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in Journal of the American Veterinary Medical Association

Abstract

Objective

To evaluate megakaryocyte size and ploidy, using Feulgen staining and microspectrophotometry, in adult dogs with normal platelet count.

Animals

Group A contained 8 and group B contained 11 adult dogs.

Procedure

Megakaryocytes were evaluated by light microscopy and staged according to maturation status. Stage-III megakaryocytes were measured and mapped for future relocation. Bone marrow aspirates were destained and restained, using the Feulgen method. Previously identified stage-III megakaryocytes were measured for DNA content, using microspectrophotometry.

Results

Megakaryocyte size correlated with ploidy values, and mean sizes within ploidy groups were significantly (P < 0.05) different from each other for both groups. The modal ploidy value of stage-III megakaryocytes, which represented 18% of the total megakaryocyte population of the combined groups, was 32N. This is in contrast to results of flow cytometric studies, which indicated that the modal ploidy value for all canine megakaryocytes was 16N.

Conclusions

Reasons for the disparate results between microspectrophotometric techniques and flow cytometry include maturation stage of the megakaryocyte population evaluated and percentage of megakaryocytes within that maturation stage. Flow cytometric methods, which evaluate all megakaryocytes detectable by antibody, may include cells still capable of DNA synthesis, resulting in a shift in the observed modal ploidy value.

Recognition of the difference between canine and human megakaryocyte ploidy distribution is important, particularly in studies in which the dog is used as an animal model for human megakaryocytopoiesis. (Am J Vet Res 1996;57:1434–1437)

Free access
in American Journal of Veterinary Research

Abstract

Objectives—To determine the molecular and genetic basis for thrombasthenic thrombopathia in Otterhounds and establish whether the defect would be best classified as type-I Glanzmann's thrombasthenia.

Animals—57 dogs, including 13 affected Otterhounds, 23 carrier Otterhounds, 17 unaffected Otterhounds, and 4 clinically normal unrelated dogs of other breeds.

Procedure—Functional (platelet aggregation, clot retraction, buccal mucosa bleeding time) and biochemical (electrophoresis, flow cytometry, fibrinogen content) analyses were conducted. In addition, firststrand cDNA synthesis from platelet total RNA was performed. Exons of the genes encoding for glycoproteins (GP) IIb and IIIa were amplified in overlapping fashion. The resulting products were excised from agarose gels and sequenced. The sequences obtained were compared with known cDNA sequences for canine GPIIb and GPIIIa.

Results—A single nucleotide change at position G1193 (1100) was detected in exon 12 of the gene encoding for platelet GPIIb in 2 affected Otterhounds. Carrier Otterhounds were heterozygous at this position, and 2 unaffected Otterhounds were unchanged. This nucleotide change would result in substitution of histidine for aspartic acid at position 398 (367) within the third calcium-binding domain of GPIIb.

Conclusions and Clinical Relevance—These studies suggest that thrombasthenic thrombopathia of Otterhounds is homologous phenotypically and has a similar molecular basis to type-I Glanzmann's thrombasthenia in humans. (Am J Vet Res 2001;62:1797–1804)

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in American Journal of Veterinary Research

Abstract

Objective—To determine the nucleotide sequence of the α IIb gene from canine platelet-derived cDNA.

Animals—3 adult dogs.

Procedure—First-strand cDNA was prepared from total RNA isolated from canine platelets. The cDNA was amplified, using specific primers in polymerase chain reaction (PCR), and the nucleotide sequence was obtained from purified PCR products.

Results—Except for the nucleotide at position 694, results of all sequencing reactions of α IIb were identical for canine platelet-derived cDNA. Canine α IIb had 3 fewer codons than α IIb of humans. The nucleotide and deduced amino acid sequences of full-length canine α IIb shared ≥ 83% similarity with the sequences established for humans. Segments of canine α IIb nucleotide and deduced amino acid sequences were ≥ 78% similar to α IIb associated with 7 functional domains (extracellular, transmembrane, cytoplasmic, and 4 calcium-binding domains) in humans, with the highest degree of similarity correlating with the sequences of the 4 calcium-binding domains. Amino acid residues associated with development of alloantibodies in humans (Met837, Val837, Ile843, Ser843) are not encoded by canine α IIb .

