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Effects of clopidogrel and prednisone on platelet function in healthy dogs.

Abstract Background Glucocorticoids cause hypercoagulability, but it is unknown if they counteract clopidogrel's antiplatelet effects. Hypothesis/Objectives Determine the effects of clopidogrel and prednisone on platelet function. Animals Twenty‐four healthy dogs. Methods Double‐blinded, placebo‐controlled randomized trial. Platelet function was evaluated using a platelet function analyzer and impedance aggregometry (days 0, 14, and 28) for dogs treated with placebo, clopidogrel (2‐3 mg/kg/d), prednisone (2 mg/kg/d), or prednisone with clopidogrel PO for 28 days. Results were categorized as nonresponder versus responder (platelet function analyzer), and inadequate, ideal, or excessive response (aggregometry). Results were compared using mixed model, split‐plot repeated measures analysis of variance and generalized estimating equation proportional odds models. P < .05 was considered significant. Results Closure times differed by treatment (F [3, 20] = 10.5; P < .001), time (F [2, 40] = 14.3; P < .001), and treatment‐by‐time (F [6, 40] = 3.4; P = .01). Area under the curve (AUC) differed by treatment (F [3, 20] = 19.6; P < .001), time (F [2, 40] = 35.4; P < .001), and treatment‐by‐time (F [6, 40] = 13.5; P < .001). Based on closure times, 5/6 dogs each in the clopidogrel and prednisone/clopidogrel groups were responders. All dogs in the prednisone/clopidogrel group were overcontrolled based on AUC (days 14 and 28), whereas 5/6 (day 14) and 2/6 (day 28) dogs treated with clopidogrel were overcontrolled. Compared to clopidogrel, dogs receiving prednisone/clopidogrel were 11 times (P = .03) more likely to have an excessive response. Conclusions and Clinical Importance Administration of clopidogrel/prednisone increases platelet dysfunction in healthy dogs.


| INTRODUCTION
Mortality rates in dogs with primary immune-mediated hemolytic anemia (IMHA) historically have been high (50%-70%), 1,2 primarily as a consequence of fatal thromboembolism. [1][2][3] Standard treatment for dogs with IMHA includes immunosuppression, thromboprophylaxis, and supportive care. 1,4,5 Glucocorticoids are the most commonly used immunosuppressive agent for the treatment of IMHA in dogs. 5 Unfortunately, glucocorticoid administration can cause hypercoagulability in healthy dogs, [6][7][8] and it has been identified as a risk factor for thromboembolism in clinical patients. 7,9,10 Although commonly administered to dogs with IMHA, many thromboprophylactic medications are either cost prohibitive, administered by injection or both, leaving PO antiplatelet medications a more affordable and practical option for long-term prophylactic treatment.
Clopidogrel has become a popular thromboprophylactic agent for use in dogs with IMHA. 5 In a prospective trial, 11 no difference was found in short-term survival between dogs with IMHA receiving lowdose aspirin or clopidogrel. However, it is unknown if clopidogrel counteracts glucocorticoid-induced hypercoagulability or, alternatively, if glucocorticoid-induced hypercoagulability offsets the antiplatelet effects of clopidogrel.
The objective of this randomized-controlled double-blinded study was to determine the platelet function of healthy dogs receiving placebo, clopidogrel (2-3 mg/kg/d), prednisone (2 mg/kg/d), or prednisone with clopidogrel. Our hypothesis was that sustained administration of clopidogrel would consistently inhibit platelet function when administered singly or concurrently with prednisone.

