Responses to adenosine diphosphate in human platelets loaded with the fluorescent calcium indicator quin2.
ADP produces a rapid elevation in the concentration of cytoplasmic free calcium, [Ca2+]i, in quin2-loaded human platelets which begins within 1 s of stimulation and peaks after 10 s. In the presence of 1 mM-extracellular calcium, [Ca2+]i peaks at 670 +/- 50 nM in the absence and 610 +/- 30 nM in the presence of a cyclo-oxygenase inhibitor. The production of prostaglandin endoperoxides and thromboxane A2 are not required for stimulation of Ca2+ fluxes by ADP but appear to have a supportive role. In the absence of extracellular calcium ions and with 1 mM-extracellular EGTA, stimulation with ADP caused [Ca2+]i to peak at 160 +/- 20 nM in the absence and 150 +/- 10 nM in the presence of a cyclo-oxygenase inhibitor. ADP can cause the discharge of calcium ions from internal stores and does not require the prior formation of prostaglandin endoperoxides or thromboxane A2. The rise in [Ca2+]i in the presence of extracellular Ca2+ is sixfold larger than in the absence of extracellular Ca2+. This suggests that the major component of the ADP-stimulated rise in [Ca2+]i is caused by the influx of Ca2+ ions across the plasma membrane. Diltiazem, D600, nimodipine and nifedipine had little or no effect on resting or ADP-stimulated [Ca2+]i levels. Depolarization with potassium-rich media alone or in conjunction with valinomycin had no effect on basal [Ca2+]i and only a partial inhibitory effect on ADP-stimulated increases in [Ca2+]i. Depolarization had no effect on the ADP-stimulated rise in [Ca2+]i in Ca2+-free media. Hyperpolarization had no marked effect on the rise in [Ca2+]i produced by ADP in the presence of extracellular calcium. These results are consistent with there being no voltage-dependent channels in the platelet plasma membrane. Using ionomycin, a selective Ca2+ ionophore, and measuring both quin2 fluorescence and optical density of the suspension simultaneously, the threshold [Ca2+]i for shape change was determined to be 300 nM with half-maximal effect at 500 nM and maximal shape change at 800 nM. ADP produced maximal shape change confirmed by scanning electron microscopy with corresponding [Ca2+]i at below 200 nM. The level of [Ca2+]i required to produce aggregation using ionomycin was approximately 1 microM. ADP alone, or following a smaller rise in [Ca2+]i produced by ionomycin to disguise the effect of ADP, produced an aggregatory response at concentrations below 1 microM. These data indicate that excitatory mechanisms are involved producing shape change and aggregation to ADP other than a stimulated rise in [Ca2+]i.
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000634:Aminoquinolines; D001241:Aspirin / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects* / Q000648:ultrastructure; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D004987:Ethers / Q000494:pharmacology; D005456:Fluorescent Dyes; D006801:Humans; D066298:In Vitro Techniques; D015759:Ionomycin; D007476:Ionophores / Q000494:pharmacology; D008855:Microscopy, Electron, Scanning; D010974:Platelet Aggregation / Q000187:drug effects; D011188:Potassium / Q000494:pharmacology
D000634:Aminoquinolines; D002121:Calcium Channel Blockers; D004987:Ethers; D005456:Fluorescent Dyes; D007476:Ionophores; D015759:Ionomycin; D000244:Adenosine Diphosphate; C035738:Quin2-acetoxymethyl ester; D001241:Aspirin; D011188:Potassium; D002118:Calcium