Baseline and activated platelet cytoplasmic ionized calcium in acute ischemic stroke. Effect of aspirin.

Abstract

O ne reason for the limited usefulness of aspirin in the treatment of ischemic stroke may be that aspirin does not suppress platelet responsiveness to all in vivo thrombogenic stimuli. Among the known in vivo mediators of platelet activation, thrombin, collagen, and platelet activating factor (PAF) are believed to be important. Cytoplasmic ionized calcium is the pivotal second messenger coupling platelet stimulation and response.1 Therefore, we studied the effect of aspirin treatment on cytoplasmic ionized calcium concentration ( [Ca,2+ ]) following stimulation with thrombin, collagen, and PAF. Baseline and stimulated [Caj 2+ ] is reliably measured by loading washed platelets with aequorin, a Ca 2+ -sensitive photoprotein.2 -4 Using this technique, we show that the increase in [Ca,2+ ] following stimulation with PAF is not suppressed by aspirin treatment. Subjects and MethodsWe studied 25 healthy people (nine men, 16 women) ranging in age from 20 to 74 (mean±SD 45 ±14) From years. These controls were free of all antiplatelet medication (aspirin, nonsteroidal anti-inflammatory drugs) for at least 10 days before the study. We also studied 26 patients with acute ischemic stroke (13 men, 13 women) ranging in age from 24 to 88 (mean±SD 55 ±17) years. The patients were also free of all antiplatelet medications for at least 10 days before the study (untreated). Twelve patients had large, cortical infarcts attributed to majorvessel occlusion and 14 had small, subcortical infarcts attributed to small-vessel occlusion; the diagnosis was based on clinical examination and was supported by computed tomography, magnetic resonance imaging, or four-vessel cerebral angiography. Nine patients were studied ^24 hours, nine 36-72 hours, and eight 4-35 days after the onset of neurologic dysfunction. Sixteen patients suffered from essential hypertension, one had insulindependent diabetes mellitus and hypertension, and nine had neither; treatment of hypertension was limited to thiazide diuretics and/or clonidine.In addition, we studied 12 patients with acute ischemic stroke (nine men, three women) treated with 325 mg/day aspirin for 1-4 days. These aspirintreated patients ranged in age from 39 to 82 (mean±SD 64 ±15) years and were studied within 96 hours after the onset of neurologic dysfunction.Sixty milliliters of blood from the antecubital vein was collected from all subjects in the resting state by guest on May 9, 2018 http://stroke.ahajournals.org/ Downloaded from

A plasmatic factor may cause platelet activation in acute ischemic stroke.

“…10 -11 This blue luminescence can be detected by a highly sensitive photomultiplier tube. A platelet-ionized calcium aggregometer (Chrono-Log, Havertown, Pennsylvania), which contains such a photomultiplier tube, and a strip chart recorder (model SE-120, Brown Boveri, Vienna, Austria) were used to measure [Ca,2+ ]. To allow estimation of the rate of decay of the aequorin luminescence in platelets, the equivalent of 1 ml platelet suspension (7.5 x 10 7 platelets) in HEPES-Tyrode's buffer containing 1 mM Ca 2+ was wanned (2-3 minutes at 37° C), stirred (1,200 rpm with a Teflon-coated magnetic stirrer bar), and lysed with Triton X-100 (0.1%).…”

“…Plasma obtained by centrifuging blood (I,66%xl5 minutes) from patients or controls was kept frozen (-70° C) for up to 30 days before study until platelets from healthy controls for these experiments became available. Seven experiments were done to measure changes in platelet basal [Ca,2+ ], and six experiments were performed to measure the release of serotonin from platelets.…”

“…The samples were then incubated in a water bath (at 37° C for 45 minutes), loaded with aequorin, and processed by gel-filtration; platelet count was adjusted to 7.5xlO 7 /ml as described above. Platelet basal [Ca,2+ ] was measured as previously described. Subsequently, samples were centrifuged at 31,00% for 15 seconds, and the supernatant was removed and passed through a 0.22-/im syringe filter.…”

“…1 - 2 The release of granular contents and formation of aggregates on platelet activation may together contribute to the pathogenesis and propagation of thrombosis in cerebral ischemia. 3 However, the mechanism for the initiation of platelet activation in acute ischemic stroke is uncertain.…”

“…3 However, the mechanism for the initiation of platelet activation in acute ischemic stroke is uncertain. Ionized calcium ( [Ca,2+ ]) is the pivotal second messenger that couples platelet stimulation and activation. 4 Changes in basal and stimulated [Cai 2+ ] transients are reliably measured by loading washed platelets with aequorin, a Ca 2+ -sensitive photoprotein.…”