Platelets play a critical role in physiologic hemostasis and mediate pathological thrombosis in many diseases.1,2 In addition, platelets participate in innate immunity, inflammation, neovascularization, wound healing, and cancer metastasis.3,4 Platelets perform these diverse functions through a series of activation responses characterized by shape change, adhesion, aggregation, secretion, membrane lipid remodeling, and microvesiculation. Numerous soluble and cell membrane–bound agonists interact with platelet membrane receptors to initiate distinct signaling and effector pathways. Studies5,6 of the molecular mechanisms of platelet activation have provided a variety of targets for pharmacological manipulation of platelet reactivity.
Clopidogrel is an orally administered thienopyridine prodrug; its active metabolite is a specific antagonist of the platelet P2Y12 ADP receptor.7,8 The prodrug undergoes hepatic conversion to a thiol-containing moiety that irreversibly binds to cysteine residues on P2Y12 and thereby inhibits ADP-induced or enhanced platelet aggregation. Clopidogrel is used widely for thromboprophylaxis in humans with acute coronary syndromes, including patients undergoing percutaneous coronary interventions, and those with aspirin intolerance.9–11 Its efficacy, however, is related to each patient's activity of 4 hepatic cytochrome P450s (cytochrome P450, family 2, subfamily C, polypeptide 19; cytochrome P450, family 3, subfamily A; cytochrome P450, family 2, subfamily B, polypeptide 6; and cytochrome P450, family 1, subfamily A, polypeptide 2) required for prodrug metabolism.12 The resultant variability in bioconversion has prompted consideration of individual monitoring to assess the degree of platelet inhibition and thereby improve patient outcomes.13–16
Laboratory evaluation of platelet function is challenging because of the complexity of platelet activation response and the limited ex vivo viability of platelets. Light transmission aggregometry is the traditional gold-standard method to assess platelet reactivity for monitoring the inhibitory effects of antiplatelet drugs.6,9,16,17 Because LTA requires special instrumentation, expertise, and isolation of nonactivated platelets from large volumes of blood, point-of-care monitors and whole blood assays are under investigation as alternate methods to facilitate routine patient screening in medical and veterinary practice.
The recent availability of a generic form of clopidogrel will increase interest in the drug for veterinary patients. Horses at risk for endotoxemia and laminitis secondary to gastrointestinal tract, surgical, and obstetric disorders are patient populations that may benefit from antiplatelet drugs.18–22 However, species differences in platelet reactivity further complicate the inherent difficulties in monitoring antiplatelet drugs in both research and clinical settings.23,24 Therefore, the purpose of the study reported here was to evaluate a variety of test methods to assess equine platelet activation response and to detect inhibitory effects of clopidogrel. We considered LTA the gold-standard assay and hypothesized that a diagnostically useful test to monitor clopidogrel would detect platelet inhibition in all horses with abnormal platelet aggregation in LTA.
Materials and Methods
Animals—The study involved 12 healthy adult mares (5 Thoroughbreds, 4 warmbloods, 2 Quarter Horses, and 1 Oldenburg) from an academic teaching facility.a Their ages ranged from 8 to 25 years (median, 11.5 years), and weights ranged from 527 to 650 kg (median, 555 kg). The horses were judged healthy on the basis of prior history, physical examination, and CBC values and had not received any medication or supplements for at least 3 weeks prior to the study. After completion of the study, serum biochemical profiles to rule out preexisting hepatic disease were performed on frozen-banked, pretreatment plasma samples. The horses were transported from a paddock to box stalls and allowed to acclimate for 24 hours before baseline sample collection. The study protocol was approved by an Institutional Laboratory Animal Care and Use Committee. The in vivo studies and laboratory assays were performed between April and December 2011 at Cornell University's College of Veterinary Medicine.
Study design and drug administration—In a randomized, placebo-controlled trial, the treated horses were given a loading dose (4 mg/kg) of clopidogrelb orally followed by a 2 mg/kg/d maintenance dose for 3 days total drug administration. The dosage scheme was based on recent studies of the effects of fixed-dose clopidogrel on equine platelet function25 and the use of loading dose regimens in human medicine.26 Treatment with drug or placebo was determined with an online randomization software application.c Each subject was first nonrandomly assigned to receive clopidogrel or placebo, and then each assignment was exchanged 3 times with a randomly chosen subject. One author (AEW) performed randomization and test compound preparation. The remaining authors administered the test compounds and performed physical examinations, sample collection, and laboratory analyses. On each treatment day, approximately 15 minutes before administration, the appropriate dose of clopidogrel tablets or the placebo (corn starch with 2 to 3 drops of red food coloring and red cake sprinkles) were suspended in 200-mL water such that both treatments were visually indistinguishable. The drug or placebo suspension was administered via a nasogastric tube, followed by 750 mL of water to ensure gastric delivery of the entire dose. After the initial loading dose of 4 mg of clopidogrel/kg, the maintenance dose of 2 mg/kg (q 24 h) was given for an additional 48 hours. The horses were examined, baseline blood samples collected, and vital signs recorded (rectal temperature, pulse, and respiratory rate) before any treatment (time T0) and after 3 days of treatment (T72). Horses were examined at least once daily for any signs of bleeding, petechiae, bruising, or swelling at venipuncture sites, and vital signs were assessed and recorded twice daily. The horses’ nares were carefully examined for signs of irritation or hemorrhage after each nasal tube passage.
Blood collection and processing—Blood samples were collected via jugular venipuncture into evacuated glass EDTA- and heparin-containing anticoagulant tubesd with a 17-gauge needle attached to a plastic catheter with a stopper-piercing needle.e The piercing needle was then clipped off, and a plastic syringe containing 2.0 mL of 3.8% sodium citrate was attached to the catheter tubing to gently withdraw exactly 18.0 mL of blood. A tube of EDTA blood (3.0 mL) was submitted for determination of CBCf including measurement of total solids concentration via refractometry. Heparinized blood (3.0 mL) was used for thrombelastographg analyses. The citrated blood was transferred to a plastic tube, mixed by gentle inversion, and divided into aliquots for subsequent hemostasis testing as follows: 12.0 mL was centrifuged at 450 × g for 5 minutes at 21°C to isolate PRP for optical aggregometry; 3.0 mL was used with no further processing for determination of CT in a tabletop whole blood platelet function analyzerh and for thrombelastography; 3.0 mL remained undisturbed at room temperature (20° to 23°C) for 20 minutes, and then 0.5 mL of the supernatant PLRP was carefully aspirated for flow cytometric assays; and the remaining 2.0 mL was centrifuged at 14,500 × g for 1 minute, and the supernatant PPP was harvested and stored frozen at −50°C for batch fibrinogen and VWF:Ag determination. Whole blood remained at 20° to 23°C throughout processing, which was complete within 1 hour after blood collection.
Platelet aggregation—The isolated PRP was allowed to rest for 30 minutes at 20° to 23°C before use in optical aggregometry. Platelet counts of the PRP were determinedf to confirm platelet concentration > 150,000/μL, with no adjustment to standardize platelet count. Reactions were performed in 450-μL volumes in siliconized glass cuvettes at 37°C, with agitation at 1,000 cycles/min. The PRP was transferred to a cuvette and warmed for 1 minute in the aggregometeri prior to the addition of the agonists ADPj at final concentrations of 5μM and 10μM and collagenk at 6 μg/mL. Spontaneous platelet aggregation was detected by use of saline (0.85% NaCl) solution as a vehicle control. Aggregation was monitored for a 6-minute observation time. The maximal percentage aggregation and AUC (Figure 1) were calculated and recorded by a software program.1
Equine platelet aggregation over time in response to activation with 5μM ADP (arrowhead) in stirred PRP. The maximum amplitude was measured at 6 minutes after the addition of ADP. The AUC (shaded region) represents the area bounded by the time point of the aggregation tracing's return to baseline from the negative deflection (denoting shape change) to the end of the 6-minute reaction time.
