• 1. Sykes BW, Furr MO. Equine endotoxaemia—a state-of-the-art review of therapy. Aust Vet J 2005; 83: 4550.

  • 2. Werners AH, Bull S, Fink-Gremmels J. Endotoxaemia: a review with implications for the horse. Equine Vet J 2005; 37: 371383.

  • 3. Angus DC, van der Poll T. Severe sepsis and septic shock. N Engl J Med 2013; 369: 840851.

  • 4. Huisse MG, Pease S & Hurtado-Nedelec M, et al. Leukocyte activation: the link between inflammation and coagulation during heatstroke. A study of patients during the 2003 heat wave in Paris. Crit Care Med 2008; 36: 22882295.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Joop K, Berckmans RJ & Nieuwland R, et al. Microparticles from patients with multiple organ dysfunction syndrome and sepsis support coagulation through multiple mechanisms. Thromb Haemost 2001; 85: 810820.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Weber GF, Swirski FK. Immunopathogenesis of abdominal sepsis. Langenbecks Arch Surg 2014; 399: 19.

  • 7. Berg RM, Taudorf S & Bailey DM, et al. Effects of lipopolysaccharide infusion on arterial levels and transcerebral exchange kinetics of glutamate and glycine in healthy humans. APMIS 2012; 120: 761766.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Brooks AC, Menzies-Gow NJ & Wheeler-Jones C, et al. Endotoxin-induced activation of equine platelets: evidence for direct activation of p38 MAPK pathways and vasoactive mediator production. Inflamm Res 2007; 56: 154161.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9. Guha M, O'Connell MA & Pawlinski R, et al. Lipopolysaccharide activation of the MEK-ERK1/2 pathway in human monocytic cells mediates tissue factor and tumor necrosis factor alpha expression by inducing Elk-1 phosphorylation and Egr-1 expression. Blood 2001; 98: 14291439.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. McGovern KF, Lascola KM & Smith SA, et al. The effects of hyperglycemia and endotoxemia on coagulation parameters in healthy adult horses. J Vet Intern Med 2013; 27: 347353.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Michaeli B, Martinez A & Revelly JP, et al. Effects of endotoxin on lactate metabolism in humans. Crit Care 2012; 16: R139.

  • 12. Moore JN, Morris DD. Endotoxemia and septicemia in horses: experimental and clinical correlates. J Am Vet Med Assoc 1992; 200: 19031914.

    • Search Google Scholar
    • Export Citation
  • 13. Nieto JE, MacDonald MH & Braim AE, et al. Effect of lipopolysaccharide infusion on gene expression of inflammatory cytokines in normal horses in vivo. Equine Vet J 2009; 41: 717719.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Poulin Braim AE, MacDonald MH & Bruss ML, et al. Effects of intravenous administration of pirfenidone on horses with experimentally induced endotoxemia. Am J Vet Res 2009; 70: 10311042.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15. Tadros EM, Frank N. Effects of continuous or intermittent lipopolysaccharide administration for 48 hours on the systemic inflammatory response in horses. Am J Vet Res 2012; 73: 13941402.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16. Morris DD. Endotoxemia in horses. A review of cellular and humoral mediators involved in its pathogenesis. J Vet Intern Med 1991; 5: 167181.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Morris DD, Crowe N, Moore JN. Correlation of clinical and laboratory data with serum tumor necrosis factor activity in horses with experimentally induced endotoxemia. Am J Vet Res 1990; 51: 19351940.

    • Search Google Scholar
    • Export Citation
  • 18. Barton MH, Collatos C. Tumor necrosis factor and interleukin-6 activity and endotoxin concentration in peritoneal fluid and blood of horses with acute abdominal disease. J Vet Intern Med 1999; 13: 457464.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19. 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: 915924.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20. Moore JN, Barton MH. Treatment of endotoxemia. Vet Clin North Am Equine Pract 2003; 19: 681695.

  • 21. Piccin A, Murphy WG, Smith OP. Circulating microparticles: pathophysiology and clinical implications. Blood Rev 2007; 21: 157171.

  • 22. Schouten M, Wiersinga WJ & Levi M, et al. Inflammation, endothelium, and coagulation in sepsis. J Leukoc Biol 2008; 83: 536545.

  • 23. Brown GT, McIntyre TM. Lipopolysaccharide signaling without a nucleus: kinase cascades stimulate platelet shedding of proinflammatory IL-1beta-rich microparticles. J Immunol 2011; 186: 54895496.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24. Soop A, Hallstrom L & Frostell C, et al. Effect of lipopolysaccharide administration on the number, phenotype and content of nuclear molecules in blood microparticles of normal human subjects. Scand J Immunol 2013; 78: 205213.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25. 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: 167173.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26. Kornerup KN, Salmon GP & Pitchford SC, et al. Circulating platelet-neutrophil complexes are important for subsequent neutrophil activation and migration. J Appl Physiol (1985) 2010; 109: 758767.