| Study population
Blood was collected from 24 healthy dogs from the University of Tennessee, College of Veterinary Medicine teaching and research colony during a related study assessing gastrointestinal effects of clopidogrel and prednisone treatment. 12 Sample size calculation was performed using data from a previous study that measured platelet dysfunction in healthy dogs treated with clopidogrel. 13 Based on these results, and assuming a SD of 13.5 and 7.5 for pretreatment and posttreatment samples, respectively, 6 dogs per treatment group would be needed to have 95% power to find a difference of 25% in platelet aggregation significant with an alpha of .05. Animal use was approved by the University of Tennessee, College of Veterinary Medicine Institutional Animal Care and Use Committee (protocol number 2335) and was in compliance with the requirements of a facility accredited by the American Association for Accreditation of Laboratory Animal Care.
Blood was collected at the conclusion of baseline and on days 14 and 28. Blood was collected by jugular venipuncture using a 20-gauge needle directly into Vacutainer tubes containing EDTA (hematocrit and manual platelet count), 3.2% sodium citrate (whole blood platelet analyzer), and hirudin (impedance aggregometry). For both platelet function analyses, blood was kept at room temperature without disturbance until analysis, and all samples were analyzed within 4 hours of collection.  For analysis, the blood samples were gently inverted 3 to 5 times by hand and 800 μL of citrated whole blood was placed into a cartridge and analyzed. The collagen/ADP cartridge (PFA Collagen/ADP Test Cartridge, Siemens Healthcare Diagnostics, Duluth, Georgia) was used before administration of clopidogrel to ensure normal platelet function and during drug administration to assess drug-associated platelet dysfunction. Two cartridges were analyzed concurrently, and closure times were averaged. Platelet response to clopidogrel was categorized as "responder" if closure time increased ≥30% compared to baseline, and "nonresponder" if closure time increased by ≤29% compared to baseline. 16 A multiple electrode impedance aggregometer (Multiplate Analyzer, Verum Diagnostica GmbH, Munich, Germany) was used according to the manufacturer's instructions (Multiplate Analyzer Manual, Verum Diagnostica GmbH, Munich, Germany) and previously has been validated for use in dogs. [17][18][19] Briefly, blood was transferred into a single-use test cell that contained warmed 0.9% sodium chloride, a dual sensor unit, and a Teflon-coated magnetic stir bar (1200 revolutions/min). The electrical resistance between the wires within the sensor unit was recorded. Aggregation was assessed using ADP (6.5 μM; ADPtest, Roche Diagnostics GmbH, Mannheim, Germany) at a temperature of 37 C for 6 minutes. Platelet aggregation was recorded as area under the curve (AUC). The dual sensor unit generated 2 separate results, which were averaged to yield a single AUC value for each sample. Additionally, deviation from the mean was calculated for the 2 measurements that were used to create the final AUC value. Platelet response to clopidogrel was categorized as inadequate (AUC > 46 U) versus adequate (AUC ≤46 U), with positive response also subdivided into ideal (19-46 U) versus excessive control (<19 U). 20 Based on these classification criteria, dogs were classified as poorly controlled, adequately controlled, or overcontrolled.

| Study population
Details of the study population have been reported elsewhere. 12 Briefly, there were 9 intact females, 8 intact males, and 7 neutered males. There were 15 beagles and 9 hounds, which were evenly distributed among the treatment groups. Median age was 3 years (range, 2-7 years), and median body weight was 13 kg (range, 8.1-30.4 kg).

| Hematocrit and platelet count
The hematocrit results were within reference intervals (RI) at all time points for all dogs (Table 1). Hematocrit did not differ significantly by treatment group, sampling time, or treatment-by-time. Platelet count differed significant by treatment-by-time (F [6, 40] = 3.25; P = .01) but not treatment group or time ( Table 1). The platelet counts were within RI at all time-points for all but 2 dogs. At baseline, 1 dog in the placebo group had a platelet count of 167 000/μL (RI, 170 000-400 000/μL), but platelet clumping was present. Both automated and manual evaluation performed the next day were within RI. On day 28, 1 dog in the prednisone/clopidogrel group had a platelet count of 136 000/μL without evidence of platelet clumping.  Table 2.

