Platelet function in clinically healthy cats and ... - Wiley Online Library

Veterinary Clinical Pathology ISSN 0275-6382

B R I E F C O M M U N I C AT I O N

Platelet function in clinically healthy cats and cats with hypertrophic cardiomyopathy: analysis using the Platelet Function Analyzer-100 Karl E. Jandrey1, Jeffrey W. Norris2, Kristin A. MacDonald3, Mark D. Kittleson3, Fern Tablin2 Departments of 1Surgical and Radiological Sciences, 2Anatomy, Physiology, and Cell Biology, and 3Medicine and Epidemiology, School of Veterinary Medicine, University of California–Davis, Davis, CA

Key Words Cardiology, echocardiography, feline, hemostasis, platelets, reference interval Correspondence Karl E. Jandrey, Department of Surgical and Radiological Sciences, University of California–Davis, 1104C Tupper Hall, Davis, CA 95616, USA E-mail: [email protected] DOI:10.1111/j.1939-165X.2008.00062.x

Background: There is currently no simple analytical tool for the evaluation of hypercoagulability in cats. The Platelet Function Analyzer-100s (PFA100; Dade Behring Inc., Deerfield, IL, USA) is a bench-top machine that evaluates platelet function by measuring closure time (CT) in citrated whole blood under high shear conditions. We hypothesized that cats with hypertrophic cardiomyopathy (HCM) have up-regulated platelet function, which shortens their CT and increases their risk for thromboembolic events. Objectives: The goals of this study were to: (1) establish a feline reference interval for CT using the PFA-100, (2) measure CT in blood from cats with HCM, and (3) determine if there is a measurable difference between the CT of healthy cats compared with cats with HCM. Methods: Citrated blood samples from 42 clinically healthy cats and 30 cats with HCM were analyzed according to manufacturer’s specifications. CT was measured in triplicate and the mean value was used for analysis. Transformed data were compared between clinically healthy cats and cats with HCM using a Student’s t-test, and among cats with mild, moderate, or severe HCM using ANOVA. Results: The median CT of clinically healthy cats was 64 seconds (range 43–176 seconds). The median CT of cats with HCM was 74 seconds (range 48–197 seconds). There was no significant difference in CT between cats with HCM and clinically healthy cats. There also were no significant differences in cats with mild, moderate, or severe HCM. Conclusions: A feline reference interval for PFA-100 CT will be useful in future studies of platelet function in cats. Cats with HCM do not have shorter CTs when compared with clinically healthy cats.

Introduction Virchow’s triad suggests intravascular thrombosis is due to endothelial damage, stagnant blood flow, or hypercoagulability.1 Aortic thromboembolism secondary to hypertrophic cardiomyopathy (HCM) is a relatively common veterinary emergency that has minimal clinically effective therapy and is frequently fatal.2–5 The pathophysiology of arterial thromboembolism secondary to HCM may involve alterations in any or all of the above mechanisms, resulting in activation of the coagulation system. The specific contribution of platelets to this process remains to be defined.

Platelet aggregation in vitro has been studied in clinically normal cats,6 yet the mechanism of feline platelet activation is not well defined.7 Antiplatelet and anticoagulant therapeutic investigations also have been conducted in normal cats,8–11and have been the subject of retrospective studies in cats with HCM.3–5,12,13 The Platelet Function Analyzer-100s (PFA-100) is a bench-top instrument that evaluates platelet function in whole blood as determined by closure time (CT), the time it takes for a platelet plug to form and occlude flow. The CT is sensitive to defects in platelet receptors that mediate adhesion (GPIb/IX) and aggregation (GPIIb/IIIa).14,15 The PFA-100 has most commonly

c 2008 American Society for Veterinary Clinical Pathology Vet Clin Pathol 37/4 (2008) 385–388

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been used for analysis of primary hemostatic disorders (eg, von Willebrand’s disease) in dogs, horses, and humans as well as for assessing aspirin and fluid therapy.16–18 The extent to which the PFA-100 can detect increased platelet function is currently unknown. Studies have shown that the PFA-100 is a precise and reliable indicator of platelet function and dysfunction in humans14,15 and dogs17,19 that are not anemic or thrombocytopenic. To our knowledge, the PFA-100 has not been validated for use in cats. Using the PFA-100, our goals were to (1) establish a feline reference interval for CT, (2) measure CT in cats with HCM, and (3) determine if there is a measurable difference between the CT of clinically healthy cats compared with cats with HCM. We hypothesized that cats with HCM have up-regulated platelet function that is reflected in a shortened CT when compared with clinically healthy cats.

Materials and Methods This study was approved by the Institutional Animal Care and Use Committee at the University of California–Davis. Signed owner consent was obtained for all cats used in the investigation. Clinically healthy cats were owned by staff and students at the University of California–Davis School of Veterinary Medicine. The cats were 41 year old, had no current or historical cardiac disease, were not on medication, and had no abnormalities on complete general physical and echocardiographic examinations. Cats with HCM were obtained either from clinical cases at the Veterinary Medical Teaching Hospital (VMTH) or from a colony of cats with heritable HCM.20 Cats with HCM were classified into 3 groups based on severity of cardiac measurements from standard 2-dimensional echocardiography (Sonos 5500 Echocardiography System, Hewlett-Packard, Andover, MA, USA) and/or thoracic radiographs. Group 1 (mild HCM) cats had a left atrium of normal size and left ventricular wall thickness of 6–7 mm (regional or global). Group 2 (moderate HCM) cats had mild to moderate left atrial enlargement and/or wall thickness Z7 mm. Group 3 (severe HCM) cats had moderate to severe left atrial enlargement and congestive heart failure. In a quiet environment, blood from a jugular vein of each cat was drawn atraumatically using a 20-gauge needle and 12 mL plastic syringe and placed into 3.2% sodium citrate (0.109 M; Vacutainer, Becton-Dickinson, Franklin Lakes, NJ, USA) with a ratio of 9 parts blood to 1 part citrate. If chemical restraint was needed to relieve anxiety in any cat, acepromazine maleate