Citation: American Journal of Veterinary Research 74, 9; 10.2460/ajvr.74.9.1212
Equine platelet aggregation over time in response to activation with 5μM ADP (arrowhead) in stirred PRP. The maximum amplitude was measured at 6 minutes after the addition of ADP. The AUC (shaded region) represents the area bounded by the time point of the aggregation tracing's return to baseline from the negative deflection (denoting shape change) to the end of the 6-minute reaction time.
Citation: American Journal of Veterinary Research 74, 9; 10.2460/ajvr.74.9.1212
Equine platelet aggregation over time in response to activation with 5μM ADP (arrowhead) in stirred PRP. The maximum amplitude was measured at 6 minutes after the addition of ADP. The AUC (shaded region) represents the area bounded by the time point of the aggregation tracing's return to baseline from the negative deflection (denoting shape change) to the end of the 6-minute reaction time.
Citation: American Journal of Veterinary Research 74, 9; 10.2460/ajvr.74.9.1212
Closure time—Platelet aggregation and adhesion in citrated blood under high shear were evaluated in the platelet function analyzerh with collagen and ADP (Col/ADP)m cartridges according to the manufacturer's instructions. After gentle mixing, 800 μL of blood was pipetted into a cartridge sample reservoir, and the cassette was loaded into the instrument for incubation and measurement of Clt. Assays were performed in duplicate, serially, with a single lot of cartridges. The mean values of the duplicate values were used for statistical analyses.
Thrombelastography—A 2-channel thrombelastographg with associated software was used with the manufacturer's test kit in an assay designed to assess the effects of platelet inhibitory drugs. The ADP platelet mapping assayn is performed by comparing MAthrombin, MAADP, and MAfibrin.27 The assay assumes that MAthrombin represents the total maximal clot strength, combining the contributions of fibrin formed via the coagulation cascade and activated platelets. The variable MAfibrin represents only the fibrin generated by reptilase cleavage of fibrinogen and is determined by use of heparinized blood to inhibit thrombin generation and prevent thrombin's action on fibrinogen. The MAADP reflects a further increment in clot strength resulting specifically from ADP-induced platelet activation. Preliminary experiments revealed that unlike humans, horses had similar or greater MAfibrin values, compared with MAthrombin (Figure 2). This lack of differential rendered the assay an insensitive measure of platelet function. We therefore undertook a series of dilution experiments with blood samples from 2 healthy horses (not included in either treatment group) and found that a 1:4 dilution of the reptilase-factor XIII reagent consistently generated MAfibrin values no greater than half that of each horse's paired MAthrombin values. For the study, the supplied reptilase-factor XIII reagent was first reconstituted in 50 μL of distilled water according to the manufacturer's instructions, then further diluted 1:4 in distilled water just before use. The assay was then performed according to the manufacturer's directions.27 The thrombelastograph'sg software calculates the contribution of ADP-induced platelet activation to maximal amplitudes as follows: ([MAADP – MAfibrin/MAthrombin – MAfibrin) × 100) and then subtracts this value from 100 to derive percentage platelet inhibition. In addition to the derived percentage platelet inhibition variable and MAthrombin, the routine kaolin-activated thrombelastographic parameters R, K, and alpha were calculated from the qualitative tracings.
Thrombelastographic profiles of the effects of undiluted and diluted reptilase used for activation of equine blood on the platelet mapping assay variable MAfibrin. A—Superimposed reference profiles of human blood depicting the expected relative maximal clot strength of kaolin-activated blood (MAthrombin; gray line) versus reptilase-activated blood (MAfibrin; black line). B—Superimposed profiles of equine kaolin-activated blood (MAthrombin; gray line) and undiluted-reptilase–activated blood (MAfibrin; black line). Unlike human blood, equine MAfibrin is greater than MAthrombin. C—The same equine blood sample as in B activated with diluted reptilase. Note that MAfibrin is now smaller than MAthrombin and follows the pattern of the human tracing.
Citation: American Journal of Veterinary Research 74, 9; 10.2460/ajvr.74.9.1212
Thrombelastographic profiles of the effects of undiluted and diluted reptilase used for activation of equine blood on the platelet mapping assay variable MAfibrin. A—Superimposed reference profiles of human blood depicting the expected relative maximal clot strength of kaolin-activated blood (MAthrombin; gray line) versus reptilase-activated blood (MAfibrin; black line). B—Superimposed profiles of equine kaolin-activated blood (MAthrombin; gray line) and undiluted-reptilase–activated blood (MAfibrin; black line). Unlike human blood, equine MAfibrin is greater than MAthrombin. C—The same equine blood sample as in B activated with diluted reptilase. Note that MAfibrin is now smaller than MAthrombin and follows the pattern of the human tracing.
Citation: American Journal of Veterinary Research 74, 9; 10.2460/ajvr.74.9.1212
Thrombelastographic profiles of the effects of undiluted and diluted reptilase used for activation of equine blood on the platelet mapping assay variable MAfibrin. A—Superimposed reference profiles of human blood depicting the expected relative maximal clot strength of kaolin-activated blood (MAthrombin; gray line) versus reptilase-activated blood (MAfibrin; black line). B—Superimposed profiles of equine kaolin-activated blood (MAthrombin; gray line) and undiluted-reptilase–activated blood (MAfibrin; black line). Unlike human blood, equine MAfibrin is greater than MAthrombin. C—The same equine blood sample as in B activated with diluted reptilase. Note that MAfibrin is now smaller than MAthrombin and follows the pattern of the human tracing.
Citation: American Journal of Veterinary Research 74, 9; 10.2460/ajvr.74.9.1212
Flow cytometry sample activation and labeling—Platelet activation status and PMP were evaluated in samples of PLRP with no exogenous stimulation and after the ex vivo addition of a series of agonists. All reactions were carried out in 5-mL polystyrene tubeso in a labeling bufferp containing final concentrations of 2mM ionized calcium and 0.4mM gly-pro-arg-pro-NH2.q The activation mixtures contained 1 μL of PLRP in a total reaction volume of 100 μL and a final concentration of one of the following agonist treatments: thrombinr (0.15 U/mL), convulxins (50 ng/mL), thrombin and convulxin (0.15 U/mL of thrombin and 50 ng/mL of convulxin), calcium ionophoret (3μM), or the buffer alone as an unstimulated control. In preliminary experiments, ADP proved to be a weak agonist and even at high concentration (40μM) failed to evoke a consistent response. The reaction tubes were set up in triplicate, activated at 20° to 23°C for 15 minutes, and double-labeled by the addition of a PE-conjugated fluorescent MAB directed against the platelet membrane antigen CD61 (GpIIIa)u and 1 of 3 activation markers. Platelet surface–bound fibrinogen was detected with an FITC-conjugated polyclonal antifibrinogen antibody,v P-selectin (CD62P) externalization was detected with a monoclonal anti-CD62P antibody conjugated with a green fluorescent dye,w and FITC-conjugated annexin Vx was used as a probe to detect outer membrane PS exposure. In preliminary experiments, the concentration of each marker was adjusted to center the population of interest between the second and third decade of a 4-decade log fluorescence scale while an isotype or unstimulated control was centered in the first decade (Figure 3). On the basis of these results, the manufacturer's supplied CD61-PE was diluted 1:4, fibrinogen-FITC was diluted 1:10, conjugated CD62P was diluted 1:100, and annexin V–FITC was diluted 1:40. Ten microliters of each dilution was used for labeling. To minimize nonspecific binding, the fibrinogen-FITC label was added to the reaction tubes at the same time as the agonists, and the remaining fluorescent markers were added after the 15-minute activation time. After addition of the labels, sample tubes were held in the dark for 30 minutes at 20° to 23°C, quench-diluted by the addition of 600 μL of the reaction buffer, and analyzed within 1 hour of quenching.
