Abstract
Objectives
Cats with hyperthyroidism have been reported to develop thromboembolism, with and without echocardiographic abnormalities consistent with hyperthyroidism. The objective of this study was to compare platelet function in cats with hyperthyroidism with euthyroid age-matched cats. We hypothesized that cats with hyperthyroidism have shortened collagen and adenosine diphosphate (C-ADP) closure times as measured with the platelet function analyzer (PFA-100) in comparison with healthy, age-matched controls.
Methods
Sixteen hyperthyroid and nine euthyroid healthy cats >7 years of age were recruited from the hospital population. Platelet function, measured using the C-ADP closure times by the PFA-100, and platelet count were measured in healthy euthyroid cats and cats with hyperthyroidism.
Results
Mean ± SD closure times were not significantly different between control (66.3 ± 9.6 s) and hyperthyroid cats (65.9 ± 11.5 s; P = 0.75). The mean ± SD closure times of hyperthyroid cats that either were untreated or received methimazole for ⩽3 weeks (n = 6; mean 68.5 ± 15.4 s) was not different than that of cats treated for >3 weeks (n = 10; mean 64.3 ± 8.9 s; P = 0.57). The mean automated platelet count was higher in the hyperthyroid group than in the control group (P = 0.023).
Conclusions and relevance
Platelet function, as measured by closure time under high shear conditions using C-ADP as an agonist, was not affected by hyperthyroidism in this group of cats. Further research is needed to determine if a hypercoagulable state exists in hyperthyroid cats and the potential roles platelets and von Willebrand factor may have.
Introduction
Cats with hyperthyroidism have been reported to develop thromboembolism.1,2 It is unclear if this is the result of cardiovascular changes that occur secondary to hyperthyroidism or if a hypercoagulable state is induced through another mechanism. Many affected cats have concurrent, potentially reversible myocardial disease. 3 However, aortic thromboembolism has been documented in hyperthyroid cats without echocardiographic abnormalities. 2
Abnormalities of platelet function can contribute to aortic thromboembolism. The platelet function analyzer (PFA-100; Siemens Healthcare Diagnostics) measures platelet function in citrated whole blood, effectively mimicking the high shear forces that influence platelet behavior in arterioles. Agonists such as adenosine diphosphate (ADP) and collagen, or epinephrine and collagen initiate platelet plug formation and occlusion of the PFA-100’s cartridge aperture. The time taken for blood to form a platelet plug that occludes the aperture is reported as the closure time. This test has been used to assess platelet function in healthy cats and those with cardiac disease, as well as the response to administration of antiplatelet drugs.4–7
The risk of venous or pulmonary arterial thromboembolism in people with hyperthyroidism is more than twice that of euthyroid individuals.8,9 Systemic arterial thromboembolism is also a complication of hyperthyroidism as evidenced by the increased risk of stroke in patients with thyrotoxicosis. 10 Factors contributing to a hypercoagulable and hypofibrinolytic state in hyperthyroidism include increases in plasma von Willebrand factor, factor VIII, factor IX, fibrinogen and plasminogen activator inhibitor 1. 11 In addition, platelet hyperactivity is found in hyperthyroid humans, including in studies documenting shortened closure time using the PFA-100.12,13 To our knowledge, the only study to evaluate platelet function in hyperthyroid cats found platelet aggregation to be decreased in response to ADP and unchanged in response to collagen. 14 We hypothesized that cats with hyperthyroidism have shortened closure times with the PFA-100 than healthy, age-matched controls.
Materials and methods
A prospective, case-controlled design compared platelet function in cats with hyperthyroidism with platelet function in healthy euthyroid cats. The study design and client consent form were approved by the Veterinary Teaching Hospital (VTH) Board, and informed, written consent was obtained from each client. The control group comprised client-owned cats aged ⩾8 years that were presented to the VTH for preventive care. These cats were determined to be healthy based on physical examination, complete blood count (CBC), serum biochemistries, urinalysis and serum total thyroxine (T4) concentrations. CBCs (including automated platelet counts) were performed on EDTA anticoagulated blood (Siemens ADVIA 2120), while chemistries (Beckman-Coulter AU480) and total T4 concentrations (Siemens Immulite 1000) were measured on serum.
