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Potentiation of platelet P2Y receptor responses by K+; a physiological mechanism to accelerate haemostasis?
Significant increases in extracellular K+ ([K+]o) occur under a variety of physiological and pathophysiological situations. [K+]o is known to influence a number of membrane conductances and exchangers, whereas direct effects on the activation of G-protein-coupled receptors are less well described. We have recently shown (Pitt et al., 2005) that Ca2+ release evoked by P2Y receptors in cell lines and native megakaryocytes is markedly potentiated by [K+]o , including levels observed under physiological conditions. The underlying mechanism is novel in that K+ enhances agonist-mediated, IP3-dependent Ca2+ release, without a requirement for Ca2+ influx and is in part independent of changes in membrane potential. Given the importance of P2Y receptors in platelet activation, we have examined the effect of increasing [K+]o on ADP-dependent activation of [Ca2+]i and functional responses in human platelets. Blood was drawn from consenting adult volunteers with local ethical committee approval and citrated platelet-rich-plasma (PRP) or washed platelet suspensions were prepared as previously described (Heptinstall et al., 1998; Rolf et al., 2001) . Population measurements of [Ca2+]i were made from Fluo4-loaded platelets (2 µM, fluo4AM, 45 min at 37 oC) using a Flexstation II fluorimete r. Aggregation was measured using standard turbidimetric techniques. The effect of a 10 mM [K+]o increase was studied following pre-addition of KCl, or during co-addition of agonist and KCl with an equimolar reduction of NaCl. Control samples were exposed to an identical increase in NaCl (pre-exposure) or an identical volume of saline (co-addition). The concentration-response curve for the ADP-stimulated peak [Ca2+]i increase was shifted to the left by [K+]o without a significant change in slope or maximum response compared to controls; the average EC50 shifted 2.3-fold (pEC50 values of 6.20 ± 0.06 in 5mM K+ and 6.56 ± 0.04 in 15 mM K+, n = 6, p < 0.001). A [K+]o increase also significantly enhanced both the initial rate and peak of the aggregation responses to 1 µM ADP (1.80 ± 0.2 and 1.98 ± 0.5 fold increase over controls, respectively; n = 9, p < 0.05). This enhancement of P2Y receptor-induced platelet activation was independent of an alteration in ectonucleotidase activity, as it was also observed during stimulation by ADPβS, a hydrolysis-resistant form of ADP. These data support recent evidence from cell lines that small increases in [K+]o potentiate P2Y receptor signalling. This phenomenon may enhance platelet activation at the sites of tissue damage, where nucleotides and K+ will be co-elevated, and thus represents a potential physiological means of accelerating haemostasis. The extent to which the two platelet P2Y receptors, P2Y1 and P2Y12, contribute to this phenomenon is currently under investigation. P2Y-receptors display a widespread distribution in adult and developing tissues, therefore their modulation by small changes in extracellular K+ may influence cellular responses in a variety of physiological and pathophysiological situations.
Heptinstall, S. et al. (1998). Br. J. Haematol. 103, 1023-1030. |