Fluorescence histograms of platelet populations gated on the basis of forward scatter and side scatter properties for optimization of fluorescent labels used to characterize platelets and variables of the platelet activation response in equine platelet– and leukocyte-rich plasma. The concentration of each fluorescent-conjugated probe was adjusted to center the histogram of positive events between the second and third decades of the log fluorescence scale and the corresponding isotype antibody and relevant unstimulated cell population within the scale's first decade. FL2-H = Height of fluorescence intensity signal in FL2 channel. FL1-H = Height of fluorescence intensity signal in FL1 channel. A—Unstimulated platelets labeled with murine MAb anti-human CD61-PE (solid line) or murine IgG-PE isotype control (gray fill). B—Thrombin-stimulated platelets (solid line) and unstimulated platelets (dashed line) labeled with rabbit polyclonal anti-human fibrinogen-FITC (Fib-FITC) antibody and unstimulated platelets labeled with a rabbit IgG-FITC isotype control (gray fill). C—Thrombin-stimulated platelets (solid line) and unstimulated platelets (dashed line) labeled with murine MAB anti-human CD62P-D488 and unstimulated platelets labeled with a murine IgG-D488 isotype control (gray fill). D—Ionophore-stimulated platelets (solid line) and unstimulated platelets (dashed line) labeled with annexin V–FITC (AnnV-FITC).
Citation: American Journal of Veterinary Research 74, 9; 10.2460/ajvr.74.9.1212
Fluorescence histograms of platelet populations gated on the basis of forward scatter and side scatter properties for optimization of fluorescent labels used to characterize platelets and variables of the platelet activation response in equine platelet– and leukocyte-rich plasma. The concentration of each fluorescent-conjugated probe was adjusted to center the histogram of positive events between the second and third decades of the log fluorescence scale and the corresponding isotype antibody and relevant unstimulated cell population within the scale's first decade. FL2-H = Height of fluorescence intensity signal in FL2 channel. FL1-H = Height of fluorescence intensity signal in FL1 channel. A—Unstimulated platelets labeled with murine MAb anti-human CD61-PE (solid line) or murine IgG-PE isotype control (gray fill). B—Thrombin-stimulated platelets (solid line) and unstimulated platelets (dashed line) labeled with rabbit polyclonal anti-human fibrinogen-FITC (Fib-FITC) antibody and unstimulated platelets labeled with a rabbit IgG-FITC isotype control (gray fill). C—Thrombin-stimulated platelets (solid line) and unstimulated platelets (dashed line) labeled with murine MAB anti-human CD62P-D488 and unstimulated platelets labeled with a murine IgG-D488 isotype control (gray fill). D—Ionophore-stimulated platelets (solid line) and unstimulated platelets (dashed line) labeled with annexin V–FITC (AnnV-FITC).
Citation: American Journal of Veterinary Research 74, 9; 10.2460/ajvr.74.9.1212
Fluorescence histograms of platelet populations gated on the basis of forward scatter and side scatter properties for optimization of fluorescent labels used to characterize platelets and variables of the platelet activation response in equine platelet– and leukocyte-rich plasma. The concentration of each fluorescent-conjugated probe was adjusted to center the histogram of positive events between the second and third decades of the log fluorescence scale and the corresponding isotype antibody and relevant unstimulated cell population within the scale's first decade. FL2-H = Height of fluorescence intensity signal in FL2 channel. FL1-H = Height of fluorescence intensity signal in FL1 channel. A—Unstimulated platelets labeled with murine MAb anti-human CD61-PE (solid line) or murine IgG-PE isotype control (gray fill). B—Thrombin-stimulated platelets (solid line) and unstimulated platelets (dashed line) labeled with rabbit polyclonal anti-human fibrinogen-FITC (Fib-FITC) antibody and unstimulated platelets labeled with a rabbit IgG-FITC isotype control (gray fill). C—Thrombin-stimulated platelets (solid line) and unstimulated platelets (dashed line) labeled with murine MAB anti-human CD62P-D488 and unstimulated platelets labeled with a murine IgG-D488 isotype control (gray fill). D—Ionophore-stimulated platelets (solid line) and unstimulated platelets (dashed line) labeled with annexin V–FITC (AnnV-FITC).
Citation: American Journal of Veterinary Research 74, 9; 10.2460/ajvr.74.9.1212
Flow cytometry data collection and analysis—Data were collected with an optical flow cytometery with the manufacturer's softwarez and a calibration bead setaa for daily standardization of forward and side scatter and fluorescence parameters. Data were acquired by use of logarithmic gain settings, and after initial optimization and compensation, the same settings were used throughout the study. For acquisition, 7,500 platelet events were collected in a gate defined by forward and side scatter properties. The cytometry data were analyzed with a software application.bb A gate to discriminate platelets and PMP was first set on dot plots derived from positive CD61-PE fluorescence. The subset of CD61-positive events that were below the first decade of the log forward scatter scale on these plots was enumerated as small platelets and PMP. The percentage of total CD61-positive events that were dual-labeled was compiled for each of the activation markers.
Fibrinogen and von Willebrand factor assays—The frozen samples of platelet-poor plasma were stored at −50°C until thawing at 37°C immediately before batch assay of fibrinogen and VWF:Ag. Fibrinogen was measured by use of a Clauss method and VWF:Ag, was measured in a multispecies ELISA, as described.28,29 A pooled equine plasma sample (prepared from 10 healthy horses) was used as a standard for both assays, with the fibrinogen content of the standard determined by a gravimetric method and its VWF:Ag value assigned as 100%.
Statistical analysis—In planning the study, it was assumed that 90% of horses that received a loading dose of clopidogrel would have inhibition of ADP-induced platelet aggregation and that a sample size of 12 horses (6 treatment and 6 control horses) would yield an 85% chance of detecting a significant (P < 0.05) difference between groups. Results obtained for the various assays were tested for normality by means of the D'Agostino-Pearson test. Parametric data were summarized as mean and SD values. Values were compared between treatment groups with nonpaired t tests and for individual horses between time points with paired t tests. The median and range were calculated for nonparametric data, with values compared between treatment groups by use of Mann-Whitney tests and among time points by use of Wilcoxon signed rank tests. The correlations between activation variables in cytometric tests were determined with the Spearman coefficient of rank correlation. Values of P < 0.05 were considered significant. All statistical analyses were performed with a software program.cc
Results
Clinical signs—All horses remained clinically healthy and had no abnormalities in vital signs during the study period. None developed spontaneous petechiae or ecchymoses, and none had abnormal bleeding from venipuncture sites or hemorrhage associated with passage of the nasogastric tube for drug administration.
CBC—No significant differences in mean CBC variables were detected between the 2 treatment groups at either time point; however, the clopidogrel-treated horses had a slight but significant decrease in hemoglobin concentration, Hct, WBC count, and RBC count at the T72 time point, compared with their pretreatment values (Table 1). The decrease in the group mean was attributed to mild reduction in cell counts for most horses, rather than a pronounced decrease in counts for 1 or 2 horses.
