Thrombin and activators of protein kinase C modulate secretory responses of permeabilised human platelets induced by Ca2+
Authors: KNIGHT Derek E. , NIGGLI Verena , SCRUTTON Michael C. 
Published: March 2005

Addition of thrombin enhances secretion of both [14C]serotonin and β‐N‐acetylglucosaminidase induced by Ca2+ in human platelets rendered permeable by exposure to intense electric fields.

Enhancement of β‐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 EC50 for Ca2+. In contrast, thrombin shifts the dose/response curve for Ca2+‐induced [14C]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‐.

The relationship between extent of response and [MgATP2‐] is similar for secretion of [14C]serotonin and of β‐N‐acetylglucosaminidase in the presence or absence of thrombin. Similar nucleotide specificities are also observed.

Activators of protein kinase C have previously been shown to mimic quantitatively the effect of thrombin on [14C]serotonin secretion induced by Ca2+ [D. E. Knight & M. C. Scrutton (1984) Nature (Lond.) 309, 66–68]. Such activators have the same qualitative effect as thrombin on the properties of β‐N‐acetylglucosaminidase secretion induced by Ca2+ but are less effective.

The EC50 for thrombin observed for enhancement of [14C]serotonin and β‐N‐acetylglucosaminidase secretion is in the same range as that obtained for intact platelets under comparable conditions [D. E. Knight, T. J. Hallam & M. C. Scrutton (1982) Nature (Lond.) 296, 256–257]. The EC50, and the specificity of response, observed for activators of protein kinase C in these systems are consistent with those reported previously for the purified enzyme.

Addition of 1 – 10 μM Ca2+ to permeabilised platelets in the presence of [γ‐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 μM Ca2+, but some increase is observed in the extent of phosphorylation of the 45‐kDa polypeptide. Addition of 1‐oleyl‐2‐acetylglycerol + 1 – 2 μM 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 Ca2+‐dependent [14C]serotonin secretion under these conditions.

At 0.01 μM Ca2+ addition of either thrombin or 1‐oleyl‐2‐acetylglycerol in the presence of [γ‐32P] ATP causes enhanced phosphorylation of the 45‐kDa polypeptide but not of the 20‐kDa or 66‐kDa polypeptides without significant release of [14C]serotonin.

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‐diacyiglycerol and hence activating protein kinase C, but that this mechanism is not adequate to explain the enhancement of β‐N‐acetylglucosaminidase secretion caused by addition of thrombin in this preparation.