Acetylsalicylic acid enhances purinergic receptor-mediated outward currents in rat megakaryocytes

Abstract

Young JP, Beckerman J, Vicini S, Myers A. Acetylsalicylic acid enhances purinergic receptor-mediated outward currents in rat megakaryocytes. Am J Physiol Cell Physiol 298: C602-C610, 2010. First published December 30, 2009 doi:10.1152/ajpcell.00422.2009.-Purinergic receptor activation increases cytosolic Ca 2ϩ concentration in a fluctuating fashion, triggering oscillatory outward Ca 2ϩ -activated K ϩ currents in rat megakaryocytes (MKs). Whole cell and nystatinperforated patch-clamp techniques were used to analyze changes in ionic conductance in MK with acetylsalicylic acid (ASA), a cyclooxygenase-1 inhibitor and antithrombotic agent. MKs are a model for platelet reactivity, particularly in ASA treatment failure (ASA resistance). Freshly isolated MKs were incubated 30 min in the absence or presence of 1 mM ASA. Using a K ϩ -rich internal solution, we recorded outward currents in response to 10 M ATP, 10 M ADP, and 5 M 2-methyl-thio-ADP (2MeSADP) in the voltage-clamp mode. Agonist-induced currents decreased in amplitude over time, but this decline was attenuated by ASA in both continuous and repeated agonist challenge, indicating increased MK reactivity with ASA treatment. In separate experiments, heterologous desensitization was observed when MKs were stimulated with ADP after exposure to a thromboxane receptor agonist (U46619), indicating cross talk between thromboxane and purinergic pathways. Different cells, treated with ASA or MRS2179 (P2Y1 receptor antagonist), were stimulated with 2MeSADP. The dose-response curve was shifted to the left in both cases, suggesting increased MK reactivity. ASA also caused an increased interval between currents (delay). ASA attenuated desensitization of purinergic receptors and increased delay, again suggesting cross talk between purinergic and thromboxane pathways. These findings may be relevant to ASA resistance, because individual variations in sensitivity to the multiple effects of ASA on signaling pathways could result in insensitivity to its antiplatelet effects in some patients.platelets; patch clamp; ADP; ATP; thromboxane CARDIOVASCULAR DISEASE IS the leading cause of morbidity and mortality in the United States and developed countries (17, 54). Platelet increased reactivity is a key factor in the development of stroke and ischemic heart disease. Therefore, platelets have become a target for pharmacological treatment of cardiovascular complications after percutaneous coronary intervention (angioplasty and stent implant) (37) as well as in stroke prevention and management. Aspirin has been shown to be effective in the acute phase of recurrent stroke (49), whereas purinergic receptor blockade in the CAPRIE study showed more effectiveness than aspirin with lower side effects such as bleeding (1). The use of drug-eluting stents and dual therapy of cyclooxygenase (COX) inhibitors and purinergic receptor P2Y 12 blockers reduces the incidence of thromboembolism and ischemic episodes but with the adverse outcome of more bleeding occurrence (47, 57). Common platelet evaluation...

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