Mean (SD) CBC values and total plasma solids concentration for horses before (T0) and after (T72) clopidogrel or placebo administration.
| Variable | Clopidogrel | P value* | Placebo | P value* |
|---|---|---|---|---|
| Hemoglobin | 0.04 | 0.20 | ||
| T0 | 14.8 (1.4) | 14.3 (0.7) | ||
| T72 | 13.8 (1.6) | 13.9 (1.1) | ||
| Hct (%) | 0.04 | 0.16 | ||
| T0 | 42.9 (3.9) | 41.4 (2.3) | ||
| T72 | 39.9 (4.7) | 40.5 (3.2) | ||
| RBC (× 106/μL) | 0.03 | 0.08 | ||
| T0 | 8.6 (0.8) | 8.3 (0.3) | ||
| T72 | 7.9 (1.0) | 7.9 (0.4) | ||
| WBC (× 103/μL) | 0.03 | 0.61 | ||
| T0 | 8.9 (0.8) | 8.2 (1.7) | ||
| T72 | 8.2 (0.7) | 8.0 (1.0) | ||
| Platelets (× 103/μL) | 0.36 | 0.70 | ||
| T0 | 183 (71) | 156 (32) | ||
| T72 | 163 (24) | 153 (21) | ||
| Total solids (g/dL) | 0.05 | 0.90 | ||
| T0 | 6.9 (0.4) | 6.6 (0.4) | ||
| T72 | 6.5 (0.4) | 6.5 (0.5) |
P value for comparison between T0 and T72 values for each treatment group.
No significant between-group differences were found for any variable at T0 or T72.
Platelet aggregation—No significant between-group differences were detected at T0 for any of the mean aggregation variables in response to ADP or collagen stimulation. At T72, the mean AUC for 5μM ADP stimulation was significantly different between groups, and the clopidogrel group alone had significantly reduced posttreatment values for mean percentage aggregation in response to 5 and 10μM ADP and mean AUC for 5μM ADP (Table 2). The relative intensity of inhibition varied among the 6 clopidogrel-treated horses, with 4 having > 50% reduction in pretreatment values in response to both 5 and 10μM ADP stimulation, 1 horse having only mild reduction in aggregation response to both dosages, and 1 horse having no evidence of inhibition to either dose (Figure 4). The placebo group had no significant change in ADP response variables from T0 to T72. Neither treatment group had significant differences in the collagen aggregation response between the 2 time points, and posttreatment values did not differ between groups for this agonist (Table 2).
Maximal platelet aggregation (%) and AUC after the addition of ADP to PRP samples from 6 horses before and after treatment with clopidogrel. The symbols in each dot plot represent individual horses, and the lines interconnect their result values before clopidogrel treatment (T0) and after treatment (T72). The horse identification numbers were determined by order of entry into the study. A and B—Maximal aggregation and AUC in response to 5μM ADP. C and D—Maximal aggregation and AUC in response to 10μM ADP.
Citation: American Journal of Veterinary Research 74, 9; 10.2460/ajvr.74.9.1212
Maximal platelet aggregation (%) and AUC after the addition of ADP to PRP samples from 6 horses before and after treatment with clopidogrel. The symbols in each dot plot represent individual horses, and the lines interconnect their result values before clopidogrel treatment (T0) and after treatment (T72). The horse identification numbers were determined by order of entry into the study. A and B—Maximal aggregation and AUC in response to 5μM ADP. C and D—Maximal aggregation and AUC in response to 10μM ADP.
Citation: American Journal of Veterinary Research 74, 9; 10.2460/ajvr.74.9.1212
Maximal platelet aggregation (%) and AUC after the addition of ADP to PRP samples from 6 horses before and after treatment with clopidogrel. The symbols in each dot plot represent individual horses, and the lines interconnect their result values before clopidogrel treatment (T0) and after treatment (T72). The horse identification numbers were determined by order of entry into the study. A and B—Maximal aggregation and AUC in response to 5μM ADP. C and D—Maximal aggregation and AUC in response to 10μM ADP.
Citation: American Journal of Veterinary Research 74, 9; 10.2460/ajvr.74.9.1212
Mean (SD) values of platelet aggregation variables for horses before (T0) and after (T72) clopidogrel or placebo administration.
| Variable | Clopidogrel | P value* | Placebo | P value* |
|---|---|---|---|---|
| Aggregation to 5μM ADP (% of maximal) | 0.03 | 0.34 | ||
| T0 | 65 (10.9) | 66.3 (11.9) | ||
| T72 | 39.5 (24.1) | 60.2 (9.0) | ||
| AUC to 5μM ADP | 0.02 | 0.51 | ||
| T0 | 261 (48.7) | 268 (54.6) | ||
| T72 | 154 (82.4) | 244 (30.8) | ||
| Aggregation to 10μM ADP (% of maximal) | 0.04 | 0.45 | ||
| T0 | 72.5 (11.9) | 67.3 (8.9) | ||
| T72 | 45.7 (22.4) | 63.2 (5.3) | ||
| AUC to 10μM ADP | 0.06 | 0.42 | ||
| T0 | 335.7 (111.0) | 278.8 (46.7) | ||
| T72 | 189.4 (85.2) | 254.3 (29.1) | ||
| Aggregation to collagen (% of maximal) | 0.38 | 0.28 | ||
| T0 | 62.3 (8.1) | 58.2 (2.9) | ||
| T72 | 57.3 (8.1) | 57.8 (4.9) | ||
| AUC to collagen | 0.20 | 0.61 | ||
| T0 | 220.3 (79.6) | 196.4 (23.1) | ||
| T72 | 161.3 (46.6) | 182.0 (26.4) |
The only significant between-group difference was for AUC to 5μM ADP at T72 (P = 0.04).
See Table 1 for remainder of key.
Closure time—At T0, the mean ± SD CT for the clopidogrel- and placebo-treated groups was 94.5 ± 28.5 seconds and 83.5 ± 8.2 seconds, respectively. The corresponding T72 values were 89.0 ± 18.6 seconds and 93.1 ± 15.6 seconds, respectively. No significant differences were detected between the T0 and T72 mean values for either group, and no differences were detected between groups at either time point.
Kaolin-activated thrombelastography and platelet mapping—No significant between-group differences were detected at T0 or T72 for the kaolin-activated parameters R, K, or angle or for MAthrombin, MAfibrin, and MAADP (Table 3). The data for these parameters were normally distributed, and the mean ± SD values for all 12 study horses at baseline were 18.0 ± 4.1 minutes for R, 5.9 ± 1.9 minutes for K, 21.0 ± 5.5 degrees for angle, 54.9 ± 8.3 mm for MAthrombin, 25.0 ± 12.1 mm for MAfibrin, and 32.1 ± 9.3 mm for MAADP. Although mean MAADP was greater than mean MAfibrin, the difference was not significant (P = 0.129). Comparisons between time points within each group revealed significant differences in mean MAthrombin for the clopidogrel-treated horses and mean K for the placebo group. The contribution of ADP-induced platelet aggregation to final clot strength was detected by subtracting the value for MAfibrin from MAADP. Although MAADP is expected to be greater than MAfibrin, the observed difference between these variables varied widely among horses. The median (range) for the difference at T0 for the clopidogrel-treated horses was 8.5 (–1.6 to 19.6) and at T72 was 4.1 (–1.5 to 15.2). The corresponding values for the placebo group at T0 were 2.6 (1.1 to 17.4) and at T72 were 2.6 (–0.6 to 7.8). These median values did not differ significantly between groups or between time points for either group. The calculated platelet mapping parameter incorporates all 3 maximal amplitude values and represents a percentage reduction in maximal amplitude attributed to inhibition of the ADP receptor. This parameter also varied widely among horses. The median (range) for the clopidogrel-treated horses at T0 was 78.5% (55.6% to 100%) and at T72 was 85% (39% to 100%). The placebo group median value at T0 was 86.5% (43.7% to 97.5%) and at T72 was 81.3% (0% to 100%). The differences in median ADP receptor inhibition were not significant between treatment groups or between time points for each treatment group.
