteins involved in haemostasis. Human and mouse platelet α-granules are packaged selectively to contain different molecules and platelets can differentially release these proteins on specific receptor activation. Confocal laser scanning microscopy was used to analyse vWF and fibrinogen localization within canine platelet α-granules. In resting canine platelets, the majority of platelet vWF and fibrinogen is located in separate α-granule populations. These findings provide evidence that canine platelets contain different α-granule subtypes, suggesting that selective protein packaging occurs during canine platelet ontology.

Platelets Inhibit Migration of Canine Osteosarcoma Cells

The interaction between platelets and tumour cells is important for tumour growth and metastasis. Thrombocytopenia or antiplatelet treatment negatively impact on cancer metastasis, demonstrating potentially important roles for platelets in tumour progression. To our knowledge, there is no information regarding the role of platelets in cancer progression in dogs. This study was designed to test whether canine platelets affected the migratory behaviour of three canine osteosarcoma cell lines and to give insights of molecular mechanisms. Intact platelets, platelet lysate and platelet releasate inhibited the migration of canine osteosarcoma cell lines. Addition of blood leucocytes to the platelet samples did not alter the inhibitory effect on migration. Platelet treatment also significantly downregulated the transcriptional levels of SNAI2 and TWIST1 genes. The interaction between canine platelets or molecules released during platelet activation and these tumour cell lines inhibits their migration, which suggests that canine platelets might antagonize metastasis of canine osteosarcoma. This effect is probably due to, at least in part, downregulation of genes related to epithelial-mesenchymal transition.

Frequency of the MDR1 mutant allele associated with multidrug sensitivity in dogs from Brazil

To date, a 4-bp deletion in the MDR1 gene has been detected in more than ten dog breeds, as well as in mixed breed dogs, in several countries, however information regarding this mutation in dogs from Brazil is lacking. For this reason, 103 Collies, 77 Border Collies, 76 Shetland Sheepdogs, 20 Old English Sheepdogs, 55 German Shepherds, 16 Australian Shepherds, and 53 Whippets from Brazil were screened for the presence of the mutation. The heterozygous mutated genotype, MDR1 (+/−), frequency found for Collies, Australian Shepherd, and Shetland Sheepdog was 50.5% (95% CI =41.1%–59.9%), 31.3% (95% CI =8.6%–53.2%), and 15.8% (95% CI =7.7%–23.9%), respectively. Homozygous mutated genotype, MDR1 (−/−), was detected only in Collies 35.9%. The MDR1 allele mutant frequency found for Collies, Australian Shepherd, and Shetland Sheepdog was 61.2% (95% CI =54.8%–67.5%), 15.6% (95% CI =3.1%–28.2%), and 7.9% (95% CI =3.7%–12.1%), respectively. Additionally, even free of the mutant allele, the maximum mutant prevalence (MMP) in that population, with 95% CI, was 3.8%, 5.2%, 5.4%, and 13.8% for Border Collies, German Shepherds, Whippets, and Old English Sheepdogs, respectively. In this way, this information is important, not only for MDR1 genotype-based breeding programs and international exchange of breeding animals of predisposed breeds, but also for modification of drug therapy for breeds at risk.

Identification of canine platelet proteins separated by differential detergent fractionation for nonelectrophoretic proteomics analyzed by Gene Ontology and pathways analysis

Abstract During platelet development, proteins necessary for the many functional roles of the platelet are stored within cytoplasmic granules. Platelets have also been shown to take up and store many plasma proteins into granules. This makes the platelet a potential novel source of biomarkers for many disease states. Approaches to sample preparation for proteomic studies for biomarkers search vary. Compared with traditional two-dimensional polyacrylamide gel electrophoresis systems, nonelectrophoretic proteomics methods that employ offline protein fractionation methods such as the differential detergent fractionation method have clear advantages. Here we report a proteomic survey of the canine platelet proteome using differential detergent fractionation coupled with mass spectrometry and functional modeling of the canine platelet proteins identified. A total of 5,974 unique proteins were identified from platelets, of which only 298 (5%) had previous experimental evidence of in vivo expression. The use of offline prefractionation of canine proteins by differential detergent fractionation resulted in greater proteome coverage as compared with previous reports. This initial study contributes to a broader understanding of canine platelet biology and aids functional research, identification of potential treatment targets and biomarkers, and sets a new standard for the resting platelet proteome.

Detection of heterozygous MDR1 nt230(del4) mutation in a mixed-breed dog: case report of possible doxorubicin toxicosis

1Department of Veterinary Clinics, School of Veterinary Medicine and Animal Science, Sao Paulo State University, Botucatu, Brazil; 2Department of Clinical Sciences and Animal Health Center, College of Veterinary Medicine, Mississippi State University, MS, USA; 3Department of Microbiology and Immunology, Biosciences Institute, Sao Paulo State University, Botucatu, Brazil; 4Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, MS, USA