Conclusions and Clinical Relevance—The nucleotide variation at position 694 of canine α IIb may represent a polymorphism. The species differences in the α IIb sequence may contribute to variations in receptor-li gand interactions. The high degree of α IIb sequence conservation of the 4 calcium-binding domains implies functional importance. Some disorders associated with α IIb β3 in dogs are clinically analogous to diseases in humans, and results indicate that dogs are an appropriate model for the evaluation of gene therapy and other treatments of platelet-associated disorders. (Am J Vet Res 2001;62:1486–1492)

Full access
in American Journal of Veterinary Research

Summary

Platelet function, antithrombin and plasminogen activities, and fibrinolytic capabilities in 11 cats with acquired heart disease were compared with results in 4 healthy cats. Of 11 cats with heart disease, 9 had hyperthyroidism with secondary cardiac dysfunction. One cat with hyperthyroidism had renal disease and heart failure, and of 2 cats with idiopathic hypertrophic cardiomyopathy, 1 also had renal disease. At the time of testing, 3 cats had thromboembolic events associated with the disease. Compared with healthy cats, cats with acquired heart disease had increased activity of antithrombin III, a protein that behaves as an acute-phase reactant. Plasminogen activity was decreased, although not significantly, in cats with acquired heart disease, compared with results in healthy cats. In cats with left ventricular dysfunction, clot retraction was decreased (marginal significance, P = 0.058) and might be attributed, in some cases, to the medications received by the cats. Dilute whole blood clots from all cats failed to lyse in vitro. This observation, at present, lacks adequate explanation. Platelets from cats with acquired heart disease, compared with platelets from healthy cats, had decreased responsiveness (aggregation and [14C]serotonin release) to adenosine diphosphate and increased responsiveness to collagen. Hyperthyroid cats were receiving various drugs (propranolol, atenolol, or diltiazem) to empirically treat clinical signs of disease attributable to cardiac dysfunction. Although numbers of cats in each group were small, definite trends were observed in the results of tests. Platelets from cats receiving atenolol had decreased responsiveness to adenosine diphosphate and unaltered responsiveness to collagen, compared with platelets from healthy cats, and may have decreased risk of thrombus formation. Cats receiving propranolol and diltiazem had platelets with markedly increased responsiveness to collagen; however, these drugs appeared to provide sufficient cardioprotective benefits to counter the prothrombotic effects.

Free access
in American Journal of Veterinary Research

Summary

Cats with cardiomyopathy, especially dilated cardiomyopathy associated with taurine deficiency, often develop systemic thrombi. To investigate the relation of taurine deficiency to formation and persistence of thrombi, cats were made taurine-deficient by consumption of a casein-based taurine-deficient diet, then were evaluated for anticoagulant and profibrinolytic activities and platelet function. The cats served as their own controls in the taurine-replete state; then, values were compared for the taurine-deficient state. Plasma (P < 0.01), blood (P < 0.05), and platelet (P < 0.05) taurine concentrations were decreased markedly after cats consumed the taurine-deficient diet for 6 weeks, compared with baseline concentrations before diet. Compared with the taurine-replete state, taurine deficiency induced significantly (P < 0.05) increased mean antithrombin III activity, no significant change in plasminogen and fibrinolytic activities, and similar clot retraction/lysis test results. Decreased (P < 0.01) adenosine diphosphate (adp)-induced platelet aggregation and [14C]serotonin release, and slightly increased (P < 0.05) collagen-induced platelet [14C]serotonin release, but unchanged collagen-induced platelet aggregation were observed in taurine-deficient cats, compared with taurine-replete cats.

Changes in antithrombin III activity most likely reflected hepatocellular acute-phase reaction, which indicates that taurine deficiency may induce a stress-responsive state. Results of platelet function testing indicate that taurine may modulate platelet responsiveness to physiologic agonists, but not in consistent manner. That platelets from the taurine-deficient cats had decreased responsiveness to adp, but increased responsiveness to collagen is surprising, because irreversible aggregation is mediated by release of granule-associated ADP after sufficient initial stimulus.

All cats had normal clot retraction in dilute blood, which indicated adequate platelet numbers and function; however, clots failed to lyse in vitro. To the authors knowledge, this observation, at present, lacks adequate explanation.

Development of marked taurine deficiency and altered in vitro results of anticoagulant activities and some platelet function tests did not result in clinical manifestations in our cats. Results of our study do not conclusively document a pathophysiologic role of taurine depletion in the formation or persistence of thrombi.