Mean (SD) values of thrombelastographic variables for horses before (T0) and after (T72) clopidogrel or placebo administration.
| Variable | Clopidogrel | P value* | Placebo | P value* |
|---|---|---|---|---|
| TEG-R (min) | 0.9 | 0.1 | ||
| T0 | 20.2 (5.1) | 15.9 (0.8) | ||
| T72 | 19.9 (2.7) | 18.0 (2.34) | ||
| TEG-K (min) | 0.7 | 0.02 | ||
| T0 | 6.9 (2.3) | 5.0 (0.6) | ||
| T72 | 6.5 (2.5) | 6.2 (0.7) | ||
| TEG-angle (degrees) | 0.06 | 0.07 | ||
| T0 | 18.6 (6.6) | 23.5 (3.1) | ||
| T72 | 25.5 (8.7) | 20.9 (2.5) | ||
| MAthrombin (mm) | 0.03 | 0.67 | ||
| T0 | 52.3 (9.7) | 57.6 (6.4) | ||
| T72 | 60.8 (11.0) | 58.3 (6.2) | ||
| MAfibrin (mm) | 0.08 | 0.12 | ||
| T0 | 20.6 (11.6) | 29.5 (11.9) | ||
| T72 | 28.5 (9.5) | 39.5 (7.9) | ||
| MAADP (mm) | 0.17 | 0.67 | ||
| T0 | 29.8 (7.9) | 34.3 (10.8) | ||
| T72 | 34.0 (8.3) | 42.5 (7.5) |
See Table 1 for key.
Flow cytometric analyses of activation parameters for unstimulated platelets—No significant differences were detected between treatment groups at T0 or T72 for any variables of platelet activation in the unstimulated platelet reactions (Table 4). These data were not normally distributed, and the median (range) for all 12 horses at baseline that had positive results for the activation markers was 2.1% (1.0% to 7.7%) for fibrinogen, 1.8% (0.3% to 4.9%) for CD62P, and 1.1% (0.5% to 3.5%) for annexin V. The number of PMP/7,500 CD61-positive events was 119 (range, 49 to 250). The values for these markers at T72 were similar to the values at T0 and did not differ significantly between time points or between treatment groups.
Median (range) values for activation variables of unstimulated platelets for horses before (T0) and after (T72) clopidogrel or placebo administration.
| Variable | Clopidogrel | P value* | Placebo | P value* |
|---|---|---|---|---|
| Fibrinogen positive (%) | 1.0 | 0.44 | ||
| T0 | 2.8 (1.0–7.7) | 1.8 (1.0–6.0) | ||
| T72 | 3.5 (1.3–8.3) | 1.8 (1.1–3.9) | ||
| CD62P positive (%) | 0.43 | 0.22 | ||
| T0 | 2.0 (0.6–4.9) | 1.6 (0.3–2.4) | ||
| T72 | 1.7 (0.8–6.5) | 1.2 (0.7–1.4) | ||
| Annexin V positive (%) | 0.52 | 0.43 | ||
| T0 | 0.9 (0.8–2.1) | 1.2 (0.5–3.5) | ||
| T72 | 0.8 (0.3–14.0) | 0.9 (0.5–1.2) | ||
| PMP (per 7,500 CD61-positive events) | 0.56 | 0.84 | ||
| T0 | 113 (75–213) | 119 (49–250) | ||
| T72 | 80 (59–1,186) | 134 (51–211) |
P value for comparison between T0 and T72 values for each treatment group.
See Table 1 for remainder of key.
Flow cytometric analyses of activation parameters on agonist-stimulated platelets—The first 2 horses enrolled in the study (clopidogrel group) had data only for thrombin stimulation; the remaining 10 horses had data for stimulation with 4 agonist treatments: thrombin, convulxin, combined thrombin plus convulxin, and ionophore. The pattern of platelet activation was generally consistent for all horses but varied by agonist. The agonists evoked qualitative differences in labeling intensity and the light scatter properties of platelets (Figure 5), in addition to quantitative differences in the percentage of platelet events that expressed the activation markers (Figure 6). Most thrombin-stimulated platelets had CD62P labeling and labeling for fibrinogen, but < 20% bound annexin V. Convulxin stimulation alone induced an intermediate proportion of platelets labeled with fibrinogen and CD62P but induced minimal annexin V binding. The combination of thrombin and convulxin resulted in a new population of small platelets and PMP (denoted by a decrease in forward scatter). The CD62P and fibrinogen markers bound platelets and PMP equally; however, annexin V was bound almost exclusively to activation-induced populations of small platelets and PMP. Ionophore treatment generated mostly small platelets and PMP that demonstrated high-intensity annexin V fluorescence but relatively low intensity and few platelets with CD62P or fibrinogen labeling. Convulxin stimulation alone induced the most variable interindividual response in CD62P and fibrinogen labeling, and the values for these 2 variables were correlated (r = 0.69; P = 0.02). No significant differences were detected between treatment groups at either time point for any of the activation variables in response to the agonist stimuli, and no significant differences were detected between time point T0 and T72 for either treatment group.
Flow cytometric analyses of equine platelet activation in response to a series of agonists. Platelet- and leukocyte-rich plasma samples were incubated with a vehicle control (NS) or activated with thrombin alone (Th, 0.15 U/mL), convulxin alone (CVX, 50 ng/mL), combined thrombin and convulxin (TC), or calcium ionophore (Iono, 3μM). The dual-labeled samples were first gated on the CD61-positive events for analysis. The rectangular gate on each of the plots encloses the subpopulation of activated cells. The y-axis arrow represents increasing forward light scatter properties, and the x-axis arrows represent increasing fluorescence intensity of 3 activation markers: fibrinogen (top row), CD62P (middle row), and annexin V (AV; bottom row).
Citation: American Journal of Veterinary Research 74, 9; 10.2460/ajvr.74.9.1212
Flow cytometric analyses of equine platelet activation in response to a series of agonists. Platelet- and leukocyte-rich plasma samples were incubated with a vehicle control (NS) or activated with thrombin alone (Th, 0.15 U/mL), convulxin alone (CVX, 50 ng/mL), combined thrombin and convulxin (TC), or calcium ionophore (Iono, 3μM). The dual-labeled samples were first gated on the CD61-positive events for analysis. The rectangular gate on each of the plots encloses the subpopulation of activated cells. The y-axis arrow represents increasing forward light scatter properties, and the x-axis arrows represent increasing fluorescence intensity of 3 activation markers: fibrinogen (top row), CD62P (middle row), and annexin V (AV; bottom row).
Citation: American Journal of Veterinary Research 74, 9; 10.2460/ajvr.74.9.1212
Flow cytometric analyses of equine platelet activation in response to a series of agonists. Platelet- and leukocyte-rich plasma samples were incubated with a vehicle control (NS) or activated with thrombin alone (Th, 0.15 U/mL), convulxin alone (CVX, 50 ng/mL), combined thrombin and convulxin (TC), or calcium ionophore (Iono, 3μM). The dual-labeled samples were first gated on the CD61-positive events for analysis. The rectangular gate on each of the plots encloses the subpopulation of activated cells. The y-axis arrow represents increasing forward light scatter properties, and the x-axis arrows represent increasing fluorescence intensity of 3 activation markers: fibrinogen (top row), CD62P (middle row), and annexin V (AV; bottom row).
Citation: American Journal of Veterinary Research 74, 9; 10.2460/ajvr.74.9.1212
Flow cytometric assessment (median percentage of platelets labeled) of variables of equine platelet activation (A = fibrinogen binding; B = P-selectin externalization; and C = PS externalization) before (T0) and after (T72) clopidogrel or placebo administration in 12 horses. Error bars represent the 25th and 75th percentiles. Th = Thrombin (0.15 U/mL). CVX = Convulxin (50 ng/mL). Iono = Calcium ionophore (3μM). TC = Combined thrombin and convulxin (0.15 U/mL and 50 ng/mL).
