Objective—To develop a flow cytometric assay to quantify platelet-derived microparticles (PMPs) in equine whole blood and plasma. Sample—Citrate-anticoagulated whole blood from 30 healthy adult horses. Procedures—Platelet-poor plasma (PPP) was prepared from fresh whole blood by sequential low-speed centrifugation (twice at 2,500 × g). Samples of fresh whole blood and PPP were removed and stored at 4° and 24°C for 24 hours. Platelet-derived microparticles were characterized in fresh and stored samples on the basis of the forward scatter threshold (log forward scatter < 10(1)) and labeling with annexin V (indicating externalized phosphatidylserine) and CD61 (a constitutive platelet receptor). A fluorescent bead–calibrated flow cytometric assay was used to determine microparticle counts. Platelet counts, prothrombin time, and activated partial thromboplastin time were measured in fresh samples. Results—Significantly more PMPs were detected in fresh whole blood (median, 3,062 PMPs/μL; range, 954 to 13,531 PMPs/μL) than in fresh PPP (median, 247 PMPs/μL; range, 104 to 918 PMPs/μL). Storage at either temperature had no significant effect on PMP counts for whole blood or PPP. No significant correlation was observed between PMP counts and platelet counts in fresh whole blood or PPP or between PMP counts and clotting times in fresh PPP. Conclusions and Clinical Relevance—Results indicated that the described PMP protocol can be readily used to quantify PMPs in equine blood and plasma via flow cytometry. Quantification can be performed in fresh PPP or whole blood or samples stored refrigerated or at room temperature for 24 hours.

Objective—To evaluate the effects of clopidogrel on clinical and clinicopathologic variables in healthy horses with experimentally induced endotoxemia. Animals—12 adult mares. Procedures—Horses were assigned with a randomization procedure to receive clopidogrel (4 mg/kg, once, then 2 mg/kg, q 24 h; n = 6) or a placebo (6) through a nasogastric tube. After 72 hours of treatment, horses received lipopolysaccharide (LPS; 30 ng/kg, IV). Heart rate, respiratory rate, rectal temperature, CBC variables, plasma fibrinogen concentration, serum tumor necrosis factor-α concentration, plasma von Willebrand factor concentration, and measures of platelet activation (including ADP- and collagen-induced platelet aggregation and closure times, thrombelastography variables, and results of flow cytometric detection of platelet membrane P-selectin, phosphatidylserine, and microparticles) were determined at various times before and after LPS administration by investigators unaware of the treatment groups. Statistical analyses were performed with repeated-measures ANOVA. Results—4 of 6 clopidogrel-treated horses had significant decreases in ADP-induced platelet aggregation before and after LPS administration. Heart rate increased significantly after LPS administration only for the placebo group. No significant differences were detected between groups for CBC variables, closure time, and plasma concentration of fibrinogen or serum concentration of tumor necrosis factor-α, and no clinically relevant differences were detected for other hemostatic variables. Conclusions and Clinical Relevance—In this study, administration of LPS did not induce platelet hyperreactivity in horses on the basis of measures of platelet adhesion, aggregation, degranulation, and procoagulant activity. Administration of clopidogrel was associated with variable platelet antiaggregatory activity and attenuated some clinical signs of endotoxemia.