Different calcium pools in human platelets and their role in thromboxane A2 formation.
Activation of human platelets by diverse receptor-transduced signals is followed by an intracellular calcium increase. Calcium liberation from an inositol 1,4,5-trisphosphate-sensitive compartment is recognized to be one of the prime events, followed by further mechanisms to amplify the signal. Among these, the formation of prostaglandin endoperoxides and thromboxane A2 are part of the self-amplificating activation system. Two inhibitors of intracellular Ca(2+)-ATPases, thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone have been reported to deplete the intracellular inositol 1,4,5-trisphosphate-responsive stores. In human platelets with EGTA present, we found that these inhibitors of the microsomal Ca2+ sequestration generate quite different Ca2+ transients due to an inherent cyclooxygenase inhibition by the benzohydroquinone derivative compared to thapsigargin, and, therefore, only one-half of the fura-2 signal is generated. For a maximal calcium release, Ca(2+)-ATPase inhibitors depend on the self-amplification system involving thromboxane formation. Following the thapsigargin-induced [Ca2+]i transient, thrombin was unable to raise [Ca2+]i, indicating that thapsigargin mobilizes calcium from the thrombin-responsive store, as long as the self-amplifying system of platelets is intact. With the thromboxane receptor blocked, thapsigargin releases only one-half of the calcium, and, hence, thrombin was able to release additional calcium. Interestingly, in the converse experiment, thrombin did not prevent a raise of [Ca2+]i by thapsigargin at all, although applying thrombin a second time was without any effect. Therefore, we propose two calcium pools in human platelets: receptor activation transiently releases calcium from an inositol-sensitive pool including the thapsigargin-sensitive compartment, followed by reuptake within minutes. Sequestration occurs into the thapsigargin-sensitive compartment from where it can be released even when the endoperoxide/thromboxane receptor is blocked. Calcium release from both compartments allows the formation of thromboxane B2, but not if only the Ca(2+)-ATPase inhibitor-sensitive pool is emptied. In the presence of a protonophor, a calcium accumulation in the Ca(2+)-ATPase-sensitive pool could be observed.
Brüne
B
B
University of Konstanz, Faculty of Biology, Federal Republic of Germany.
Ullrich
V
V
D000975:Antioxidants; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D005343:Fibrinolytic Agents; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D009550:Nigericin / Q000494:pharmacology; D015539:Platelet Activation; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D013917:Thrombin / Q000494:pharmacology; D013928:Thromboxane A2 / Q000096:biosynthesis*; D013929:Thromboxane B2 / Q000096:biosynthesis
D000975:Antioxidants; D005343:Fibrinolytic Agents; D006873:Hydroquinones; D013449:Sulfonamides; D013729:Terpenes; C019359:2,5-di-tert-butylhydroquinone; D013929:Thromboxane B2; D013928:Thromboxane A2; D019284:Thapsigargin; C040624:sulotroban; D011451:Prostaglandin-Endoperoxide Synthases; D013917:Thrombin; D000252:Calcium-Transporting ATPases; D009550:Nigericin; D002118:Calcium