Free access
in American Journal of Veterinary Research

Abstract

Objective—To identify subclinical Babesia gibsoni infection in American Pit Bull Terriers from the southeastern United States and to determine the genetic sequence of parasite DNA isolated from these dogs.

Design—Case series.

Animals—33 American Pit Bull Terriers and 87 dogs of various other breeds.

Procedure—Blood smears were examined for microscopic evidence of the parasite, and DNA was extracted from blood samples and used in a polymerase chain reaction (PCR) assay designed to amplify the small subunit ribosomal RNA gene sequence of B gibsoni. Amplification products of the expected size were sequenced, and sequences were compared with published sequences for B gibsoni isolates. Hematocrit, platelet count, mean platelet volume, WBC count, and eosinophil count were compared between dogs with positive PCR assay results and dogs with negative results.

Results—Results of the PCR assay were positive for 18 of the 33 (55%) American Pit Bull Terriers, including all 10 dogs with microscopic evidence of parasitemia. Only 1 of these dogs was clinically ill at the time blood samples were collected. Results of microscopic evaluation of blood smears and of the PCR assay were negative for the 87 other dogs. Hematocrit and platelet count were significantly lower in dogs with positive PCR assay results than in dogs with negative results.

Conclusions and Clinical Relevance—Results suggest that American Pit Bull Terriers in the southeastern United States may be subclinically infected with B gibsoni. However, subclinical infection was not identified in dogs of other breeds from the same geographic area. (J Am Vet Med Assoc 2002;220: 325–329)

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in Journal of the American Veterinary Medical Association

Abstract

Objective—To determine whether a polymerase chain reaction (PCR) assay could be used to detect Eperythrozoon wenyoniin the blood of cattle.

Design—Prospective study.

Animals—95 cattle from various herds in Alabama and Georgia and 96 bulls enrolled in Auburn University's Alabama Beef Cattle Improvement Association Bull Test program.

Procedure—Blood samples were collected by means of venipuncture of the median caudal vein and submitted for a CBC and PCR assay. Blood smears were made immediately after blood collection and examined by means of light microscopy.

Results—Three of 95 cattle from herds in Alabama and Georgia and 5 of 96 bulls enrolled in the Bull Test program had positive PCR assay results. Organisms were seen in blood smears from only 5 of these 8 animals. Organisms were not seen in blood smears from any animals for which results of the PCR assay were negative.

Conclusions and Clinical Relevance—Results suggest that a PCR assay may be an effective method for detecting E wenyoni infection in cattle and that the PCR assay may be a more sensitive test than evaluation of blood smears. (J Am Vet Med Assoc 2001; 219:1432–1434)

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in Journal of the American Veterinary Medical Association

Abstract

Objective

To establish the existence of platelet-derived proteins in equine plasma, with the future goal of developing an assay for the detection of in vivo platelet activation.

Animals

5 mature healthy horses.

Procedure

Platelet-rich plasma and platelet-poor plasma were prepared from anticoagulated blood. Platelets were separated from plasma proteins by gel filtration, then activated with 0.5 μM platelet-activating factor. Protease inhibitors were added, and the released platelet proteins were harvested. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on the released platelet proteins and platelet-poor plasma, and the resultant silver-stained bands were compared. Immunoblot analysis was performed on released platelet proteins, using an antibody to human thrombospondin; human platelet-derived proteins served as the positive control for the antibody.

Results

Released platelet proteins in the presence of β-mercaptoethanol (reduced samples) contained several proteins that were not observed in plasma including (mean ± SEM) 194 ± 2, 159 ± 2, 151 ±2, 104 ± 2, and 95 ± 1 kd. Immunoblots of released platelet proteins had a prominent 180 ± 2-kd protein in reduced samples that was recognized by an antibody to human thrombospondin, and with prolonged color development, 2 additional less prominent proteins (166 ± 1 and 155 ± 1 kd) were observed.

Conclusions

Several proteins are released from activated equine platelets that are not detectable in normal equine plasma. Thrombospondin is one of the high molecular mass proteins released by activated equine platelets.

Clinical Relevance

An assay can be developed for detection of thrombospondin in equine plasma and may be useful for detection of in vivo platelet activation in horses. (Am J Vet Res 1997;58:954–960)

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in American Journal of Veterinary Research