Cats presented to the VTH were included in the hyperthyroid group if clinical findings were consistent with hyperthyroidism (including a history of weight loss, hyperactivity, polyphagia, polyuria and/or polydipsia, palpable thyroid enlargement, tachycardia and heart murmur) and a serum T4 concentration was above the reference interval (16–38 nmol/l). Systolic blood pressure was measured only in the hyperthyroid group using Doppler ultrasonography with cats in lateral recumbency. The mean of five consecutive measurements is reported at a single point in time. Hypertension was defined as a systolic blood pressure ⩾160 mmHg. 15 Cats with concurrent disease based on physical examination findings or clinically relevant abnormalities on CBC, serum biochemistries (including anemia or azotemia) or urinalysis, were excluded from the study. None of the cats included in the study were receiving any medication for at least 2 weeks prior to evaluation.
Approximately 2.7 ml of blood was collected by jugular venipuncture using a 20 G or 21 G needle into a plastic syringe and immediately placed into a 3.2% sodium citrate tube (BD Vacutainer Plus Plastic Citrate Tubes). The tube was inverted 3–4 times after collection and allowed to sit undisturbed at room temperature for at least 30 mins prior to analysis, which was completed within 120 mins of sample collection. Cats that struggled substantially during venipuncture or required sedation were excluded from the study. Platelet numbers were assessed for each sample by one of the authors (KMB). Estimation of unclumped platelet numbers (×103/μl) was determined by multiplying the average number of platelets in a × 100 objective field over 10 fields by 15 and then by 1.1. A correction factor of 1.1 was used to account for dilution caused by the volume of citrate in the blood collection tubes. 16 Cats with microscopic platelet clumping falsely lowering the platelet count (estimated platelet count < 150 × 103/μl) were excluded.
Platelet function was assessed using the PFA-100 utilizing a combined collagen and adenosine diphosphate (C-ADP) cartridge. After inverting the tube containing blood 3–4 times, 800 μl of the sample was pipetted into the test cartridge channel and samples were run in duplicate simultaneously using the A and B channels of the instrument. If the coefficient of variance (CV) was >9% between the two channels, the assay was repeated once. Samples were excluded from the study if triplicate testing had a greater than 9% CV.
Statistical analysis
Data were assessed for normality by inspection of normal probability plots. Student’s t-test was used to compare results of the hyperthyroid group with the healthy control group. Data were expressed as mean ± SD. The level of significance was set at P <0.05.
Results
Four of 20 cats in the hyperthyroid group were excluded because of platelet clumping that falsely lowered the platelet count. One of 10 cats in the control group was excluded because a blood smear was not evaluated.
The control group consisted of four neutered male and five spayed female domestic shorthair (DSH) cats, while the hyperthyroid group consisted of seven neutered male and nine spayed female DSH cats. The mean age and body weight in control and hyperthyroid groups was not different (Table 1). The median time from diagnosis of hyperthyroidism to evaluation was 5.5 weeks (range 0–104 weeks). Three cats received no treatment for hyperthyroidism prior to evaluation. Of the 13 hyperthyroid cats receiving methimazole prior to evaluation, three had been treated ⩽3 weeks, four for 5–6 weeks and the remaining six for >6 weeks. Methimazole administration was discontinued 2, 3, 4 and 13 weeks before the study in eight, three, one and one cats, respectively. Two cats received a restricted iodine diet that was discontinued 2 and 4 weeks before evaluation, respectively, both of which had also been treated with methimazole.
