Chronic degenerative atrioventricular valve disease is the leading cause of heart failure in dogs, accounting for 70% of cardiovascular diseases in this species. Although the mitral valve is most frequently affected, isolated myxomatous degeneration of the tricuspid, aortic, and pulmonic valves is less common in veterinary practice (11). In human studies, patients with valvular heart disease have shown platelet (PLT) function defects that are detectable under high shear stress conditions (9,17). Valvular heart disease may influence PLT activation and function due to the turbulent, high-velocity blood flow and resulting shear stress on blood cells (24). Therefore, an increase in PLT activation and reactivity is initially anticipated.

PLT function testing has been explored as a potential method for predicting clinical outcomes in cardiovascular diseases and monitoring the efficacy of antiplatelet therapy (4,13). The Platelet Function Analyzer-200 (PFA-200) evaluates PLT function by assessing the time required for a PLT plug to occlude a small orifice in a coated membrane under high shear stress (4,5,13). This device uses anticoagulated whole blood and simulates in vivo blood flow at high shear rates, thus providing an alternative to PLT aggregometry, which assesses PLT function under lower shear conditions in plasma. Additionally, the PFA-200 device is user-friendly and does not require specialized laboratory facilities (5). This study aimed to investigate the potential differences in coagulation time—measured using the PFA-200—in dogs with myxomatous mitral valve disease (MMVD) in relation to the presence of cardiac remodeling (10). We hypothesized that the closure times (CT) for collagen-adenosine diphosphate (C/ADP) and collagen-epinephrine (C/EPI) in the PFA test results would be prolonged in the cardiac remodeling group.

Animals

This study was approved by our Institutional Animal Care and Use Committee. In total, 55 client-owned dogs were assessed at the Jeonbuk National University Animal Center between October 2023 and August 2024. Dogs with MMVD were classified into the control (n = 18), stage B1 (n = 12), stage B2 (n = 11), and stage C (n = 14) groups, following the guidelines established by the American College of Veterinary Internal Medicine. The dogs were further divided into the non-cardiac remodeling (normal, MMVD stage B1; n = 30) and cardiac remodeling groups (MMVD stages B2 and C; n = 25). Additionally, dogs receiving medications unrelated to MMVD management were excluded from this study. Also, the MMVD stage C patients in the cardiac remodeling group were included only if they were under chronic management. The non-cardiac remodeling group included 11 mixed-breed dogs, one Bichon Frisé, six Cocker Spaniels, three Schnauzers, three Pomeranians, one Chihuahua, four Maltese, and one Poodle. The cardiac remodeling group included five mixed-breed dogs, 12 Maltese, two Poodles, five Chihuahuas, and one Shih Tzu.

Blood collection

Blood samples were collected from the jugular vein using a vacutainer blood collection set with a 21-gauge needle. The samples were drawn into 4.5-mL tubes containing 3.2% sodium citrate and tubes containing ethylenediaminetetraacetic acid as an anticoagulant (25). If complications occurred during venipuncture, an additional sample was obtained from another jugular vein. PLT aggregation tests were performed within 2 h of blood collection.

PFA-200

The PFA-200 (Siemens Canada, Mississauga, Ontario, Canada) was used to assess PLT activation in 800 μL of citrated blood samples with cartridges containing C/ADP- and C/EPI-coated membranes (4,18). CT, measured in seconds, represents the duration required for PLTs to occlude the orifice and halt whole blood flow (13). Prolonged CTs were defined according to the manufacturer’s recommended ranges (71-118 and 85-165 for C/ADP and C/EPI, respectively). Any CT exceeding 300 s was recorded as 300 s (5,18).

Thoracic radiography

Thoracic radiographs were obtained from both ventrodorsal and right lateral views. The vertebral heart score (VHS) and vertebral left atrial size (VLAS) were measured from the right lateral view to assess the cardiac size, as in previous studies (3,14).

The likelihood of congestive heart failure was assessed by correlating the lung parenchymal condition with the clinical symptoms.

Echocardiography

All echocardiographic examinations were performed by a single trained investigator (MS) using ultrasound systems (Philips EPIC 7C, Philips Medical System, Bothell, WA, USA) with 3-8- and 4-12-MHz phased transducers, under the supervision of the corresponding author. The left atrial-to-aortic root ratio (LA:Ao) and left ventricular end-diastolic diameter normalized to body weight (LVIDdN) were measured following a standardized protocol to assess cardiac remodeling and function, as outlined in previous studies (22).

Statistical analysis

Statistical analyses were conducted using commercially available statistical software (IBM SPSS Statistics 29.0, SPSS Inc., USA). The Shapiro-Wilk test was used to evaluate data distribution. The results were expressed as the median and interquartile range based on data distribution. The Mann-Whitney U test was used to compare the values between the non-cardiac remodeling and cardiac remodeling groups. The Spearman’s correlation coefficients (r) were calculated to examine the associations between the CTs of C/ADP and C/EPI and other variables.

