Decreased platelet function in Cavalier King Charles Spaniels with mitral valve regurgitation

With aggregometry, increased platelet activity has been reported in Cavalier King Charles Spaniels (CKCS) without mitral regurgitation (MR). In contrast, dogs with MR have been found to have decreased platelet activity. The purpose of this study was to test an easy bedside test of platelet function (the Platelet Function Analyzer [PFA-100]) to see if it could detect an increase in platelet activity in CKCS without MR and a decrease in platelet activity in CKCS with MR. This study included 101 clinically healthy dogs > 1 year of age: 15 control dogs of different breeds and 86 CKCS. None of the dogs received medication or had a history of bleeding. The PFA-100 evaluates platelet function in anticoagulated whole blood under high shear stress. Results are given as closure times (CT): the time it takes before a platelet plug occludes a hole in a membrane coated by agonists. The CT with collagen and adenosine-diphosphate as agonists was similar in control dogs (median 62 seconds; interquartile interval 55-66 seconds) and CKCS with no or minimal MR (55; 52-64 seconds). The CT was higher in CKCS with mild MR (regurgitant jet occupying 15-50% of the left atrial area) (75; 60-84 seconds; P = .0007) and in CKCS with moderate to severe MR (jet > 50%) (87: 66-102 seconds; P < .0001). CKCS with mild, moderate, and severe, clinically inapparent MR have decreased platelet function. The previous finding of increased platelet reactivity in nonthrombocytopenic CKCS without MR could not be reproduced with the PFA-100 device.     

Increased platelet aggregation response in Cavalier King Charles Spaniels with mitral valve prolapse

Mitral valve prolapse (MVP) is a fundamental feature of myxomatous mitral valve disease in the dog. In humans, primary MVP is associated with increased platelet reactivity. In Cavalier King Charles Spaniels (CKCS), a breed predisposed to myxomatous mitral valve disease, there is a high prevalence of hypomagnesemia and platelet anomalies, such as thrombocytopenia and macrothrombocytosis. The objective of this study was to evaluate platelet aggregation responses in CKCS and to determine the relationship between the platelet aggregation response and serum magnesium concentration, MVP, mitral regurgitation (MR), and platelet count. In 19 CKCS with MVP and 7 control dogs (not CKCS), the platelet aggregation response to 3 different agonists was compared. The CKCS with >100,000 platelets/microL (n = 10) had a significantly higher maximum aggregation response with regard to all tested agonists than the CKCS with <100,000 platelets/microL (n = 9) and control dogs (n = 7). The CKCS with <100,000 platelets/microL had a platelet aggregation response similar to the control dogs. There was no correlation between degree of MVP and platelet aggregation response. Platelet diameter increased (P = .006) and serum magnesium concentration decreased (P = .04) with lower platelet concentration. In conclusion, CKCS with MVP appeared to separate into 2 groups--1 group with <100,000 platelets/microL, normal platelet aggregation, low serum magnesium concentration, and enlarged platelets, and another group with >100,000 platelets/microL, increased platelet aggregation, and normal serum magnesium concentration and platelet size.     

Assessment of changes in hemostatic markers in Cavalier King Charles Spaniels with myxomatous mitral valve disease

OBJECTIVE: To evaluate markers of hemostasis and their relationship to the degree of mitral regurgitation (MR) and platelet function in Cavalier King Charles Spaniels (CKCSs) with myxomatous mitral valve disease. ANIMALS: 76 clinically healthy CKCSs and 24 control dogs. PROCEDURE: All dogs underwent echocardiographic examination; various hemostatic, hematologic, and biochemical variables were evaluated in blood. The CKCSs were allocated to 1 of 3 groups on the basis of MR severity. In 8 control dogs and 8 CKCSs, plasma von Willebrand factor (vWF) multimer analysis was performed. RESULTS: Compared with control dogs, plasma fibrinogen concentration was higher in all CKCSs and related to left ventricular end diastolic diameter and left atrial-to-aortic root ratio among all CKCSs. The activated partial thromboplastin times and plasma D-dimer concentration were similar among the 4 groups. Plasma vWF concentration was lower in CKCSs with moderate to severe MR, compared with that of CKCSs with no MR and control dogs. There was a relationship between plasma vWF concentration and platelet function in CKCSs but not in control dogs. In 4 CKCSs with moderate to severe MR and low plasma vWF concentration, amounts of vWF high-molecular-weight multimers (HMWMs) were low. CONCLUSIONS AND CLINICAL RELEVANCE: In CKCSs, MR appeared to be associated with a low plasma vWF concentration and likely a loss of vWF HMWMs (possibly through their destruction via shear stress to the blood). The importance of the changes in plasma fibrinogen concentration and the thromboembolic risk in dogs with MR remain to be investigated.     

Idiopathic thrombocytopenia in Cavalier King Charles Spaniels

OBJECTIVE: To determine the prevalence of asymptomatic idiopathic macrothrombocytopenia in the population of Cavalier King Charles Spaniels (CKCS) in New South Wales (NSW) and to determine if it exhibits an autosomal recessive inheritance pattern. We also aimed to determine if significant differences existed when counting platelets manually, by auto analyser or by blood smear estimation in CKCS and mixed breed dogs. METHODS: Blood was collected from 172 dogs (152 CKCS and 20 mixed breed) and placed into sodium-citrate anticoagulant. Platelet counts were performed manually, by auto analyser and by blood smear estimates in CKCS and mixed breed dogs. Blood smears were also examined for platelet clumping and erythrocyte, leukocyte and platelet morphology. Pedigree analysis was performed to determine if an autosomal recessive inheritance pattern was supported. RESULTS: A statistically significant difference was found in platelet counts between CKCS and mixed breed dogs (P < 0.0001). CKCS had a platelet count that was 32% that of the controls (95% confidence interval, 28 to 37%). There was no significant difference between methods used to count platelets. Thirty percent of CKCS had macrothrombocytes. Pedigree analysis and examination of obtained and expected segregation ratios from 17 CKCS families supported an autosomal recessive pattern of Mendelian inheritance. CONCLUSIONS: A high prevalence of idiopathic macrothrombocytopenia exists in CKCS in NSW and automated or blood smear estimates are sufficient to count platelet numbers. Data supports an autosomal recessive inheritance pattern.     

Immunoglobulin deficiency in Cavalier King Charles Spaniels with Pneumocystis pneumonia

Serum concentrations of immunoglobulin G (IgG), IgM, and IgA were measured in 9 Cavalier King Charles Spaniels with pneumonia caused by Pneumocystis sp that were examined at 4 veterinary surgeries in the United Kingdom (UK) between September 2001 and November 2002. Pneumocystis pneumonia was confirmed in all dogs by visualization of the organism in bronchoalveolar lavage fluid or a transthoracic lung aspirate. Two dogs had a history of demodicosis. Immunoglobulin concentrations also were measured in breed-and age-matched dogs sampled over the same period. IgG concentrations were significantly (P = .000) lower in the affected dogs (median 3.2 mg/mL) than in the control dogs (median 8.5 mg/mL). IgM concentrations were significantly (P = .002) higher in the affected dogs (median 1.95 mg/mL) than in the control dogs (median 1.12 mg/mL). One affected dog had no change in IgG concentration more than 3 months after resolution of infection or vaccination, but did have reduction in IgM concentration after resolution of infection and vaccination. Control dogs had low serum IgG and IgM concentrations, compared with the reference interval for all dogs. Lymphocyte count in blood was normal or high in 7 of 8 affected dogs. The results of this study suggest that there is a defect in immunity in Cavalier King Charles Spaniels that underlies the susceptibility of these dogs to pneumocystosis. Further studies are indicated to elucidate the mechanisms behind the defect, the prevalence within the breed, and the potential mode of inheritance of the problem.     

Mitral valve prolapse in 3-year-old healthy Cavalier King Charles Spaniels. An echocardiographic study.

Clinical studies have shown that Cavalier King Charles Spaniels (CKCS) have a high prevalence of mitral valvular insufficiency (MVI). Echocardiography has the potential to disclose early valvular changes, and the present prospective study was designed to investigate the occurrence of mitral valve prolapse (MVP) in young CKCS without heart murmurs, and to correlate the degree of MVP with the clinical status of the dogs by including CKCS with MVI as well. The study was based on blinded evaluations of echocardiographic recordings of mitral valves from 34 CKCS and 30 control dogs. Thirteen (87%) of 15 three-year-old CKCS without heart murmurs had MVP (2 total and 11 partial), as compared with 1 (7%) of 15 three-year-old normal Beagle dogs (P < 0.0001), and none of 15 three-year-old normal Medium Size Poodles (P < 0.0001). Of 19 CKCS with MVI, MVP was found in 84% of the entire group and in 100% of dogs with pulmonary congestion or edema. The occurrence of total MVP tended to be higher in the group with MVI (47%, 9/19), when compared with the younger CKCS without heart murmurs (13%, 2/15, P = 0.06). MVP was positively associated with excessive heart rate variability (P = 0.003). The radius of curvature of the anterior mitral valve leaflet in systole was significantly reduced in dogs with MVP when compared with those without (P < 0.0001). In conclusion, this study shows that CKCS at an early age have a high occurrence of MVP. This suggests: 1) A genetic predisposition of CKCS to MVP; and 2) That MVP is a pathogenetic factor in the development of mitral valvular insufficiency. Follow up studies may add further support to these proposals, and clarify whether echocardiography may be an aid in selecting CKCS for future breeding.     

Idiopathic asymptomatic thrombocytopenia in Cavalier King Charles Spaniels is an autosomal recessive trait

Cavalier King Charles Spaniels (CKCS) often have idiopathic asymptomatic thrombocytopenia. In affected dogs, the thrombocytes often are large, and it has been speculated that the condition could be an inherited macrothrombocytopenia. The aim of this study was to examine the inheritance of idiopathic, asymptomatic thrombocytopenia in CKCS. Sixteen families (both parents and > or = 3 offspring) of privately owned CKCS were included. There were 105 clinically healthy dogs (50 from Denmark and 55 from Sweden): 81 offspring and 26 parents (2 dogs had both roles). Because autoanalyzers have difficulty counting large platelets, the platelets were counted manually, with a counting chamber. Platelet counts were not influenced by age, gender, or heart murmur status. Thrombocytopenia (< or = 100,000 platelets/microL) was found in 46% of the parents. The pedigrees indicated that thrombocytopenia segregated as an autosomal recessive trait and that 100,000 platelets/microL was appropriate as a lower limit of normal. Affected offspring were found in all families, showing that all of the included parents were at least carriers. Therefore, the expected segregation ratios (which were in good accordance with the observed ones) were 1:0, 1:1, and 1:3 for the 3 crosses: affected x affected, normal x affected, and normal x normal. Within a given cross, the mean parental platelet count had no influence on the platelet counts of the offspring. We conclude that idiopathic, asymptomatic thrombocytopenia in CKCS is inherited in an autosomal recessive manner. The condition most likely constitutes an inherited macrothrombocytopenia in dogs.     

Mutation in beta1-tubulin correlates with macrothrombocytopenia in Cavalier King Charles Spaniels.

BACKGROUND: Cavalier King Charles Spaniels (CKCS) have a high prevalence of inherited macrothrombocytopenia. The purpose of this study was to determine if a mutation in beta1-tubulin correlated with presumptive inherited macrothrombocytopenia. HYPOTHESIS: A mutation in beta1-tubulin results in synthesis of an altered beta1-tubulin monomer. alpha-beta tubulin dimers within microtubule protofilaments are unstable, resulting in altered megakaryocyte proplatelet formation. ANIMALS: Blood samples were obtained from CKCS and non-CKCS dogs. METHODS: DNA was used in polymerase chain reaction (PCR) assays to evaluate beta1-tubulin. Platelet numbers and mean platelet volume (MPV) were evaluated for a correlation with the presence or absence of a mutation identified in beta1-tubulin. Platelets obtained from homozygous, heterozygous, and clear CKCS were further evaluated using electron microscopy and immunofluorescence. RESULTS: A mutation in the gene encoding beta1-tubulin correlated with macrothrombocytopenia in CKCS. Electron microscopy and immunofluorescence studies suggest that platelet microtubules are present but most likely are unstable and decreased in number. CONCLUSIONS AND CLINICAL IMPORTANCE: The macrothrombocytopenia of CKCS correlated with a mutation in beta1-tubulin. alpha-beta tubulin dimers within protofilaments most likely are unstable, leading to altered proplatelet formation by megakaryocytes. This information will aid in distinguishing inherited from acquired thrombocytopenia. It also provides insight into the mechanism of platelet production by megakaryocytes, and also may prove useful in understanding heart-related changes in macrothrombocytopenic CKCS with concurrent mitral valve regurgitation.     

Pulmonary blood volume in mitral regurgitation in Cavalier King Charles spaniels

Background: Pulmonary edema and venous congestion are well‐recognized signs of congestive heart failure (CHF) in advanced canine chronic mitral regurgitation (MR). However, little is known about pulmonary blood volume (PBV), blood pulmonary transit time (PTT), and the regulation of these. Objectives: To measure and evaluate the relationships of PBV, forward stroke volume (FSV), and heart rate normalized blood pulmonary transit time (nPTT) in healthy dogs and dogs with MR. Animals: Thirty‐three Cavalier King Charles Spaniels; 11 healthy, 4 in modified New York Heart Association (NYHA) class I, 11 in class II, and 7 in CHF. Methods: Heart rate normalized PTTs were measured by radionuclide angiocardiography. Left ventricular end diastolic and systolic diameter, left atrial/aortic root ratio, and FSV were measured by echocardiography. PBV and pulmonary blood volume index (PBVI) were calculated by established formulas. Results: PBVI was 308 ± 56 (mean ± SD) mL/m² for healthy dogs, 287 ± 51 mL/m² in NYHA class I, 360 ± 66 mL/m² in Class II, and 623 ± 232 mL/m² in CHF (P= .0008). Heart rate normalized PTT, not FSV, was a predictor of PBV (r= 0.92 and 0.02, respectively). Conclusions and Clinical Importance: Increased PBV, not decreased FSV, is the main cause of increased nPTT in MR. Increased nPTT can be used as an indicator of abnormal cardiopulmonary function in dogs with MR.     

Plateletcrit is superior to platelet count for assessing platelet status in Cavalier King Charles Spaniels

Background: Many Cavalier King Charles Spaniel (CKCS) dogs are affected by an autosomal recessive dysplasia of platelets resulting in fewer but larger platelets. The IDEXX Vet Autoread (QBC) hematology analyzer directly measures the relative volume of platelets in a blood sample (plateletcrit). We hypothesized that CKCS both with and without hereditary macrothrombocytosis would have a normal plateletcrit and that the QBC results would better identify the total circulating volume of platelets in CKSC than methods directly enumerating platelet numbers.
Objectives: The major purpose of this study was to compare the QBC platelet results with platelet counts from other automated and manual methods for evaluating platelet status in CKCS dogs.
Methods: Platelet counts were determined in fresh EDTA blood from 27 adult CKCS dogs using the QBC, Sysmex XT-2000iV (optical and impedance), CELL-DYN 3500, blood smear estimate, and manual methods. Sysmex optical platelet counts were reanalyzed following gating to determine the number and percentage of normal- and large-sized platelets in each blood sample.
Results: None of the 27 CKCS dogs had thrombocytopenia (defined as <164 × 10⁹ platelets/L) based on the QBC platelet count. Fourteen (52%) to 18 (66%) of the dogs had thrombocytopenia with other methods. The percentage of large platelets, as determined by regating the Sysmex optical platelet counts, ranged from 1% to 75%, in a gradual continuum.
Conclusions: The QBC may be the best analyzer for assessing clinically relevant thrombocytopenia in CKCS dogs, because its platelet count is based on the plateletcrit, a measurement of platelet mass.     

Distribution of syringomyelia along the entire spinal cord in clinically affected Cavalier King Charles Spaniels

Chiari-like malformation (CM) and syringomyelia (SM) is an important disease complex in the Cavalier King Charles Spaniel (CKCS) but data about the anatomical distribution of SM along the spinal cord are lacking in veterinary medicine. The objective of this study was to define the anatomic distribution of SM in CKCS clinically affected by CM/SM. Magnetic resonance imaging (MRI) of the brain and the entire spinal cord of 49 dogs was performed and different morphological parameters compared.Syrinx formation was present in the C1–C4 region and in other parts of the spinal cord. The maximal dorsoventral syrinx size can occur in any region of the spinal cord and the total syrinx size was positively correlated with age. Seventy-six per cent of CKCS with a cranial cervical syrinx also have a syrinx affecting more caudal spinal cord regions. MRI restricted to the cervical region may underestimate the extent of SM and the severity of the disease process in the majority of dogs.     

Mutation in β1-Tubulin Correlates with Macrothrombocytopenia in Cavalier King Charles Spaniels

Background: Cavalier King Charles Spaniels (CKCS) have a high prevalence of inherited macrothrombocytopenia. The purpose of this study was to determine if a mutation in β1-tubulin correlated with presumptive inherited macrothrombocytopenia.
Hypothesis: A mutation in β1-tubulin results in synthesis of an altered β1-tubulin monomer. α-β tubulin dimers within microtubule protofilaments are unstable, resulting in altered megakaryocyte proplatelet formation.
Animals: Blood samples were obtained from CKCS and non-CKCS dogs.
Methods: DNA was used in polymerase chain reaction (PCR) assays to evaluate β1-tubulin . Platelet numbers and mean platelet volume (MPV) were evaluated for a correlation with the presence or absence of a mutation identified in β1-tubulin. Platelets obtained from homozygous, heterozygous, and clear CKCS were further evaluated using electron microscopy and immunofluorescence.
Results: A mutation in the gene encoding β1-tubulin correlated with macrothrombocytopenia in CKCS. Electron microscopy and immunofluorescence studies suggest that platelet microtubules are present but most likely are unstable and decreased in number.
Conclusions and Clinical Importance: The macrothrombocytopenia of CKCS correlated with a mutation in β1-tubulin . α–β tubulin dimers within protofilaments most likely are unstable, leading to altered proplatelet formation by megakaryocytes. This information will aid in distinguishing inherited from acquired thrombocytopenia. It also provides insight into the mechanism of platelet production by megakaryocytes, and also may prove useful in understanding heart-related changes in macrothrombocytopenic CKCS with concurrent mitral valve regurgitation.     

Increased NT-proANP predicts risk of congestive heart failure in Cavalier King Charles spaniels with mitral regurgitation caused by myxomatous valve disease

ObjectivesTo evaluate the predictive value of plasma N-terminal pro-atrial natriuretic peptide (NT-proANP) and nitric oxide end-products (NOx) as markers for progression of mitral regurgitation caused by myxomatous mitral valve disease.AnimalsSeventy-eight privately owned Cavalier King Charles spaniels with naturally occurring myxomatous mitral valve disease.MethodsProspective longitudinal study comprising 312 measurements over a 4.5 year period. Clinical values were recorded, NT-proANP concentrations were measured by radioimmunoassay, and NOx were analyzed colorimetrically. To predict congestive heart failure (CHF), Cox proportional hazards models with time-varying covariates were constructed.ResultsThe hazard ratio for NT-proANP (per 1000 pmol/l increase) to predict future CHF was 6.7 (95% confidence interval, 3.6–12.5; p < 0.001). The median time to CHF for dogs with NT-proANP levels >1000 pmol/l was 11 months (95% confidence interval, 5.6–12.6 months), compared to 54 months (46 – infinity) for dogs with concentrations ≤1000 pmol/l (p < 0.001). Due to intra- and inter-individual variability, most corresponding analyses for NOx were insignificant but dogs reaching CHF had a lower mean NOx concentration than dogs not reaching CHF (23 vs. 28 μmol/l, p = 0.016). Risk of CHF increased with increase in heart rate (>130 beats per minute) and grade of murmur (≥3/6).ConclusionsThe risk of CHF due to mitral regurgitation is increased in dogs with blood NT-proANP concentrations above 1000 pmol/l. Measurement of NT-proANP can be a valuable tool to identify dogs that may develop CHF within months.