Citation: American Journal of Veterinary Research 74, 9; 10.2460/ajvr.74.9.1212
Flow cytometric assessment (median percentage of platelets labeled) of variables of equine platelet activation (A = fibrinogen binding; B = P-selectin externalization; and C = PS externalization) before (T0) and after (T72) clopidogrel or placebo administration in 12 horses. Error bars represent the 25th and 75th percentiles. Th = Thrombin (0.15 U/mL). CVX = Convulxin (50 ng/mL). Iono = Calcium ionophore (3μM). TC = Combined thrombin and convulxin (0.15 U/mL and 50 ng/mL).
Citation: American Journal of Veterinary Research 74, 9; 10.2460/ajvr.74.9.1212
Flow cytometric assessment (median percentage of platelets labeled) of variables of equine platelet activation (A = fibrinogen binding; B = P-selectin externalization; and C = PS externalization) before (T0) and after (T72) clopidogrel or placebo administration in 12 horses. Error bars represent the 25th and 75th percentiles. Th = Thrombin (0.15 U/mL). CVX = Convulxin (50 ng/mL). Iono = Calcium ionophore (3μM). TC = Combined thrombin and convulxin (0.15 U/mL and 50 ng/mL).
Citation: American Journal of Veterinary Research 74, 9; 10.2460/ajvr.74.9.1212
Fibrinogen and VWF—At T0, the mean ± SD fibrinogen concentrations for the clopidogrel- and placebo-treated groups were 357 ± 149 mg/dL and 402 ± 62 mg/dL, respectively. The corresponding T72 values were 385 ± 97 mg/dL and 412 ± 39 mg/dL, respectively. At T0, the mean VWF:Ag for the clopidogrel- and placebo-treated groups were 137 ± 45% and 150 ± 23%, respectively. The corresponding T72 values were 143 ± 43% and 154 ± 20%, respectively. No significant differences in mean values for these proteins were detected between groups at either time point, and no differences were detected between the T0 and T72 time points for either group.
Discussion
Results indicated that oral clopidogrel administration at an initial loading dose of 4 mg/kg, followed by once-daily administration of 2 mg/kg, caused significant inhibition of ADP-induced aggregation as detected by LTA in most healthy horses. The drug was well tolerated and did not cause clinical signs of hemorrhage. A slight reduction in posttreatment mean RBC and WBC counts occurred, but all horses’ values remained within reference intervals, and the group mean did not differ significantly from placebo-treated horses. Four of the 6 treated horses had > 50% platelet inhibition from baseline and had residual aggregation < 70%. These measures of posttreatment response have been determined to be clinically beneficial in human treatment trials13,16 and are comparable to results of a recent pharmacodynamic study25 of clopidogrel in horses. Unlike the previous equine study,25 2 horses in the study reported here appeared to be poorly responsive to clopidogrel's antiplatelet effect. Rather than 15μM ADP, we used 5 and 10μM ADP agonist stimulation in the LTA experiments. It is unlikely, however, that suboptimal agonist concentration hindered the ability to detect platelet inhibition. A robust aggregation response occurred to both concentrations of ADP, and values were similar to the mean percentage aggregation in a previous report.25 Moreover, the use of a lower-dose agonist concentration is expected to enhance, rather than reduce, LTA sensitivity for detection of the presence of antiplatelet agents.6,30 A recent study31 performed to standardize LTA for clopidogrel monitoring in humans found highly concordant results across a dosage range of 10 to 20μM ADP, and the authors ultimately recommended use of 10μM ADP to identify patients with high residual posttreatment platelet activity.
The present study subjects included horses of several breeds in addition to Quarter Horses as used in the previous equine study.25 Both of the clopidogrel-refractory horses in the present study were warmbloods; however, a third warmblood in the treatment group had > 50% platelet inhibition. Evaluation of more horses and more breeds will be required to determine whether breed-specific factors influence equine clopidogrel response. Polymorphisms at the hepatic cytochrome P450, family 2, subfamily C, polypeptide 19 locus are the most common cause of ineffective prodrug metabolism, which in turn causes clopidogrel nonresponsiveness in humans.12 It is possible that variations among horses in activity of this or other cytochrome P450s affect clopidogrel metabolism and consequently its antiplatelet effects.
The whole blood assays, CT determined by platelet function analyzer,h and thrombelastographic platelet mappingn were not useful to detect clopidogrel's platelet inhibitory action in horses. The platelet function analyzer's assay cartridges and the instrument's vacuum mechanism are not modifiable; therefore, we could not determine whether manipulating agonist concentration or shear force might improve assay sensitivity.32 The collagen-ADP cartridges that were evaluated are most widely used to screen for aggregation defects, von Willebrand disease, and, in combination with a collagen-epinephrine cartridge, to monitor aspirin effect in humans.17,33 Although some studies34,35 have associated CT results with patient outcomes, the platelet function analyzer's diagnostic usefulness for clopidogrel monitoring has not been established.16,30,36 The manufacturer, however, has recently developed a modified cartridge for a second-generation analyzerdd that is undergoing evaluation for this purpose.
The platelet mapping assayn is complex and based on a differential in maximal amplitude values obtained in the presence and absence of platelet agonists in reptilase-treated heparinized whole blood. That difference is then compared with the maximal amplitude of a clot generated via contact pathway–activated thrombin formation. The reptilase–factor XIIIa components of the manufacturer's supplied reagent generated clots with much higher MAfibrin in horses than in humans, and those high values precluded detection of the influence of ADP-induced platelet activation on clot strength. The modified assay with diluted reptilase reagent generated thrombelastographic profiles that were qualitatively similar to human profiles. However, the difference between MAfibrin and MAADP varied widely among horses, including 1 of 12 study horses that had no detectable increment in maximal amplitude in the ADP-activated sample. Further reducing the reptilase reagent concentration could be attempted to generate a more pronounced MAADP increment, but too low a reptilase concentration risks rendering the assay nonspecific (because of inadequate fibrin generation). An alternate strategy would be to increase the ADP concentration in the final assay mixture from 2μM to a concentration that consistently generates higher MAADP values. This strategy to modify the platelet mapping assayn was used in a recent study37 of the antiplatelet effects of acepromazine in dogs. High concentrations of ADP, however, were not required in a second canine study38 that detected clopidogrel's inhibitory action on the basis of posttreatment reduction in MAADP. Together, the observed variations in MAfibrin and MAADP among species suggest that functional differences in the underlying processes of fibrin formation and polymerization and platelet activation limit direct application of the platelet mapping assayn in veterinary studies.
In the present study, previously described flow cytometric methods of analyzing equine platelets39–41 were adapted with the goals of minimizing ex vivo platelet activation, detecting distinct variables of the activation response, and simplifying and standardizing the process to use commercially available fluorescent-conjugated labels. In the protocol, platelets from small-volume samples of PLRP were recovered without centrifugation and then activated, labeled, and analyzed in a single tube with no washing steps. The gating strategy for analyses was based on threshold intensity of a cross-reactive CD61 label, a high-density antigen constitutively expressed on platelets and PMP. This allowed identification of platelets in cell suspensions and differentiation of small platelets and PMP from machine noise and debris. Detection of P-selectin (CD62P), an α-granule membrane protein, on the outer membrane surface denotes the process of degranulation and is the most widely used cytometric marker of platelet activation. Unlike a previous study40 that found high (20% to 25%) constitutive expression of P-selectin on equine platelets, a median value of < 2% expression on unstimulated platelets was detected in the present study. Low basal expression was not caused by platelet refractoriness because thrombin stimulation generated > 80% of the platelets positive for the P-selectin marker. Differences in CD62P antibodies and preanalytic processing may account for the observed variation between studies.
