Heterozygosity for P2Y12 receptor gene mutation associated with postoperative hemorrhage in a Greater Swiss Mountain dog

A 3‐year‐old, female Greater Swiss Mountain dog developed a hemoperitoneum following an exploratory laparotomy and ovariohysterectomy. Platelet count, PT, APTT, and plasma von Willebrand factor antigen concentration were within RIs. A buccal mucosal bleeding time (BMBT) was prolonged. Given the probability of a hereditary thrombopathia, the dog was administered desmopressin, fresh platelet transfusions, and aminocaproic acid to control hemorrhage. Subsequently, DNA testing for the P2Y12 receptor gene mutation identified the dog as being a heterozygote (carrier). Further platelet function testing was performed following complete recovery. Results of a repeat BMBT and a point‐of‐care screening test using the Platelet Function Analyzer‐100 (collagen/adenosine‐diphosphate [ADP] test cartridge) were within RIs. Flow cytometric studies demonstrated a marked reduction in fibrinogen binding to the dog's platelets in response to ADP ‐ adenosine diphosphate activation. Likewise, turbidimetric aggregometry revealed a complete absence of platelet aggregation in response to ADP. However, there were a normal aggregation response to the platelet agonist convulxin and a mild reduction in amplitude in response to γ‐thrombin. This is the first report of a dog heterozygous for the P2Y12 receptor gene mutation exhibiting a bleeding tendency and having evidence of impaired platelet function in vitro in response to ADP activation. Given that the mutant allele for the P2Y12 thrombopathia appears to be widespread in the Greater Swiss Mountain dog breed, veterinarians need to be aware that both homozygotes and heterozygotes for this platelet receptor mutation are at risk of developing life‐threatening bleeding following trauma or surgery.     

Prophylactic use of a lyophilized platelet product for rhinoscopic diagnosis and treatment of sinonasal aspergillosis in a dog with a P2Y12 platelet receptor mutation

Objective

To describe the periprocedural use of a lyophilized platelet product during rhinoscopic diagnosis and treatment of sinonasal aspergillosis in a Greater Swiss Mountain Dog with a P2Y12 platelet receptor disorder.

Case Summary

After the development of severe epistaxis, a Greater Swiss Mountain Dog was diagnosed with thrombopathia secondary to a P2Y12 receptor gene mutation. Concurrent primary nasal disease was also suspected due to persistent mucopurulent nasal discharge. One month after the initial presentation for epistaxis, the dog was readmitted for workup of nasal disease. Computed tomography of the head showed turbinate lysis and regional lymphadenopathy. Because of concern for a high risk of bleeding in a thrombopathic patient subjected to rhinoscopy and nasal biopsies, a lyophilized platelet product was administered prior to the procedure. Rhinoscopic exam revealed fungal plaques consistent with Aspergillus spp. that were later confirmed on fungal culture to be Aspergillus fumigatus. Rhinoscopic biopsies were performed as well as debridement of the fungal plaques, followed by topical administration of clotrimazole solution. Bleeding was minimal during and after the procedure, and the dog recovered uneventfully.

New or Unique Information Provided

This is the first report of the prophylactic use of lyophilized platelets in a thrombopathic patient undergoing an invasive procedure with potential for significant hemorrhage. Minimal bleeding occurred during the procedure, suggesting that lyophilized platelets could be used for the prevention of bleeding in thrombopathic patients undergoing invasive procedures.     

Detection of activated platelets in canine blood by use of flow cytometry

OBJECTIVE: To evaluate whether markers of platelet activation, including P-selectin expression, phosphatidylserine exposure, platelet-leukocyte aggregates, and microparticle formation, could be measured in nonstimulated and stimulated canine blood samples and develop a standardized protocol for detection of activated platelet markers in canine blood. SAMPLE POPULATION: Blood samples from 10 dogs. PROCEDURE: Platelet activation was determined by flow cytometric measurement of platelets with P-selectin expression, platelet-leukocyte aggregates, platelet microparticles, and platelets with phosphatidylserine exposure. Changes in specific markers of platelet activation in nonstimulated versus stimulated samples were assessed by use of varying concentrations of 2 platelet agonists, platelet-activating factor (PAF) and adenosine diphosphate. Flow cytometry was used to detect platelet CD61 (glycoprotein IIIa), CD62P (P-selectin), and the leukocyte marker CD45. Annexin V was used to identify exposed phosphatidylserine. RESULTS: A significant difference was detected in the percentages of platelets with P-selectin, plateletleukocyte aggregates, microparticles, and platelets with annexin V exposure (phosphatidylserine) in samples stimulated with 10nM PAF versus the nonstimulated samples, with platelet-leukocyte aggregates having the greatest difference. CONCLUSIONS AND CLINICAL RELEVANCE: Platelet activation is essential for thrombus formation and hemostasis and may be potentially useful for evaluation of dogs with suspected thromboembolic disease. Prior to development of a thrombotic state, a prothrombotic state may exist in which only a small number of platelets is activated. Identification of a prothrombotic state by use of activated platelets may help direct medical intervention to prevent a thromboembolic episode.     