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(0.1 mg/kg, not to exceed 0.5 mg/cat IM) was administered. Colony cats received sedation with acepromazine and hydromorphone (0.1 mg/kg of each SC). An automated blood cell analyzer (Coulter ACT Diff, Beckman Coulter Inc., Fullerton, CA, USA) was used to determine HCT and platelet count. Blood smears were not evaluated. Cats were excluded from the study if they had a platelet count o 100,000/mL or a HCT o 30%. Citrated whole blood samples also were used to measure CT using the PFA-100 (Dade Behring Inc., Deerfield, IL, USA). Cartridges coated with the agonists collagen and adenosine diphosphate (C/ADP) were used for all measurements. CT was measured in triplicate in all but 7 samples, which were measured in duplicate, and the SD and mean (X) values for each cat were used for analysis. The coefficient of variation (CV) was calculated from the multiple CTs for each cat [(SD/X) 100] to determine the consistency of the measure. Because CT data were not normally distributed for either group of cats (based on visual assessment of histograms), results were expressed as median and range (minimum–maximum) and CT values were log-transformed before statistical analysis. The median CV for each group was also calculated. Minimum–maximum values from clinically healthy cats were used to define the reference interval. The CTs of clinically healthy and HCM populations were compared using a Student’s t-test. For cats with HCM, CT was compared among groups based on disease severity by ANOVA (Microsoft Excel, Version 11.3.3, Microsoft Co, Redmond, WA, USA). Statistical significance was set at P o.05.

Results and Discussion The study included 42 clinically healthy cats (24 castrated males, 17 spayed females, 1 intact female) and 30 cats with HCM (13 intact males, 3 castrated males, 13 intact females, 1 spayed female). Median age of the clinically healthy group was 7 years (range 1–14 years). Breeds represented in the clinically healthy population included: domestic shorthair (n = 29), domestic longhair (n = 8), Siamese cross (n = 2), Himalayan (n = 1), Rex (n = 1), and Burmese (n = 1). Median age of the HCM group was 6 years (range 1–16 years). Breeds represented in the HCM group were Maine Coon cross (n = 15), Maine Coon (n = 9), American Shorthair (n = 2), domestic shorthair (n = 2), Abyssinian (n = 1), and domestic longhair (n = 1). The severity of disease within the group of HCM cats was distributed as follows: Group 1 (n = 13), Group 2 (n = 13), and Group 3 (n = 4).


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Figure 1. Closure time (CT) in 42 clinically healthy cats and 30 cats with hypertrophic cardiomyopathy (HCM), including cats with mild (HCM-1, n = 13), moderate (HCM-2, n = 13), or severe (HCM-3, n = 4) HCM using collagen/ADP cartridges on the PFA-100 analyzer. The box is the 50% interquartile range, the center horizontal line is the median, the whiskers are 10% and 90% percentiles, and individual points are outliers.

The median CT for clinically healthy cats was 64 seconds (range, 43–176 seconds), and for cats affected with HCM was 74 seconds (range, 48–197 seconds). No significant difference (P =.13) was found in median CT between clinically healthy cats and cats with HCM (Figure 1). No significant difference was found in the median CT among the 3 HCM groups (P =.91). Only 4 clinically healthy cats and 3 cats with HCM had a CT greater than 120 seconds. Median CV values (13.0% for healthy cats, 16.6% for cats with HCM) fell within manufacturer recommendations for consistency ( 17%). Our median values for clinically healthy cats (64 seconds) and cats with HCM (74 seconds) were similar to published data for other species. Normal C/ADP CT reference intervals have previously been established in dogs (47–98 seconds),16,17,19 horses (60–116 seconds),21 humans (71–118 seconds),14,15,22–24 and pigs.25 Feline blood and platelets are notoriously difficult to handle and study. Care was taken to draw blood from the cats in a quiet environment under minimal restraint. Gentle handling of blood during laboratory manipulation was imperative for the prevention of platelet activation. Chemical restraint is also best avoided to prevent its potential effects on platelet function.26,27 Unpublished data from our laboratory, however, showed that cat platelets assessed by flow cytometry retained their responsiveness to agonists under the sedation protocol used in this study. Other investigators have used acepromazine (up to 1 mg IM or IV) and have not noted any effects on platelet function.9,10


Despite the fact the several studies have sought to use the CT in a prospective manner to diagnose platelet function abnormalities that could lead to thrombosis in humans, test results are highly variable and therefore limited in their ability to consistently correlate with severity of disease or to predict future cardiac events.28–30 We were unable to identify shorter CTs in cats with HCM. Individual CT measurements were unable to discriminate whether a cat was healthy or had HCM. It is possible the PFA-100 is inappropriate for analyzing platelet function or identifying hypercoagulability in cats with HCM. We did not use other methods to verify hypercoagulability or altered platelet function in the cats with HCM, and no cats with aortic thromboembolism were included in the study, so it is possible the cats with HCM were not in a hypercoagulable state. Other types of analysis, such as flow cytometry, may be more sensitive for the detection of platelet hyper-reactivity in cats with HCM.

Acknowledgments Supported by the American College of Veterinary Emergency and Critical Care and the Center for Companion Animal Health, School of Veterinary Medicine, University of California, Davis. The authors would like to thank Naomi Walker and Dr. Fiona Campbell for their technical expertise.

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