The fibrinogen assay configuration we used detected fibrinogen bound to platelets but did not define specific receptors or conformational states that supported the interaction.42–44 The fibrinogen-positive cells may have included platelets with occupied GpIIbIIIa receptors in either resting or active conformation and fibrinogen-antibody complexes nonspecifically bound to the platelet membrane Fc receptors. The pattern of fibrinogen binding differed among agonist treatments and did not match either of the other activation markers. Fibrinogen binding, although not specific for aggregation, reflected a variable of the platelet activation response distinct from degranulation or procoagulant activity. Annexin V binding, which indicates externalized PS and the procoagulant property of activated platelets, also had a distinct labeling pattern. Dual agonist stimulation with thrombin plus convulxin (an agonist that binds to the glycoprotein VI collagen receptor) generated a unique subpopulation of small platelets and PMP with high expression of PS, and ionophore treatment was the most intense stimulus to evoke this procoagulant response. Overall, equine platelets had the characteristic procoagulant response described in other species for each of these agonist stimuli.42,43,45
Inhibitory effects of clopidogrel on any of the variables of basal or poststimulation platelet reactivity were not detected in the cytometric assays. This finding was similar to that of a recent study46 that found no change in platelet P-selectin expression after thrombin stimulation in 6 clopidogrel-treated horses. In our preliminary experiments, ADP failed to induce P-selectin expression at concentrations 10-fold the concentrations that induced aggregation. Therefore, ADP could not be used as the agonist for posttreatment inhibition studies. A more sensitive and specific cytometric test to detect P2Y12 receptor inhibition has been developed to monitor thienopyridine treatment in humans.14,30,36 The assay measures the phosphorylation status of an intraplatelet phosphoprotein downstream of the P2Y12 receptor, and results have been correlated with LTA in some human studies. The direct application of these commercial cytometric assays to monitor equine clopidogrel response will depend on the cross-reactivity of the kits’ monoclonal antibody reagents directed against the human phosphoprotein (vasodilator-stimulated phosphoprotein) and human platelet GpIIIa.
The small number of horses was an important limitation of this study, and it is possible that detection of small treatment effects was not possible because of type 2 error. An inability to detect minor impairment of platelet activation, however, does not influence our interpretation of the study results. None of the whole blood or cytometric activation variables that were assessed can augment or replace LTA as a screening test of clopidogrel's platelet inhibitory action in horses.
In the present study, an interindividual variability in the antiplatelet effects of clopidogrel in healthy horses was detected that may be relevant for evaluating drug efficacy in treatment trials and clinical practice. Among the assays investigated, LTA induced by ADP was the most appropriate test to assess clopidogrel's biological action; 10μM ADP, a concentration recommended to standardize LTA in humans,31 induced a strong aggregation response in horses, but did not prevent identification of horses with high residual platelet reactivity after clopidogrel treatment. Additional investigations are needed to determine whether high-dose clopidogrel can overcome apparent drug resistance and to configure simple whole blood, cytometric, or other screening tests suitable to monitor clopidogrel in horses for future pharmacodynamic or clinical studies.
ABBREVIATIONS
| AUC | Area under the aggregation curve |
| CT | PFA-100 closure time |
| FITC | Fluorescein isothiocyanate |
| LTA | Light transmission aggregometry |
| MAB | Monoclonal antibody |
| MAADP | Maximal amplitude of reptilase/factor XIII– and ADP-activated heparinized blood |
| MAfibrin | Maximal amplitude of reptilase/factor XIII–activated heparinized blood |
| MAthrombin | Maximal amplitude of kaolin-activated citrated blood |
| PE | Phycoerythrin |
| PLRP | Platelet- and leukocyte-rich plasma |
| PMP | Platelet membrane–derived microparticles |
| PPP | Platelet-poor plasma |
| PRP | Platelet-rich plasma |
| PS | Phosphatidylserine |
| VWF:Ag | von Willebrand factor antigen |
Cornell University Equine Park, Ithaca, NY.
Plavix, 75 mg USP, Bristol-Meyers Squibb, Princeton, NJ.
QuickCalcs, GraphPad Software Inc, La Jolla, Calif.
Vacutainer K2 EDTA and Vacutainer Heparin, BD, Franklin Lakes, NJ.
Blood collection set, Hospira Inc, Lake Forest, Ill.
Advia 2120, Siemens Heathcare Diagnostics, Tarrytown, NY.
TEG 5000 Thromboelastograph Haemostasis Analyzer System, Haemonetics, Braintree, Mass.
PFA-100 System, Siemens Heathcare Diagnostics, Tarrytown, NY.
500CA, Lumi-aggregometer, Chrono-log Corp, Havertown, Pa.
ADP, Chrono-log Corp, Havertown, Pa.
Collagen (type 1, equine tendon), Chrono-log Corp, Havertown, Pa.
Aggro/Link interface and software, Chrono-log Corp, Havertown, Pa.
Col/ADP test cartridges, Siemens Heathcare Diagnostics, Tarrytown, NY.
Platelet mapping assay, ADP, Haemonetics Corp, Braintree, Mass.
Falcon, BD, Franklin Lakes, NJ.
Annexin V binding buffer, R&D Systems Inc, Minneapolis, Minn.
gly-pro-arg-pro-NH2 acetate, Sigma-Aldrich Corp, St Louis, Mo.
Bovine thrombin, Sigma-Aldrich Corp, St Louis, Mo.
Convulxin, Centerchem, Norwark, Conn.
Calcium ionophore A23187, Fisher Scientific, Pittsburgh, Pa.
CD 61PE (clone SZ21), Beckman Coulter Inc, Brea, Calif.
Fibrinogen-FITC, Dako, Carpinteria, Calif.
CD62P-DY488 (clone Psel.KO.2.7), Novus Biologicals, Littleton, Colo.
Annexin V–FITC, R&D Systems Inc, Minneapolis, Minn.
BD FACSCalibur, BD, San Jose, Calif.
CELLQUEST, BD, San Jose, Calif.
Calibrite Beads, BD, San Jose, Calif.
FloJo, version 9.5.3, Treestar Inc, Ashland, Ore.
MedCalc, version 12.3.0, MedCalc Software, Mariakerke, Belgium.
Innovance PFA P2Y for PFA-200 System, Siemens Heathcare Diagnostics, Tarrytown, NY.
References
1. Boudreaux MK, Catalfamo JL. Platelet biochemistry, signal transduction, and function. In: Weiss D, Wardrop KJ, eds. Schalm's veterinary hematology. 6th ed. Ames, Iowa: Blackwell Publishing, 2010;569–575.
2. Furie B, Furie BC. Mechanisms of thrombus formation. N Engl J Med 2008; 359:938–949.
3. Leslie M. Beyond clotting: the powers of platelets. Science 2010; 328:562–564.
4. Parise LV, Boudignon-Proudhon C, Keely PJ, et al. Platelets in hemostasis and thrombosis. In: Lee GR, ed. Wintrobe's clinical hematology. Baltimore: Williams & Wilkins, 1999;661–683.
5. Jedlitschky G, Greinacher A, Kroemer HK. Transporters in human platelets: physiologic function and impact for pharmacotherapy. Blood 2012; 119:3394–3402.
6. Kalantzi KI, Tsoumani ME, Goudevenos IA, et al. Pharmacodynamic properties of antiplatelet agents: current knowledge and future perspectives. Expert Rev Clin Pharmacol 2012; 5:319–336.
7. Hechler B, Cattaneo M, Gachet C. The P2 receptors in platelet function. Semin Thromb Hemost 2005; 31:150–161.
8. Hechler B, Magnenat S, Zighetti ML, et al. Inhibition of platelet functions and thrombosis through selective or nonselective inhibition of the platelet P2 receptors with increasing doses of NF449 [4,4′,4″,4″′-(carbonylbis(imino-5,1,3-benzenetriylbis-(carbonylimino)))tetrakis -benzene-1,3-disulfonic acid octasodium salt]. J Pharmacol Exp Ther 2005;314:232–243.