Healthy control and hyperthyroid cat findings
Data are mean ± SD
Statistically significant
T4 = thyroxine; C-ADP = collagen–adenosine diphosphate
The mean ± SD serum T4 concentration was 146 ± 65.5 nmol/l and 25 ± 5.9 nmol/l in the hyperthyroid and control groups, respectively. Hematocrit, mean cell volume, serum creatinine and urine specific gravity were significantly lower in hyperthyroid cats compared with controls (Table 1). The mean systolic blood pressure in the 15 hyperthyroid cats was 160 ± 27 mmHg; 9/15 (60%) were hypertensive. Blood pressure was not measured in one hyperthyroid cat. Blood pressure was 160–180 mmHg in seven cats and >180 mmHg in two cats.
The estimated platelet count performed on citrate-anticoagulated whole blood was not different between hyperthyroid and control cats, but the automated platelet count performed on EDTA-anticoagulated whole blood was significantly (P = 0.046) higher for hyperthyroid cats than controls (Table 1). The mean platelet volume was not different between the groups (P = 0.41; Table 1).
The closure time measured using C-ADP was not different (P = 0.9) between the control (66.3 ± 9.6 s) and hyperthyroid (65.9 ± 11.5 s) cats (Figure 1, Table 1). The closure times of hyperthyroid cats that were untreated or received methimazole for ⩽3 weeks (n = 6; mean 68.5 ± 15.4 s) was not different than that of cats treated >3 weeks (n = 10; mean 64.3 ± 8.9 s; P = 0.5).
Closure times measured by the platelet function analyzer (PFA-100) in hyperthyroid and healthy control cats. The middle horizontal bar is the median and the bars above and below indicate the interquartile range
Discussion
While thromboembolism has infrequently been reported in hyperthyroid cats, 8.4–9.4% of the population in the largest retrospective studies of cats with arterial thromboembolism had hyperthyroidism.1,2 Abnormalities in platelet function were suspected by these authors to contribute to thromboembolism in hyperthyroid cats. However, platelet function, as measured by the PFA-100, was not significantly affected by hyperthyroidism in cats in the present study. These results conflict with the previous study of primary hemostasis in hyperthyroid cats that found decreased platelet aggregation in response to ADP but not to collagen. 14 Platelet function was evaluated in only seven cats in that study, five of which were being administered beta-adrenergic antagonists or calcium channel blockers that could affect platelet function, whereas cats in the present study did not have a history of drug administration within the 2 weeks prior to sampling for hemostasis testing. Many of the hyperthyroid cats in the referenced study 14 had substantial alterations of cardiac structure and function, which could also have influenced the results.17,18
Although the present study failed to identify significantly altered platelet function, as measured by the PFA-100 in cats, it is possible that a different agonist could have detected enhanced platelet function. People with hyperthyroidism have shortened closure times when using epinephrine (adrenaline) and collagen (C-EPI) as agonists.12,13 Unfortunately, marked variability in closure time has been documented using C-EPI in cats, limiting PFA-100-derived closure times to C-ADP cartridges. 6 Other methods of assessing platelet function using different agonists and methods of activation could shed further light on the role of platelets in any prothrombotic state in hyperthyroidism.6,7 Although our results using PFA-100 differ from those in humans based on C-ADP results alone, the present study suggests that the substantial thrombophilic effects of hyperthyroidism in people may not be present in cats.
Hypercoagulability in people with hyperthyroidism is, in part, attributable to increases in von Willebrand factor (vWF) and factor VIII, with closure times in response to C-EPI that are inversely correlated with vWF concentrations.12,13 While hyperthyroidism and hypothyroidism are consistently associated with increases and decreases, respectively, of vWF in people, this is not the case in dogs and remains to be studied in cats.9,19–22 If hyperthyroidism does not lead to increased vWF concentration in cats, the lack of platelet hyperactivity in the present study could be, at least in part, explained by unaltered vWF.