    • Search Google Scholar
    • Export Citation
  • 27. Ouellette AL, Evans RJ, Heath MF. Platelets enhance endotoxininduced monocyte tissue factor (TF) activity in the horse. Res Vet Sci 2004; 76: 3135.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28. Savi P, Herbert JM. Clopidogrel and ticlopidine: P2Y12 adenosine diphosphate-receptor antagonists for the prevention of atherothrombosis. Semin Thromb Hemost 2005; 31: 174183.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29. Brooks MB, Divers TJ & Watts AE, et al. Effects of clopidogrel on the platelet activation response in horses. Am J Vet Res 2013; 74: 12121222.

  • 30. Gaffney PJ, Wong MY. Collaborative study of a proposed international standard for plasma fibrinogen measurement. Thromb Haemost 1992; 68: 428432.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31. Benson RE, Catalfamo JL, Dodds WJ. A multispecies enzyme-linked immunosorbent assay for von Willebrand's factor. J Lab Clin Med 1992; 119: 420427.

    • Search Google Scholar
    • Export Citation
  • 32. Lavoie-Lamoureux A, Maghni K, Lavoie JP. Optimization of a procedure to accurately detect equine TNFalpha in serum samples. Vet Immunol Immunopathol 2010; 138: 118123.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33. Suagee JK, Burk AO & Quinn RW, et al. Effects of diet and weight gain on circulating tumour necrosis factor-alpha concentrations in Thoroughbred geldings. J Anim Physiol Anim Nutr (Berl) 2011; 95: 161170.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34. Vick MM, Adams AA & Murphy BA, et al. Relationships among inflammatory cytokines, obesity, and insulin sensitivity in the horse. J Anim Sci 2007; 85: 11441155.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35. Gachet C. P2Y(12) receptors in platelets and other hematopoietic and non-hematopoietic cells. Purinergic Signal 2012; 8: 609619.

  • 36. Jacob F, Perez Novo C & Bachert C, et al. Purinergic signaling in inflammatory cells: P2 receptor expression, functional effects, and modulation of inflammatory responses. Purinergic Signal 2013; 9: 285306.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37. Semrad SD, Moore JN. Effects of a specific thromboxane synthetase inhibitor in equine endotoxaemia. Res Vet Sci 1987; 43: 137142.

  • 38. Moore JN, Norton N & Barton MH, et al. Rapid infusion of a phospholipid emulsion attenuates the effects of endotoxaemia in horses. Equine Vet J 2007; 39: 243248.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39. Brooks AC, Menzies-Gow NJ & Wheeler-Jones C, et al. Regulation of platelet activating factor-induced equine platelet activation by intracellular kinases. J Vet Pharmacol Ther 2009; 32: 189196.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 40. Shashkin PN, Brown GT & Ghosh A, et al. Lipopolysaccharide is a direct agonist for platelet RNA splicing. J Immunol 2008; 181: 34953502.

  • 41. Zhang G, Han J & Welch EJ, et al. Lipopolysaccharide stimulates platelet secretion and potentiates platelet aggregation via TLR4/MyD88 and the cGMP-dependent protein kinase pathway. J Immunol 2009; 182: 79978004.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42. Aktan I, Dunkel B, Cunningham FM. Equine platelets inhibit E. coli growth and can be activated by bacterial lipopolysaccharide and lipoteichoic acid although superoxide anion production does not occur and platelet activation is not associated with enhanced production by neutrophils. Vet Immunol Immunopathol 2013; 152: 209217.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43. 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): 1519.

    • Search Google Scholar
    • Export Citation
  • 44. Arora RR, Rai F. Antiplatelet intervention in acute coronary syndrome. Am J Ther 2009; 16: e29e40.

  • 45. 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: 919933.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 46. 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: 471477.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 47. 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: 344352.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 48. 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: 116122.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 49. Ferroni P, Riondino S & Vazzana N, et al. Biomarkers of platelet activation in acute coronary syndromes. Thromb Haemost 2012; 108: 11091123.

  • 50. Bowbrick VA, Mikhailidis DP, Stansby G. Value of thromboelastography in the assessment of platelet function. Clin Appl Thromb Hemost 2003; 9: 137142.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 51. Schorer AE, Moldow CF, Rick ME. Interleukin 1 or endotoxin increases the release of von Willebrand factor from human endothelial cells. Br J Haematol 1987; 67: 193197.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 52. Ho-Tin-Noé B, Demers M, Wagner DD. How platelets safeguard vascular integrity. J Thromb Haemost 2011; 9(suppl 1): 5665.

  • 53. 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: 119125.

    • Crossref
    • Search Google Scholar
    • Export Citation

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Effects of clopidogrel on horses with experimentally induced endotoxemia

Ashlee E. WattsDepartment of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850.

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Sally L. NessDepartment of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850.

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Thomas J. DiversDepartment of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850.

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Susan L. FubiniDepartment of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850.

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Amelia H. FryeDepartment of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850.

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Tracy StokolDepartment of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850.

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Kevin J. CummingsDepartment of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

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Marjory B. BrooksDepartment of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14850.

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Abstract

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.

Abstract

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.

Contributor Notes

Dr. Watts’ present address is Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

This study was performed at the College of Veterinary Medicine, Cornell University, Ithaca, NY.

Supported by the Harry M. Zweig Memorial Fund for Equine Research.

The authors thank Scott Baxendell for coordinating the use of horses for the study.

Address correspondence to Dr. Brooks (mbb9@cornell.edu).