| Whole blood platelet function analyzer
The hematocrits and platelet counts (mean ± SD) for 24 healthy dogs administered placebo, clopidogrel with placebo, prednisone with placebo, or combination prednisone and clopidogrel for 28 days

| Impedance aggregometry
The AUCs for whole-blood aggregometry are presented in Figure 2.

| DISCUSSION
Clopidogrel has become a popular thromboprophylactic agent for use in dogs with primary IMHA. 5 Our results suggest that clopidogrel not only counteracts glucocorticoid-induced platelet reactivity, but the combination of prednisone at the dosage used in our study and clopidogrel enhanced platelet dysfunction in healthy dogs. In people, platelet function testing is used to ensure that thromboprophylactic effects fall within the therapeutic window between inadequate inhibition (non-or partial responders) and excessive loss of platelet reactivity (overresponders). 20  The mechanism underlying synergistic effects of prednisone and clopidogrel on platelet dysfunction is unknown. However, in humans, the in vitro addition of prednisolone to whole blood decreases platelet aggregation and thrombus formation by inhibition of the ADP receptors (P2Y 1 and P2Y 12 ) on platelets. 23 Additionally, when prednisolone is incubated with known ADP receptor inhibitors, a decrease in the amount of platelet aggregation is observed compared to blood exposed only to ADP receptor inhibitors. Interestingly, other glucocorticoids, such as dexamethasone and triamcinolone, do not have the same effect on the platelet ADP receptor. 23 In both our study and previously published studies, 24 Although no difference was identified in gastrointestinal hemorrhage and ulceration between dogs treated with prednisone alone versus in combination with clopidogrel, actual blood loss was not quantified. 12 Further evaluation regarding the clinical impact of the synergistic effects of prednisone and clopidogrel on platelet function in hypercoagulable dogs is warranted.
Our study also is the first to identify changing clopidogrel requirements over time in dogs, although the mechanism that causes the change in platelet dysfunction is unknown. In people, clopidogrel responsiveness changes over the first week of treatment, 26  Prednisone and clopidogrel 0/6 0/6 6/6 0/6 0/6 6/6 plasma (optical aggregometry) or whole blood (impedance aggregometry). Optical aggregometry has been considered the ideal method to assess platelet function. 27 However, optical aggregometry requires additional sample processing and manipulation, creation of platelet-rich plasma and removal of blood components from the sample, and is not an accurate representation of the natural environment for the platelet. Impedance aggregometry, on the other hand, requires minimal sample preparation and manipulation, and creates a more natural environment in which to assess platelet function. Our study did not use optical aggregometry, and additional studies using this technique could provide additional information about prednisone/ clopidogrel-induced platelet dysfunction. Additionally, aggregometry does not evaluate platelet function under shear forces, which is why the whole blood platelet function analyzer was included in our analysis. In humans [28][29][30] and dogs, 31   An additional limitation of our study is that recurrent anesthesia, endoscopy performed to evaluate gastrointestinal bleeding for the related study 12 or both could have altered platelet function and confounded the results of the present study. However, blood was collected for analysis of platelet function before administration of any anesthetic medication and performance of endoscopy, and there was at least a 14-day recovery period before collection of the next blood sample. Additionally, the anesthetic protocol used in the related study (acepromazine, butorphanol, and isoflurane) has been shown to have no significant sustained effect on platelet function. [36][37][38][39] However, the instruments used to assess platelet function in these studies were different than the instruments used in our study. [36][37][38][39]

CONFLICT OF INTEREST DECLARATION
Authors declare no conflict of interest.

OFF-LABEL ANTIMICROBIAL DECLARATION
Authors declare no off-label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION
The study protocol was approved by the IACUC of the University of Tennessee, Knoxville (protocol number 2335) and performed in compliance with "The Guide for the Care and Use of Laboratory Animals" in laboratory animal facilities that are AAALAC certified.

HUMAN ETHICS APPROVAL DECLARATION
Authors declare human ethics approval was not needed for this study.