Thrombin-induced calcium movements in platelet activation.
The thrombin-induced Ca2+ fluxes and their coupling to platelet aggregation of the human platelet were studied using quin2 as a measure of the cytoplasmic Ca2+ concentration [( Ca2+]cyt) and chlorotetracycline (CTC) as a measure of internally sequestered Ca2+. Evidence is given that the CTC fluorescence change is proportional to the free internal Ca2+ concentration in the dense tubular lumen. The intracellular quin2 concentration was 1 mM and analysis showed that it did not perturb the processes reported herein. The value of [Ca2+]cyt at rest and during thrombin activation was analyzed in terms of Ca2+ influx, Ca2+ release, Ca2+ sequestration, and Ca2+ extrusion. Influx was distinguished from internal release by removing extracellular Ca2+ 1 min before thrombin activation. In the presence of 2 mM external Ca2+, the thrombin-induced Ca2+ influx accounts for most of the increase in [Ca2+]cyt (over 80%). Thrombin-induced Ca2+ influx and release have somewhat different EC50 values (0.17 U/ml vs. 0.35 U/ml). The contribution of influx can be inhibited by verapamil, bepridil and Cd2+ (IC50 values of 19 microM, 2 microM and 50 microM). The influx results were analyzed in terms of a thrombin-activated channel. Indomethacin pretreatment experiments suggest that activation of the arachidonic pathway accounts for approx. 50% of the influx-related [Ca2+]cyt elevation. Elevation of [Ca2+]cyt by intracellular release is not inhibited by verapamil or Cd2+ but is inhibited by bepridil with a high IC50 (25 microM). It is only 15-20% inhibited by indomethacin and is thus not dependent on thromboxane A2 formation. The release reaction does not require Ca2+ influx. The rate of thrombin-activated platelet aggregation is shown to have an approximately fourth-power dependence on [Ca2+]cyt with an apparent Km of 0.4 microM. Comparisons of aggregation rates of the partially thrombin-activated vs. fully thrombin-activated, partially verapamil-inhibited conditions suggest that this dependence on [Ca2+]cyt is the major determinant of the aggregation behavior. Analysis shows that calcium influx is the major pathway for elevating [Ca2+]cyt by thrombin when physiological concentrations of external Ca2+ are present.
D000634:Aminoquinolines / Q000378:metabolism; D015764:Bepridil; D001692:Biological Transport; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002751:Chlortetracycline / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D007473:Ion Channels / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D011759:Pyrrolidines / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D014700:Verapamil / Q000494:pharmacology
D000634:Aminoquinolines; D007473:Ion Channels; D011759:Pyrrolidines; D000001:Calcimycin; D015764:Bepridil; D014700:Verapamil; D013917:Thrombin; C035739:Quin2; D002118:Calcium; D002751:Chlortetracycline; D007213:Indomethacin