May-Hegglin anomaly in a dog

An 8-year-old female spayed Pug dog was presented for evaluation of cutaneous lesions occurring secondary to immunosuppressive treatment of presumed immune-mediated thrombocytopenia. Abnormal hematologic findings included persistent thrombocytopenia, macrothrombocytes, and variably shaped, often fusiform, blue cytoplasmic inclusions in neutrophils. May-Hegglin anomaly (MHA) was suspected based on the morphologic appearance of platelets and neutrophils. Examination of cells by transmission electron microscopy revealed normal platelet ultrastructure; neutrophil inclusions had features similar to those reported for inclusions in human MHA. Neutrophil function was within normal limits based on flow cytometric analysis. Thrombelastography indicated a prolonged clotting time (r), and PlateletMapping showed a lack of response to 2 μM ADP compared with a moderate response in the control dog. Immunocytochemical staining of blood smears using 2 commercially available antibodies against MYH9 protein (nonmuscle myosin heavy chain II) yielded negative results. However, genomic DNA sequencing analysis of the dog's MYH9 gene identified a single point mutation, resulting in substitution of lysine for glutamine at the 1841 amino acid position; this mutation is identical to one identified in people with MHA. To our knowledge, this is the first report of an MYH9 mutation in the dog. MHA-associated macrothrombocytopenia may be mistaken for immune-mediated thrombocytopenia.     

Alterations in normal canine platelets during storage in EDTA anticoagulated blood

Abstract: Flow cytometric detection of platelet surface-associated IgG (PSAIgG) can be used to determine whether immunologic factors are contributing to thrombocytopenia in dogs. In vitro alterations in platelet activation and morphology, however, could impact the results of this test. The purpose of this study was to determine whether the PSAIgG test for immune-mediated thrombocytopenia was valid on whole blood in EDTA anticoagulant after 24–72 hours of storage, and to characterize other alterations in canine platelets that could impact immunologic testing. Platelets were harvested and analyzed immediately after blood collection and after 24, 48, and 72 hours of storage at 4°C. Spontaneous and thrombin-induced changes in the following platelet parameters were evaluated using flow cytometric techniques: PSAIgG, platelet microparticle formation, membrane expression of P-selectin and glycoprotein CD61, exogenous IgG binding, surface-exposed phosphatidylserine, and fibrinogen binding. The amount of PSAIgG increased 6-to 9-fold in stored samples compared with fresh samples. Platelet microparticle formation was spontaneous in stored samples and increased significantly over time. Membrane phosphatidylserine, P-selectin, and fibrinogen binding were not altered by storage, indicating that platelet activation was minimal in stored samples. Although storage decreased the percentage of platelets positive for CD61 by 8-to 10-fold compared with fresh samples, activation by high-dose thrombin partially restored the percentage of CD61-positive platelets in 24-hour-old samples. In conclusion, even though platelets stored in EDTA for up to 72 hours remain in a resting state, aged platelets have an increased tendency to form microparticles and have increased surface IgG and decreased surface CD61, which may contribute to false-positive results for tests of immune-mediated thrombocytopenia.     

An inherited platelet function defect in a Simmental crossbred herd.

