Cytoplasmic Ca2+ is necessary for thrombin-induced platelet activation.
alpha-Thrombin induces a dose-dependent rapid transient increase in platelet cytosolic Ca2+ levels, coming solely from intracellular stores, since EGTA has no effect. In contrast, the post-stimulation equilibrium [Ca2+]in depends upon an influx from the extracellular milieu, and is lower in the presence of EGTA. We measured the Ca2+ transient (with Indo-1, 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2'-amino-5'-methylp henoxy)- ethane-N,N,N',N'-tetraacetic acid), cytosolic alkalinization (with BCECF, 2',7-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein), membrane depolarization (with diS-C3-(5), 3,3'-dipropylthiodi-carbocyanide iodide), and degranulation (by beta-glucuronidase release) induced in washed human platelets by 9 nM thrombin in the absence or presence of extracellular or intracellular Ca2+ chelating agents (EGTA and BAPTA, 5,5'-dimethyl-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, respectively). Platelets loaded simultaneously with 2 microM Indo-1 and 15 microM BAPTA (each as the acetoxymethyl ester) before addition of thrombin exhibited no cytoplasmic Ca2+ transient or alkalinization, no depolarization or degranulation. Replenishment of such cells with extracellular CaCl2 restored resting [Ca2+]in. Upon stimulation with 9 nM thrombin these replenished platelets exhibited no Ca2+ transient, and a slow gradual increase in [Ca2+]in from extracellular stores, a slow alkalinization and depolarization, and partial degranulation, all abolished by extracellular EGTA. Thus thrombin-induced platelet activation exhibits a biphasic Ca2+ requirement: the initial transient increase in [Ca2+]in comes from intracellular stores only, while the later steps of depolarization, alkalinization, and degranulation can proceed, albeit more slowly, if only extracellular Ca2+ is available.
Davies
T A
TA
Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118.
Drotts
D L
DL
Weil
G J
GJ
Simons
E R
ER
D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000097:blood* / Q000494:pharmacology / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology / Q000031:analogs & derivatives; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D008247:Lysosomes / Q000378:metabolism; D008564:Membrane Potentials; D015539:Platelet Activation; D013917:Thrombin / Q000502:physiology*
D004533:Egtazic Acid; D013917:Thrombin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium