SEARCH

SEARCH BY CITATION

Keywords:

  • Cardiology;
  • echocardiography;
  • feline;
  • hemostasis;
  • platelets;
  • reference interval

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgments
  7. References

Background: There is currently no simple analytical tool for the evaluation of hypercoagulability in cats. The Platelet Function Analyzer-100® (PFA-100; 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgments
  7. References

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,8–11 and have been the subject of retrospective studies in cats with HCM.3–5,12,13

The Platelet Function Analyzer-100® (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 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgments
  7. References

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 >1 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 ≥7 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 (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 <100,000/μL or a HCT <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<.05.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgments
  7. References

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).

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%).

image

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.

Download figure to PowerPoint

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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgments
  7. References

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.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgments
  7. References
  • 1
    Blann AD, Lip GY. Virchow's triad revisited: the importance of soluble coagulation factors, the endothelium, and platelets. Thromb Res. 2001;101:321327.
  • 2
    Ferasin L, Sturgess CP, Cannon MJ, Caney SM, Gruffydd-Jones TJ, Wotton PR. Feline idiopathic cardiomyopathy: a retrospective study of 106 cats (1994–2001). J Feline Med Surg. 2003; 5:151159.
  • 3
    Laste NJ, Harpster NK. A retrospective study of 100 cases of feline distal aortic thromboembolism: 1977–1993. J Am Anim Hosp Assoc. 1995;31:492500.
  • 4
    Atkins CE, Gallo AM, Kurzman ID, Cowen P. Risk factors, clinical signs, and survival in cats with a clinical diagnosis of idiopathic hypertrophic cardiomyopathy: 74 cases (1985–1989). J Am Vet Med Assoc. 1992;201:613618.
  • 5
    Rush JE, Freeman LM, Fenollosa NK, Brown DJ. Population and survival characteristics of cats with hypertrophic cardiomyopathy: 260 cases (1990–1999). J Am Vet Med Assoc. 2002;220:202207.
  • 6
    MacDonald ML, Rogers QR, Morris JG. Effects of dietary arachidonate deficiency on the aggregation of cat platelets. Comp Biochem Physiol C. 1984;78:123126.
  • 7
    Tablin F, Johnsrude JD, Walker NJ. Evaluation of glycoprotein Ib expression on feline platelets. Am J Vet Res. 2001;62:195201.
  • 8
    Bright JM, Dowers K, Powers BE. Effects of the glycoprotein IIb/IIIa antagonist abciximab on thrombus formation and platelet function in cats with arterial injury. Vet Ther. 2003;4:3546.
  • 9
    Hogan DF, Ward MP. Effect of clopidogrel on tissue-plasminogen activator-induced in vitro thrombolysis of feline whole blood thrombi. Am J Vet Res. 2004;65:715719.
  • 10
    Hogan DF, Andrews DA, Talbott KK, Green HW, Ward MP, Calloway BM. Evaluation of antiplatelet effects of ticlopidine in cats. Am J Vet Res. 2004;65:327332.
  • 11
    Behrend EN, Grauer GF, Greco DS, Rose BJ, Thrall MA. Comparison of the effects of diltiazem and aspirin on platelet aggregation in cats. J Am Anim Hosp Assoc. 1996;32:1118.
  • 12
    Smith CE, Rozanski EA, Freeman LM, Brown DJ, Goodman JS, Rush JE. Use of low molecular weight heparin in cats: 57 cases (1999–2003). J Am Vet Med Assoc. 2004;225:12371241.
  • 13
    Smith SA, Tobias AH, Jacob KA, Fine DM, Grumbles PL. Arterial thromboembolism in cats: acute crisis in 127 cases (1992–2001) and long-term management with low-dose aspirin in 24 cases. J Vet Intern Med. 2003;17:7383.
  • 14
    Kundu SK, Heilmann EJ, Sio R, Garcia C, Davidson RM, Ostgaard RA. Description of an in vitro platelet function analyzer–PFA-100. Semin Thromb Hemost. 1995;21 (Suppl 2):106112.
  • 15
    Mammen EF, Comp PC, Gosselin R, et al. PFA-100 system: a new method for assessment of platelet dysfunction. Semin Thromb Hemost. 1998;24:195202.
  • 16
    Callan MB, Giger U. Assessment of a point-of-care instrument for identification of primary hemostatic disorders in dogs. Am J Vet Res. 2001;62:652658.
  • 17
    Mischke R, Keidel A. Influence of platelet count, acetylsalicylic acid, von Willebrand's disease, coagulopathies, and haematocrit on results obtained using a platelet function analyser in dogs. Vet J. 2003;165:4352.
  • 18
    Wierenga JR, Jandrey KE, Haskins SC, Tablin F. In vitro comparison of the effects of two forms of hydroxyethyl starch solutions on platelet function in dogs. Am J Vet Res. 2007;68:605609.
  • 19
    Keidel A, Mischke R. Clinical evaluation of platelet function analyzer PFA-100 in dogs. Berl Munch Tierarztl Wochenschr. 1998;111:452456.
  • 20
    Meurs KM, Sanchez X, David RM, et al. A cardiac myosin binding protein C mutation in the Maine Coon cat with familial hypertrophic cardiomyopathy. Hum Mol Genet. 2005;14:35873593.
  • 21
    Segura D, Monreal L, Espada Y, et al. Assessment of a platelet function analyzer for horses: reference range and influence of a platelet aggregation inhibitor. Vet J. 2005;170:108112.
  • 22
    Francis JL. Platelet dysfunction detected at high shear in patients with heart valve disease. Platelets. 2000;11:133136.
  • 23
    Favaloro EJ, Facey D, Henniker A. Use of a novel platelet function analyzer (PFA-100) with high sensitivity to disturbances in von Willebrand factor to screen for von Willebrand's disease and other disorders. Am J Hematol. 1999;62:165174.
  • 24
    Harrison P, Robinson M, Liesner R, et al. The PFA-100: a potential rapid screening tool for the assessment of platelet dysfunction. Clin Lab Haematol. 2002;24:225232.
  • 25
    Escudero C, Santos M, Bujan J, et al. Optical aggregometry versus the PFA-100: experimental studies in pigs treated with propofol. Platelets. 2001;12:133137.
  • 26
    Barr SC, Ludders JW, Looney AL, Gleed RD, Erb HN. Platelet aggregation in dogs after sedation with acepromazine and atropine and during subsequent general anesthesia and surgery. Am J Vet Res. 1992;53:20672070.
  • 27
    Dwyer SD, Meyers KM. Anesthetics and anticoagulants used in the preparation of rat platelet-rich-plasma alter rat platelet aggregation. Thromb Res. 1986;42:139151.
  • 28
    Fuchs I, Frossard M, Spiel A, et al. Platelet function in patients with acute coronary syndrome (ACS) predicts recurrent ACS. J Thromb Haemost. 2006;4:25472552.
  • 29
    Frossard M, Fuchs I, Leitner JM, et al. Platelet function predicts myocardial damage in patients with acute myocardial infarction. Circulation. 2004;110:13921397.
  • 30
    Serebruany VL, Alford AB, Meister AF, et al. Clinical utility of the platelet function analyzer (PFA-100) for the assessment of the platelet status in patients with congestive heart failure (EPCOT trial). Thromb Res. 2001;101:427433.