Thrombin and activators of protein kinase C modulate secretory responses of permeabilised human platelets induced by Ca²⁺

Abstract

Addition of thrombin enhances secretion of both [14C]serotonin and P-N-acetylglucosaminidase induced by Ca2+ in human platelets rendered permeable by exposure to intense electric fields. 2. Enhancement of P-N-acetylglucosaminidase secretion by thrombin results from an increase in the maximal extent of the response with no significant change in the median effective concentration EC,o for CaZ+. In contrast, thrombin shifts the dose/response curve for Ca2+-induced ['4C]serotonin secretion to the left and has little effect on the maximal extent of this response even when this extent is reduced by use of a non-saturating concentration of MgATP2-. 6. Addition of 1-10 pM Ca2+ to permeabilised platelets in the presence of [y-32P] ATP causes marked enhancement of 32P incorporation into polypeptides of molecular mass 20 kDA, 45 kDA and 66 kDA. NO additional polypeptides become phosphorylated in this system when thrombin is added together with 10 pM Ca2+, but some increase is observed in the extent of phosphorylation of the 45-kDa polypeptide. Addition of 1 -0leyl-2-acetylglycerol + 1 -2 pM Ca2+ causes enhanced phosphorylation of the 45-kDa polypeptide and to a lesser extent of the 20-kDa polypeptide. The dose/response curves for Ca2+-dependent phosphorylation of the 45-kDa polypeptide in the presence and absence of 1 -oleyl-2-acetylglycerol are similar to those observed for CaZ +-dependent [14C]serotonin secretion under these conditions. 7. At 0.01 pM Ca" addition of either thrombin or 1 -oleyl-2-acetylglycerol in the presence of b;-32P] ATP causes enhanced phosphorylation of the 45-kDa polypeptide but not of the 20-kDa or 66-kDa polypeptides without significant release of ['4C]serotonin.8. The results are consistent with the postulate that thrombin enhances [14C]serotonin secretion induced by Ca2' in permeabilised platelets by increasing the level of 1,2-diacylglycerol and hence activating protein kinase C, but that this mechanism is not adequate to explain the enhancement of P-N-acetylglucosaminidase secretion caused by addition of thrombin in this preparation.

Effect of various excitatory agonists on the secretion of 5‐hydroxytryptamine from permeabilised human platelets induced by Ca2+ in the presence or absence of GTP

“…We have therefore performed studies in the presence of Mg CTP*-instead of Mg ATP'-since previous results have shown this nucleotide to be effective in supporting Ca'*-dependent secretion both in the presence and absence of thrombin [6]. Fig.3 demonstrates that addition of 20 PM ADP to such a system has no effect on the dose/response curve for [14C]5HT secretion induced by Ca'+.…”

“…The effect of thrombin (a), adrenaline (b) and 5HT (c) on the Ca" dependence of [r4C]5HT release from permeabilised platelets in the presence and absence of GTP. Platelets suspended in a glycine-based medium (pH 6.6) [6], but containing 10 mM Na glutamate, 5 mM K glutamate, 2 mM Mg ATP*-and 1 mM BAPTA were rendered permeable as described previously [6] and then incubated for [6][7][8][9][10][11][12] In contrast, adrenaline (20/rM), when added in the presence or absence of 10 ,uM GTP, causes no significant shift in the dose/response curve for [14C]5HT secretion induced by Ca*+ (fig-lb). Similar results are obtained in the presence of 100 pM GTP or using adrenaline concentrations in the range 5-100,~M (not shown).…”

“…Blood was obtained by antecubital venepuncture from healthy human volunteers and platelets isolated in a glycine/K glutamate medium (pH 6.6) as described previously [6]. The medium contained either Mg ATP'-or Mg CTP'-and either EGTA or bis(O-amonophenoxy)ethane-~,~,~',~'-tetraacetic acid (BAPTA) as specified in the appropriate figure legend.…”

“…fects of thrombin on secretion from the permeabilised platelet can be mimicked by addition of synthetic activators of protein kinase C, e.g., l-oleyl-2-acetylglycerol, 12 -0 -tetradecanoylphorbol -13 -acetate [2,6]. Phorbol esters which are ineffective as activators of protein kinase C are also unable to enhance the effects of Ca2+ on secretion from permeabilised platelets [6].…”

“…6.6) was added to reduce the calculated Ca2+ concentration to less than 10v9 M and the platelets were then removed by centrifugation at 8000 x g for 2 min. The 14C content of the supernatant fraction was estimated and expressed as a 910 of total 14C content in the cell suspension as described previously [6].…”