Receiver operating characteristic (ROC) curve analysis was conducted to evaluate the CTs of C/ADP and C/EPI in both the cardiac remodeling and non-cardiac remodeling groups. In addition, the area under the curve (AUC) for each indicator was assessed to determine its utility and reliability. The optimal balance of sensitivity and specificity was determined using the Youden index (J = [sensitivity + specificity] – 1). A p-value of <0.05 was considered significant.

Overall, 55 dogs were enrolled in the study, including 18 healthy and 37 dogs with MMVD. Dogs with MMVD were further classified into the stage B1 group (12 dogs), stage B2 group (11 dogs), and stage C group (14 dogs). The dogs with MMVD were further divided into the non-cardiac remodeling (comprising healthy and stage B1 dogs) and cardiac remodeling groups (comprising stage B2 and C dogs).

The characteristics and PLT functional analysis, radiographic, and echocardiographic data of the two groups are summarized in Table 1 and Table 2. In the non-cardiac remodeling group, the median age was 8.0 years (4.0-12.0), with a median PLT count of 334.0 × 10³/μL (250.0-423.5). The median CT of C/ADP was 83.5 s (61.0-101.5), while that of C/EPI was 132.0 s (84.5-152.5). The median LA:Ao was 1.3 (1.2-1.4), median LVIDdN was 1.4 (1.36-1.5), median VHS was 10.5 (10.0-10.9), and median VLAS was 2.1 (1.9-2.2). In the cardiac remodeling group, the median age was 12.0 years (10.0-13.8), with a median PLT of 411.0 × 10³/μL (342.8-594.5). The median CT of C/ADP was 106.0 s (80.5-132.0), while that of C/EPI was 166.0 s (131.0-195.8). The median LA:Ao was 1.9 (1.7-2.2), median LVIDdN was 1.9 (1.8-2.0), median VHS was 11.5 (10.8-12.0), and median VLAS was 2.4 (2.1-3.0). Significant differences were observed in PLT count and CTs of C/ADP and C/EPI values between the non-cardiac and cardiac remodeling groups (Fig. 1). A summary of the correlations between these CTs and other values is presented in Table 3. Notably, the CT values for C/ADP and C/EPI showed positive correlations with both LA:Ao and LVIDdN.

Figure 1. Mean closure times of C/ADP and C/EPI values between the cardiac remodeling and non-cardiac remodeling groups. Box plot illustrating the (A) platelet count, (B) CT of C/ADP, and (C) CT of C/EPI in the 55 dogs examined in this study. The box represents the interquartile range containing the 25th-75th percentiles of the data. The line inside the box indicates the median, while the cross within the box represents the mean value. Outliers are not shown in this figure. A comparison between the non-cardiac and cardiac remodeling groups was conducted, and the results were significant. (*p < 0.05, **p < 0.01). CT, closure time; C/ADP, collagen-adenosine diphosphate; C/EPI, collagen-epinephrine.

Table 1 . Summary characteristics for 55 client-owned dogs reviewed in this study.

	Non-cardiac remodeling group	Cardiac remodeling group
Total (n = 55)	30	25
Age (year)	8.0 (4.0-12.0)	12.0 (10.0-13.8)
Weight	4.6 (3.8-8.3)	4.4 (3.1-5.5)
Sex (M:F)	16:14	19:6

Summary of the characteristics of 55 client-owned dogs at the Jeonbuk National University Animal Center evaluated between October 2023 and August 2024. The dogs were classified, based on cardiac remodeling status, into two groups: non-cardiac remodeling and cardiac remodeling. Median (interquartile range) for continuous data. F, female; M, male..

Table 2 . Summary of PLT functional analysis, radiographic, and echocardiographic data of 55 client-owned dogs.

	Non-cardiac remodeling group	Cardiac remodeling group
CT of C/ADP	83.5 (61.0-101.5)	106.0* (80.5-132.0)
CT of C/EPI	132.0 (84.5-152.5)	166.0** (131.0-195.8)
PLT	334.0 (250.0-423.5)	411.0* (342.8-594.5)
LA:Ao	1.3 (1.2-1.4)	1.9 (1.7-2.2)
LVIDdN	1.4 (1.4-1.5)	1.9 (1.8-2.0)
VHS	10.5 (10-10.9)	11.5 (10.8-12.0)
VLAS	2.1 (1.9-2.2)	2.4 (2.1-3.0)

Summary of PLT functional analysis, radiographic, and echocardiographic data of 55 client-owned dogs at the Jeonbuk National University Animal Center evaluated between October 2023 and August 2024. Median (interquartile range) for continuous data (*p < 0.05, **p < 0.01)..

CT, closure time; C/ADP, collagen-adenosine diphosphate; C/EPI, collagen-epinephrine; PLT, platelet; VHS, vertebral heart size; VLAS, vertebral left atrial size; LA:Ao, left atrial-to-aortic ratio; LVIDdN, left ventricular end diastolic diameter normalized for body weight..

Table 3 . Correlation analysis between the closure times of C/ADP and C/EPI and other values.

	LA:Ao	LVIDdN	VHS	VLAS
C/ADP	0.345**	0.325*	0.052	0.172
C/EPI	0.449**	0.399**	0.191	0.292*
PLT	0.323*	0.235	0.344*	0.419**

Coefficient of correlation (*p < 0.05, **p < 0.01)..

PLT, platelet; LA:Ao, left atrial-to-aortic ratio; LVIDDn, left ventricular end diastolic diameter normalized for body weight; VHS, vertebral heart size; VLAS, vertebral left atrial size; C/ADP, collagen-adenosine diphosphate; C/EPI, collagen-epinephrine..

ROC curve analysis revealed that an elevated CT of C/ADP served as a moderate predictor for distinguishing between the non-cardiac remodeling and cardiac remodeling groups, with an AUC of 0.731 (95% confidence interval [CI]: 0.598-0.863, p < 0.001; Fig. 2). The cutoff value of 90.5 for C/ADP CT yielded a sensitivity of 68% and specificity of 66.7%, indicating its potential utility in identifying stage B2 dogs. Similarly, the ROC curve analysis showed that increased C/EPI CT is a fair predictor for differentiating between non-cardiac and cardiac remodeling groups, with an AUC of 0.729 (95% CI: 0.597-0.861, p < 0.001; Fig. 2). The C/EPI CT cutoff value of 141 yielded a sensitivity of 64% and specificity of 60%, suggesting its applicability in identifying dogs with cardiac remodeling associated with MMVD.

Figure 2. Receiver operator characteristic curve demonstrating the diagnostic performance of C/ADP and C/EPI in the cardiac remodeling group (stages B2 and C dogs). (A) The AUC for the CT of ADP was 0.731 (p < 0.001). (B) The AUC for the CT of EPI was 0.729 (p < 0.001). CT, closure time; C/ADP, collagen-adenosine diphosphate; C/EPI, collagen-epinephrine.

The PFA is clinically useful for evaluating primary hemostasis (1,12,19). In this test, C/ADP and C/EPI are the key stimuli that induce PLT activation and aggregation, playing critical roles in assessing PLT function (4,7,15,18). The PLT function status can be determined by measuring the CTs of C/ADP and C/EPI (7,19). A previous study found that dogs with mitral regurgitation (MR) showed reduced PLT activity, while those with mild-to-severe MR exhibited prolonged CTs (16,25). Similarly, dogs with MMVD in the cardiac remodeling group exhibited significantly increased CT for both C/ADP and C/EPI compared with those in the non-cardiac remodeling group.

Although the precise mechanism underlying the reduction in PLT function remains unclear, several possibilities should be considered. First, chronic exposure to high shear stress may activate PLTs, followed by a subsequent deactivation phase (9,20,23). This process can lead to partial degranulation, rendering the PLTs less responsive to further activation by shear stress (9,17,24). Consequently, the circulating PLT population likely comprises a mix of activated and deactivated cells, with the overall functional state determined by the dominant activation status at any given time (25). In this study, the cardiac remodeling group demonstrated prolonged CT. Our findings suggest that as MMVD progresses, the number of PLTs deactivated due to shear stress increases, thereby extending the CT. Additionally, the PLT count significantly increased in dogs with advanced MMVD. This phenomenon may be attributed to a compensatory increase in PLT production in response to the higher number of deactivated PLTs in dogs with progressive MMVD. Another potential explanation is that the increased CTs for both C/ADP and C/EPI may depend on PLT aggregation mediated by the von Willebrand factor (vWF) at high shear rates (16,24). To investigate this further, additional studies examining plasma vWF and fibrinogen concentrations in the two groups are warranted. Currently, studies investigating the coagulation system of dogs with MMVD are limited. This study assessed the PLT activation levels between the non-cardiac remodeling and cardiac remodeling groups using the PFA. A significant difference was observed, indicating PLT dysfunction in dogs with MMVD exhibiting cardiac remodeling. Therefore, it is advisable to consider reduced platelet function when assessing the coagulation system in dogs with MMVD.

Consequently, anticoagulant therapy may be more effective compared with antiplatelet therapy in dogs with MMVD and concurrent hypercoagulable conditions, such as pancreatitis, liver disease, protein-losing enteropathy, and immune-mediated hemolytic anemia (2,6,21). However, further research is required to explore this hypothesis.

This study has several limitations. First, the sample size for each group was relatively small. Hence, larger and more diverse populations are necessary to obtain more reliable data. Second, as breed-specific differences were not considered in this study, further research should be conducted to establish breed-specific reference values. Third, the non-cardiac remodeling group was not on any medications, whereas the cardiac remodeling group received treatment. This made it difficult to accurately evaluate the impact of medication on the coagulation system (8). Finally, four dogs in the cardiac remodeling group had C/ADP and C/EPI CTs exceeding 300 s, which may have led to an underestimation of the results as the maximum measurement time on the device was 300 s. Although this limitation affected the accuracy of the results, it reinforced the significance of our findings. Further research using extended-measurement CTs is necessary to obtain more accurate results.

The authors have no conflicting interests.