An inherited bleeding disorder, resembling Simmental hereditary thrombopathy (SHT), has been identified in a Simmental crossbred herd. In an affected bull calf, initially evaluated because of excessive bleeding from a vaccination site, the platelet aggregation response to the agonist, adenosine-diphosphate (ADP) was essentially absent and the aggregation response to platelet activating factor (PAF16) was reduced by at least 70%. The initial laboratory assessment of platelet function in the dam and sire yielded results which were within normal limits. The sire was not available for further testing. The dam, also a daughter of this sire, was subsequently shown to have a partially reduced aggregation response to ADP. Of 18 other offspring of the sire evaluated, 6 were also identified as having a partially impaired aggregation response. The maximum aggregation response to ADP and PAF16 in these 6 calves was approximately 50% of the level exhibited by unaffected animals. In contrast, the coagulation profiles were normal for all animals except for a heifer calf which also exhibited a partially impaired aggregation response. The plasma level of the coagulation protein, factor XI, was reduced in this heifer calf which suffered a fatal hemorrhage following dehorning. This report appears to be the 1st to have identified animals putatively heterozygous for SHT on the basis of the in vitro platelet aggregation response to ADP.     

Platelet size,platelet surface-associated IgG,and reticulated platelets in dogs with immune-mediated thrombocytopenia

Abstract: Immune-mediated thrombocytopenia (IMT) is a disorder in which bound IgG on the surface of platelets results in platelet removal and alterations in mean platelet volume. Using flow cytometry, alterations in platelet size, platelet surface-associated IgG (PSAIgG), and numbers of reticulated platelets were determined in 13 dogs with primary IMT and 4 dogs with secondary IMT induced by experimental infection with Babesia gibsoni . Effects of sample age on platelet parameters also were determined, using samples from 20 dogs with normal platelet counts analyzed within 4 hours and after 24, 48, and 72 hours of storage in EDTA. No significant changes in platelet count, platelet size, or reticulated platelet percentage were observed in samples assayed within 4 and 24 hours of blood collection; whereas PSAIgG values increased 3 to 7 fold in samples stored for 24–72 hours. Using reference values for freshly collected or 24-hour-old samples, 10 of 13 (77%) dogs with primary IMT and all B gibsoni-inf ected dogs had increased PSAIgG levels. In 12 (75%) of the 16 dogs with thrombocytopenia the percentage of reticulated platelets was increased; however, absolute numbers of reticulated platelets were within reference values. Moreover, PSAIgG level and the percentage of reticulated platelets were not always increased concurrently in dogs with primary and secondary IMT. Platelet microparticles were detected in all B gibsoni-infected dogs, 8 of 13 (62%) dogs with primary IMT, and transiently in a dog that responded to immunosuppressive treatment. The results of this study indicate that sample age and time of sampling during disease affect interpretation of platelet parameters in dogs with IMT.     

Glanzmann thrombasthenia in an Oldenbourg filly

An 18-month-old Oldenbourg filly was presented with a bleeding diathesis. Laboratory testing included platelet count, gingival bleeding time, prothrombin time (PT), activated partial thromboplastin time (aPTT), von Willebrand factor (vWf) antigen, clottable fibrinogen, clot retraction time, PFA-100 closure time, platelet aggregometry (on platelet-rich plasma), and thrombelastography (TEG). TEG was performed by using kaolin and tissue factor as coagulation activators. Expression of the platelet receptor for fibrinogen was assessed by flow cytometry by using anti CD41 (alpha(IIb) or glycoprotein IIb)/CD61 (beta(III) or glycoprotein IIIa) and anti-CD41 antibodies. Abnormal laboratory findings included prolonged oral mucosal bleeding time (>12 hours), prolonged closure time with collagen/ADP (>300 seconds), and absence of clot retraction after 60 minutes. TEG reaction times were similar with kaolin and tissue factor in the patient and a control horse. However, maximum amplitudes in the patient were decreased with both kaolin (43.7 mm; control, 63.9 mm) and tissue factor (37.7 mm; control, 57.8 mm). Platelet aggregation responses to ADP and collagen were profoundly reduced in the affected horse compared with a control. Flow cytometry showed an absence of CD41 and decreased expression of CD41/CD61-reacting antigen on the patient's platelets compared with those from a control horse. The laboratory findings supported a diagnosis of Glanzmann thrombasthenia, likely caused by a mutation in the gene encoding the GPIIb subunit.     

Evaluation of platelet function screening tests to detect platelet procoagulant deficiency in dogs with Scott syndrome

Background: Clinical diagnosis of platelet dysfunction is complex and technically challenging. The wide repertoire of platelet responses requires a test panel to assess different parameters of platelet reactivity. While “global” hemostasis analyzers and whole blood assays have potential for testing platelet function, their ability to evaluate platelet procoagulant activity is ill‐defined. Objectives: The aim of this study was to determine whether platelet procoagulant deficiency, the pathophysiologic defect of Scott syndrome, could be detected in point‐of‐care and whole blood assays. Methods: Study subjects were 4 Scott syndrome‐affected German Shepherds and 8 control dogs. We evaluated 2 point‐of‐care instruments: the platelet function analyzer (PFA‐100) and thromboelastograph (TEG). TEG analysis was performed on recalcified citrated whole blood with and without tissue‐factor activation. A whole blood flow cytometric assay was configured to detect thrombin‐induced platelet P‐selectin expression and platelet‐derived microparticle release. Cytometric samples were analyzed after 1 hour and 1 day of storage. Results: We found no significant differences between Scott and control dogs in PFA‐100 COL/ADP closure times or in any TEG parameter in tissue‐factor–activated samples. In nonactivated samples, mean clotting time (K) and time to maximal rate of thrombus generation were significantly prolonged in Scott dogs; however, values overlapped with those of control dogs. Cytometric analysis of samples from Scott dogs showed significantly diminished platelet‐derived microparticle release. Samples from all dogs reanalyzed after 1 day of storage had nonspecific increases in basal P‐selectin expression and vesiculation. Conclusion: A whole blood cytometric assay to detect stimulated platelet microparticle release can be used to screen for Scott syndrome. However, platelet activation artifacts preclude overnight storage for next‐day analysis.     

Flow cytometric detection of activated platelets in the dog

Background: Platelet activation appears to play a role in a variety of canine thrombotic disorders. At present, tests for the detection of activated platelets are not used routinely in veterinary clinical laboratories. Objective: The purpose of this study was to develop a clinically applicable method to detect activated canine platelets. Methods: A flow cytometric assay was developed to detect activated platelets, platelet aggregates, and platelet microparticles in the dog. Blood was collected from healthy dogs using EDTA or sodium citrate as the anticoagulant, and platelet‐rich plasma was prepared. Platelets were activated by adding phorbol myristate acetate. In some experiments, platelets were fixed by incubation with 0.5% paraformaldehyde. In other experiments, platelets were stored for 4 or 24 hours at 4°C before analysis. Activated platelets were detected by measuring surface expression of P‐selectin and by determining the percentages of platelet aggregates and microparticles using forward‐angle vs side‐angle light scatter plots. Results were analyzed by using 2‐way ANOVA and the SchefféF‐test. Results: Platelets collected in EDTA had minimal expression of P‐selectin, whereas platelets collected in sodium citrate had greater median fluorescence intensity. Fixation with 0.5% paraformaldehyde before labeling platelets with anti‐P‐selectin did not affect antibody binding or the percentages of platelet aggregates and microparticles. Storage of platelet‐rich plasma at 4°C for 4 hours did not affect antibody binding or the percentages of platelet aggregates or microparticles. Activation of platelets ex vivo by addition of 10 ng/mL phorbol myristate acetate resulted in a large increase in expression of P‐selectin but only slight increases in platelet aggregates and microparticles. Conclusion: Determination of platelet P‐selectin expression and percentages of platelet aggregates and platelet microparticles may provide a clinically applicable means for detection of activated platelets in dogs. The capacity to use EDTA‐anticoagulated blood samples and to fix platelets for evaluation at a later time makes the test attractive as a routine diagnostic tool.     

Two genetic defects in alphaIIb are associated with type I Glanzmann's thrombasthenia in a Great Pyrenees dog: a 14-base insertion in exon 13 and a splicing defect of intron 13

Glannzmann's thrombasthenia (GT) is an autosomal recessive bleeding disorder caused by qualitative or quantitative deficiencies of the platelet membrane glycoprotein alphaIIbbeta3. This is the first report of a molecular genetic basis for type I GT in dogs. As previously reported, a thrombasthenic Great Pyrenees dog (dog No. 1) experienced uncontrolled epistaxis despite results of coagulation screening tests, platelet quantitation, and von Willebrand factor quantitation that were within reference ranges. Platelet aggregation was minimal in response to agonists. Flow cytometry, autoradiography, and immunoblot experiments demonstrated either marked reduction or absence of glycoproteins alphaIIb and beta3. In this study, we report the presence of a 14-base insertion in exon 13 and defective splicing of intron 13 in the alphaIIb gene of two thrombasthenic dogs (Nos. 1 and 8). The insertion disrupted the fourth alphaIIb calcium-binding domain, caused a shift in the reading frame and resulted in a premature termination codon. Possible consequences of this mutation include decreased alphaIIb mRNA stability and production of truncated alphaIIb protein that lacks the transmembrane and cytoplasmic domains and a large portion of the extracellular domain. We identified the dam, sire, and three littermates of dog No. 8 as carriers of the alphaIIb mutation. Canine alphaIIb and beta3 genes share significant homology with the genes in human beings, making canine GT an excellent translational model for human GT. A defined molecular basis for canine GT will enhance ongoing gene therapy research and increase the understanding of structure-function relationships of this integrin.     

Quantitation of reticulated platelets in healthy dogs and in nonthrombocytopenic dogs with clinical disease

Background — Thrombocytopenia is a common disorder in dogs and development of an objective diagnostic assay to measure platelets newly released from bone marrow into the blood would provide a noninvasive way to predict megakaryocytopoiesis. Reticulated platelets are newly released platelets with increased concentrations of RNA that can be detected by flow cytometric analysis of blood stained with thiazole orange (TO).
Objectives — The goals of this study were to establish a reproducible method to quantitate reticulated platelets in dogs, to establish a reference interval for reticulated platelet percentages in healthy dogs, and to determine whether the percentage of reticulated platelets was nonspecifically increased in nonthrombocytopenic dogs with clinical disease.
Methods — Blood samples were obtained from healthy dogs and from nonthrombocytopenic dogs presented for a variety of disorders. An aliquot of whole blood was stained with TO and a phycoerythrin-labeled monoclonal antibody to platelet CD61, then analyzed by flow cytometry.
Results — The coefficients of variation were 7.8% to 15.6% (intra-assay precision) and 6.1% to 19.5% (interassay precision). Overnight storage for 18 to 26 hours, under variable conditions, resulted in an increase in the percentage of platelets staining with TO. The reference interval for reticulated platelets in the healthy control group was 0–4.3% (0–12,095/μL). No significant differences were found in the mean percentage of reticulated platelets or absolute concentration of reticulated platelets between control and affected dogs.
Conclusions — These studies demonstrate a reliable, noninvasive diagnostic assay for measurement of reticulated platelets in whole blood and provide a baseline for assessment of the clinical utility of the assay.     

Effects of oxypolygelatin and dextran 70 on hemostatic variables in dogs

Objective To evaluate and compare coagulation variables following the administration of oxypolygelatin and dextran 70 to clinically healthy dogs. Study design Randomized cross‐over experimental study. Animals A total of eight healthy adult female Beagles aged 2–4 years old and weighing 11.8 ± 2.7 kg. Methods The dogs received a 15‐minute intravenous (IV) infusion of 5 mL kg^?1 oxypolygelatin or 10 mL kg^?1 6% dextran 70. Before (PRE) and at 2, 5, and 24 hours after administration, packed cell volume (PCV), total solids concentration (TS), prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen concentration (FIB), platelet numbers (Plat), factor VIII coagulant activity (VIII:C), von Willebrand factor antigen concentration (vWf:Ag) and platelet function and buccal mucosal bleeding time (BMBT) were measured. Platelet function was assessed using aggregation and by measuring ATP release from aggregating platelets over 6 minutes, with 20, 10, and 5 µm ADP and 5 and 10 µg of collagen mL^?1 as platelet activation agonists. Results All baseline values were within our normal ranges, except for one dog that had low vWf:Ag PRE values prior to both dextran and oxypolygelatin administration. Following dextran and oxypolygelatin administration, the PCV and TP were significantly (p < 0.05) decreased. Plat, FIB, and vWf:Ag decreased, while BMBT and VIII:C increased following dextran administration. Dextran also caused a significant decrease in platelet aggregation in response to ADP. Oxypolygelatin caused a significant decrease in vWf:Ag, Plat, and FIB compared to PRE values. The total amount of ATP released, standardized to platelet number, did not vary significantly for either group at any sampling time from PRE values. No significant changes from PRE values were noted at any time in either group for PT or APTT. Conclusion At the doses administered, both dextran and oxypolygelatin can interfere with hemostatic variables in healthy dogs, but dextran's effect is more profound and prolonged when compared to oxypolygelatin. Clinical relevance Oxypolygelatin causes fewer hemostatic abnormalities when compared to dextran, making it a superior colloid for administration at the doses tested.     