9. Arora RR, Rai F. Antiplatelet intervention in acute coronary syndrome. Am J Ther 2009; 16: e29–e40.
10. Mullangi R, Srinivas NR. Clopidogrel: review of bioanalytical methods, pharmacokinetics/pharmacodynamics, and update on recent trends in drug-drug interaction studies. Biomed Chromatogr 2009; 23:26–41.
11. Savi P, Herbert JM. Clopidogrel and ticlopidine: P2Y12 adenosine diphosphate-receptor antagonists for the prevention of atherothrombosis. Semin Thromb Hemost 2005; 31:174–183.
12. Desta Z, Zhao X, Shin JG, et al. Clinical significance of the cytochrome P450 2C19 genetic polymorphism. Clin Pharmacokinet 2002; 41:913–958.
13. Cotton JM, Worrall AM, Hobson AR, et al. Individualised assessment of response to clopidogrel in patients presenting with acute coronary syndromes: a role for short thrombelastography? Cardiovasc Ther 2010; 28:139–146.
14. Gaglia MA, Torguson R, Pakala R, et al. Correlation between light transmission aggregometry, VerifyNow P2Y12, and VASP-P platelet reactivity assays following percutaneous coronary intervention. J Interv Cardiol 2011; 24:529–534.
15. Hochholzer W, Trenk D, Frundi D, et al. Whole blood aggregometry for evaluation of the antiplatelet effects of clopidogrel. Thromb Res 2007; 119:285–291.
16. Parodi G, Marcucci R, Valenti R, et al. High residual platelet reactivity after clopidogrel loading and long-term cardiovascular events among patients with acute coronary syndromes undergoing PCI. JAMA 2011; 306:1215–1223.
17. Favaloro EJ, Lippi G, Franchini M. Contemporary platelet function testing. Clin Chem Lab Med 2010; 48:579–598.
18. Bailey SR, Adair HS, Reinemeyer CR, et al. Plasma concentrations of endotoxin and platelet activation in the developmental stage of oligofructose-induced laminitis. Vet Immunol Immunopathol 2009; 129:167–173.
19. Dallap BL. Coagulopathy in the equine critical care patient. Vet Clin North Am Equine Pract 2004; 20:231–251.
20. Elliott J, Berhane Y, Bailey SR. Effects of monoamines formed in the cecum of horses on equine digital blood vessels and platelets. Am J Vet Res 2003; 64:1124–1131.
21. Lopes MA, Salter CE, Vandenplas ML, et al. Expression of inflammation-associated genes in circulating leukocytes collected from horses with gastrointestinal tract disease. Am J Vet Res 2010; 71:915–924.
22. Sykes BW, Furr MO. Equine endotoxaemia—a state-of-the-art review of therapy. Aust Vet J 2005; 83:45–50.
23. Boudreaux MK. Characteristics, diagnosis, and treatment of inherited platelet disorders in mammals. J Am Vet Med Assoc 2008; 233:1251–1259.
24. Brooks MB, Stokol T, Catalfamo JL. Comparative hemostasis: animal models and new hemostasis tests. Clin Lab Med 2011; 31:139–159.
25. Brainard BM, Epstein KL, LoBato D, et al. Effects of clopidogrel and aspirin on platelet aggregation, thromboxane production, and serotonin secretion in horses. J Vet Intern Med 2011; 25:116–122.
26. Savcic M, Hauert J, Bachmann F, et al. Clopidogrel loading dose regimens: kinetic profile of pharmacodynamic response in healthy subjects. Semin Thromb Hemost 1999; 25(suppl 2):15–19.
27. Bochsen L, Wiinberg B, Kjelgaard-Hansen M, et al. Evaluation of the TEG platelet mapping assay in blood donors. Thromb J [serial online]. 2007; 5:3. Available at: www.thrombosisjournal.com/content/5/1/3. Accessed Nov 8, 2012.
28. Benson RE, Catalfamo JL, Dodds WJ. A multispecies enzyme-linked immunosorbent assay for von Willebrand's factor. J Lab Clin Med 1992; 119:420–427.
29. Watts AE, Fubini SL, Todhunter RJ, et al. Comparison of plasma and peritoneal indices of fibrinolysis between foals and adult horses with and without colic. Am J Vet Res 2011; 72:1535–1540.
30. Bonello L, Tantry US, Marcucci R, et al. Consensus and future directions on the definition of high on-treatment platelet reactivity to adenosine diphosphate. J Am Coll Cardiol 2010; 56:919–933.
31. Paniccia R, Antonucci E, Maggini N, et al. Light transmittance aggregometry induced by different concentrations of adenosine diphosphate to monitor clopidogrel therapy: a methodological study. Ther Drug Monit 2011; 33:94–98.
32. Harrison P. The role of PFA-100 testing in the investigation and management of haemostatic defects in children and adults. Br J Haematol 2005; 130:3–10.
33. Shah U, Ma AD. Tests of platelet function. Curr Opin Hematol 2007; 14:432–437.
34. Chiu FC, Wang TD, Lee JK, et al. Residual platelet reactivity after aspirin and clopidogrel treatment predicts 2-year major cardiovascular events in patients undergoing percutaneous coronary intervention. Eur J Intern Med 2011; 22:471–477.
35. Tobin WO, Kinsella JA, Coughlan T, et al. High on-treatment platelet reactivity on commonly prescribed antiplatelet agents following transient ischaemic attack or ischaemic stroke: results from the Trinity Antiplatelet Responsiveness (TRAP) study. Eur J Neurol 2013; 20:344–352.
36. Geiger J, Teichmann L, Grossmann R, et al. Monitoring of clopidogrel action: comparison of methods. Clin Chem 2005; 51:957–965.
37. Conner BJ, Hanel RM, Hansen BD, et al. Effects of acepromazine maleate on platelet function assessed by use of adenosine diphosphate activated– and arachidonic acid–activated modified thromboelastography in healthy dogs. Am J Vet Res 2012; 73:595–601.
38. Brainard BM, Kleine SA, Papich MG, et al. Pharmacodynamic and pharmacokinetic evaluation of clopidogrel and the carboxylic acid metabolite SR 26334 in healthy dogs. Am J Vet Res 2010; 71:822–830.
39. Kingston JK, Bayly WM, Sellon DC, et al. Measurement of the activation of equine platelets by use of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-human thrombospondin antibody. Am J Vet Res 2002; 63:513–519.
40. Lalko CC, Deppe E, Ulatowski D, et al. Equine platelet CD62P (P-selectin) expression: a phenotypic and morphologic study. Vet Immunol Immunopathol 2003; 91:119–134.
41. Segura D, Monreal L, Perez-Pujol S, et al. Assessment of platelet function in horses: ultrastructure, flow cytometry, and perfusion techniques. J Vet Intern Med 2006; 20:581–588.
42. Dale GL. Coated-platelets: an emerging component of the procoagulant response. J Thromb Haemost 2005; 3:2185–2192.
43. Ruf A, Patscheke H. Flow cytometric detection of activated platelets: comparison of determining shape change, fibrinogen binding, and P-selectin expression. Semin Thromb Hemost 1995; 21:146–151.
44. Boudreaux MK, Panangala VS, Bourne C. A platelet activation-specific monoclonal antibody that recognizes a receptor-induced binding site on canine fibrinogen. Vet Pathol 1996; 33:419–427.
45. Brooks MB, Randolph J, Warner K, et al. Evaluation of platelet function screening tests to detect platelet procoagulant deficiency in dogs with Scott syndrome. Vet Clin Pathol 2009; 38:306–315.
46. Brainard BM, Epstein KL, LoBato DN, et al. Treatment with aspirin or clopidogrel does not affect equine platelet expression of P selectin or platelet-neutrophil aggregates. Vet Immunol Immunopathol 2012; 149:119–125.