In addition to enhanced platelet activation, hyperthyroidism in humans induces hypercoagulability due to increases in factor VIII, factor IX and fibrinogen. 11 The authors are aware of only one study assessing secondary hemostasis in hyperthyroid cats, and it failed to detect alterations of prothrombin time, activated partial thromboplastin time and proteins induced by vitamin K absence after treatment with methimazole. 23 In humans with hyperthyroidism, fibrinolysis is impaired secondary to increases in tissue plasminogen activator inhibitor-1, increases in thrombin-activated fibrinolysis inhibitor and decreases in tissue plasminogen activator.9,10,24,25 Finally, endothelial dysfunction has been documented in hyperthyroid humans, and may contribute to the hypercoagulable state in hyperthyroidism.26,27
Alterations in cardiac structure and function may also play a role in any predisposition to thrombosis in hyperthyroidism, as cardiac hypertrophy is common in affected cats. 3 The contribution of cardiac changes secondary to hyperthyroidism to any thrombophilia is difficult to separate from other effects of the primary disease. While it has been shown that closure times were not different in cats with hypertrophic cardiomyopathy (HCM) than in healthy cats, studies using other methodologies have found that platelet activation occurs in cats with heritable HCM, regardless of the presence or absence of echocardiographic changes.4,17,28 In addition, another study using platelet aggregation of platelet-rich plasma found hyperaggregability in 3/10 cats with dilated or HCM. 17 Echocardiography or thoracic radiographs were not performed in cats in the present study, so the effects of cardiac changes in hyperthyroid cats was not determined. In addition, although cats in the control group did not have overt cardiovascular abnormalities, subclinical cardiomyopathy can occur without a heart murmur. 29 Ideally, echocardiographic evaluation would have been performed on cats to assess the severity of any cardiac changes consistent with HCM or hyperthyroidism. Systemic arterial hypertension, present in 60% of the cats in the current study, could be an additional predisposing factor for thrombosis by inducing endothelial damage, as has been documented in humans. 30
The significantly higher automated platelet count in hyperthyroid cats compared with controls did not appear to alter the closure time, and samples with microscopic platelet clumping were not excluded from statistical analysis unless the estimated platelet count was low. This approach was used to ensure that platelet clumping in the blood sample on which platelet function testing was performed was not severe.
A limitation of this study is that 13/16 hyperthyroid cats were treated with methimazole or a restricted iodine diet prior to data collection. Treatment was discontinued at least 2 weeks prior to evaluation, all cats were documented to be hyperthyroid at the time of sampling and there was no difference in closure time in cats treated ⩽3 weeks with those treated for >3 weeks. While these subgroups were small, the mean closure time differed little between groups. Evaluating hyperthyroid cats at the time of initial diagnosis rather than at variable times after discontinuing treatment may have achieved different results. However, administration of levothyroxine to healthy people for 14 days to create hyperthyroidism induces a state of hypercoagulability and hypofibrinolysis, so it seems unlikely that previous methimazole treatment discontinued at least 2 weeks prior to study would abrogate the effects of hyperthyroidism on coagulation in the cats of the current study.25,31 An additional limitation is that platelet function as measured by closure time using C-ADP offers limited assessment of platelet function, and is more effective in determining reduced platelet aggregation, particularly in response to administration of antiplatelet drugs.
Conclusions
Platelet function based on closure time is not altered by hyperthyroidism in cats. The role of platelets in any thrombophilic state in hyperthyroidism may be less important than other factors based on closure time with C-ADP in the present study. Measurement of plasma vWF, individual coagulation factors, markers of fibrinolysis and thromboelastography should be considered in any future evaluations of thrombophilia in feline hyperthyroidism.
Footnotes
Conflict of interest
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
This research was funded by the Anne Hunter Professorship, Virginia-Maryland College of Veterinary Medicine, Virginia Tech.
Ethical approval
This work involved the use of experimental animals; or involved the use of non-experimental animal(s) (owned or unowned) outside of established internationally recognized high standards (‘best practice’) of individual veterinary clinical patient care. The study therefore had ethical approval from an established committee as stated in the manuscript.
Informed consent
Informed consent (either verbal or written) was obtained from the owner or legal custodian of all animal(s) described in this work for the procedure(s) undertaken. No animals or humans are identifiable within this publication, and therefore additional informed consent for publication was not required