Evaluation of canine heat shock transcription factor 4 (HSF4) as a candidate gene for primary cataracts in the Dachshund and the Entlebucher Mountain dog

OBJECTIVE: Testing of the cataract-causing insertion/deletion mutation in the canine HSF4 gene for its linkage and association with primary cataracts (CAT) in Dachshunds and Entlebucher Mountain dogs. MATERIALS: Exon 9 with flanking intronic regions of the canine HSF4 gene was sequenced in 24 Dachshunds and 20 Entlebucher Mountain dogs. The HSF4 cDNA sequence of lens tissue was analyzed in a CAT-unaffected mixed-breed dog and in three CAT-affected dogs of different breeds, including a Wire-haired Dachshund, a Dachshund-mix and a German Shepherd dog. RESULTS: In all dogs investigated here, the previously reported CAT-causing mutation did not exist. We found a single nucleotide polymorphism (SNP) in intron 9, which was neither associated nor linked with the CAT phenotype in the two dog breeds. CONCLUSION: The CAT phenotype in the two dog breeds investigated here was not caused by the same mutation found to be associated with early-onset CAT in the Staffordshire Bull Terrier and Boston Terrier. The intronic SNP may be useful to test HSF4 for linkage with CAT in further dog breeds.     

Platelet aggregation studies in acute experimental canine ehrlichiosis

BACKGROUND: Thrombocytopenia is the most common and consistent hematologic finding in patients with canine monocytic ehrlichiosis. Dogs that recover from the severe thrombocytopenia still show bleeding tendencies, which suggest that platelet dysfunction is present. OBJECTIVES: The purpose of this study was to determine the occurrence and duration of platelet dysfunction in dogs with ehrlichiosis and to assess whether dysfunction is related to thrombocytopenia. METHODS: Ten adult male and female mongrel dogs were used in the study; 7 were inoculated intravenously with whole blood containing Ehrlichia canis, and 3 were used as controls. Platelet aggregation (with collagen/epinephrine and adenosine diphosphate (ADP)/epinephrine) and platelet counts were evaluated weekly for 112 days. RESULTS: The infected group showed a decrease in platelet aggregation response to collagen/epinephrine and ADP/epinephrine on days 7, 14, 21, 28, and 35 (P <.05). Thrombocytopenia was observed in all infected animals from day 7 to 35 postinfection (P <.05). CONCLUSIONS: The tendency of dogs infected with E canis to bleed may be related not only to thrombocytopenia but also to platelet dysfunction associated with the disease.     

Identification of an Intrinsic Platelet Function Defect in Spitz Dogs

A recently identified intrinsic platelet function defect in 2 Spitz dogs is described. Both affected dogs had a history of chronic intermittent bleeding primarily from the nasal, oral, and gastrointestinal mucosa. Platelet aggregation in response to adenosine diphosphate (ADP), collagen, and platelet activating factor (PAF) was absent; however, platelet shape change did occur. Platelets aggregated in response to gamma thrombin, although a delayed onset and a reduced velocity of aggregation were present. Platelet ¹⁴C-serotonin release was diminished in response to collagen and PAF. Glycoprotein Illa was detected on the surface of platelets by flow cytometry. Platelets were morphologically normal under light and electron microscopy. Two male Spitz dogs, related to one of the affected dogs, did not have a bleeding diathesis. Collagen-induced platelet aggregation, however, was diminished in these 2 dogs. This platelet defect most closely resembles the defect described in Basset hounds.     

Evaluation of platelet function in dogs with cardiac disease using the PFA‐100 platelet function analyzer

Background: Cardiac disease has the potential to alter platelet function in dogs. Evaluation of platelet function using the PFA‐100 analyzer in dogs of multiple breeds and with a broad range of cardiac conditions would help clarify the effect of cardiac disease on platelets. Objectives: The objective of this study was to assess differences in closure time (CT) in dogs with cardiac disease associated with murmurs, when compared with that of healthy dogs. Methods: Thirty‐nine dogs with cardiac murmurs and turbulent blood flow as determined echocardiographically were included in the study. The dogs represented 23 different breeds. Dogs with murmurs were further divided into those with atrioventricular valvular insufficiency (n=23) and subaortic stenosis (n=9). Fifty‐eight clinically healthy dogs were used as controls. CTs were determined in duplicate on a PFA‐100 analyzer using collagen/ADP cartridges. Results: Compared with CTs in the control group (mean±SD, 57.6±5.9 seconds; median, 56.5 seconds; reference interval, 48.0–77.0 seconds), dogs with valvular insufficiency (mean±SD, 81.9±26.3 seconds; median, 78.0 seconds; range, 52.5–187 seconds), subaortic stenosis (71.4±16.5 seconds; median, 66.0 seconds; range, 51.5–95.0 seconds), and all dogs with murmurs combined (79.6±24.1 seconds; median, 74.0 seconds; range, 48.0–187 seconds) had significantly prolonged CTs (P<.01). Conclusions: The PFA‐100 analyzer is useful in detecting platelet function defects in dogs with cardiac murmurs, most notably those caused by mitral and/or tricuspid valvular insufficiency or subaortic stenosis. The form of turbulent blood flow does not appear to be an important factor in platelet hypofunction in these forms of cardiac disease.     

Platelet Hyperfunction in Dogs With Malignancies

In vitro platelet aggregometry was performed on whole blood samples from 59 dogs with malignancies and 24 control dogs. Three reagents were used for the aggregation studies: collagen, arachidonic acid, and adenosine diphos-phate (ADP). The parameters measured to evaluate response to collagen included delay in the aggregation response, slope of the aggregation curve, maximum aggregation, and adenosine triphosphate (ATP) secretion. The platelets of dogs with malignancies exhibited significantly ( P < .05) shorter delays in the aggregation response, higher maximum aggregation, and higher ATP secretion when compared to control dogs. For the weaker reagents, ADP and arachidonic acid, the lowest concentration resulting in aggregation was determined. Platelets of dogs with malignancies tended to aggregate in response to lower concentrations of ADP than did those of controls ( P < .05). The response of platelets to the concentrations of arachidonic acid employed in this study was poor, with few samples achieving measurable aggregation. The findings of this study suggest that dogs with malignancies have hyperaggregable platelets.     

Calcium diacylglycerol guanine nucleotide exchange factor I (CalDAG-GEFI) gene mutations in a thrombopathic Simmental calf

Simmental thrombopathia is an inherited platelet disorder that closely resembles the platelet disorders described in Basset Hounds and Eskimo Spitz dogs. Recently, two different mutations in the gene encoding calcium diacylglycerol guanine nucleotide exchange factor I (CalDAG-GEFI) were described to be associated with the Basset Hound and Spitz thrombopathia disorders, and a third distinct mutation was identified in CalDAG-GEFI in thrombopathic Landseers of European Continental Type. The gene encoding CalDAG-GEFI was sequenced using DNA obtained from normal cattle and from a thrombopathic calf studied in Canada. The affected calf was found to have a nucleotide change (c.701 T>C), which would result in the substitution of a proline for a leucine within structurally conserved region two (SCR2) of the catalytic domain of the protein. This change is likely responsible for the thrombopathic phenotype observed in Simmental cattle and underscores the critical nature of this signal transduction protein in platelets.     

Alterations in bovine platelet function and acute phase proteins induced by Pasteurella haemolytica A1.

Platelet function was assessed by aggregometry in 10 Holstein calves before and after exposure to Pasteurella haemolytica (biotype A, serotype 1) by intrabronchial challenge. At 24 h after exposure the platelets had become more reactive to stimulation with known platelet agonists such as adenosine diphosphate (ADP) and platelet-activating factor (PAF) and the platelet aggregates that formed were more resistant to disaggregation. The activation of platelets was an early response in the challenged calves as platelet function had returned to pretreatment levels 72 h after exposure to the bacteria while the acute phase reactant proteins, haptoglobin and fibrinogen, were approaching their peak values and alpha 2-macroglobulin levels had also risen significantly (P < 0.05) at this time. The plasma levels of these proteins were still elevated and albumin levels were depressed 6 d post-treatment. At post-mortem all calves exhibited pneumonic tissue damage. When P. haemolytica leukotoxin was added directly to bovine platelet suspensions both spontaneous aggregation and an increase in the aggregation response to ADP and PAF stimulation were observed. The morphological appearance of the platelet aggregates exhibited the typical pattern for bovine platelets with 2 distinct zones of cells being visible within each aggregate. One zone contained platelets in which the cytoplasmic granules were still evident and the other zone contained irregularly shaped platelets devoid of granular content. In the latter zone, discrete gaps, or pores, were evident in the plasma membrane of numerous platelets. This pore formation is characteristic of leukotoxin action and is not observed in ADP or PAF induced aggregates.