Proceedings of The Physiological Society

University of Cambridge (2008) Proc Physiol Soc 11, PC132

Poster Communications

NAADP-induced Ca2+ release is an important regulator of platelet activation

M. T. Harper1, A. Galione2, A. W. Poole1

1. Department of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom. 2. Department of Pharmacology, University of Oxford, Oxford, United Kingdom.

Although platelets have an essential role in haemostasis, inappropriate platelet activation is a major factor in pathological thrombosis, which can lead to myocardial infarction and stroke. A rise in intracellular Ca2+ concentration ([Ca2+]i) is a key step in platelet activation, regulating granule secretion, integrin activation and procoagulant activity. Platelet activators, such as collagen, thrombin and ADP, induce Ca2+ release from intracellular stores. Although inositol trisphosphate (IP3) has previously been thought to be the major Ca2+-releasing intracellular secondary messenger other second messengers such as nicotinic acid adenine dinucleotide phosphate (NAADP) have important and necessary roles in Ca2+ release. Here we have investigated the role of NAADP-induced Ca2+ release in platelet activation, using NED-14, a recently-developed inhibitor of the NAADP receptor. First, [Ca2+]i was measured in fura-2 -loaded human platelets. Ca2+ release induced by the physiological agonist ADP (10 μM) was partially decreased by NED-14 (peak height above basal was 48.9 ± 5.1 % of control; mean ± SEM; n = 4; p = 0.002, Student’s t test). Similarly, CRP (collagen-related peptide, a GPVI agonist; 5 μg/ml) -induced Ca2+ release was also decreased by NED-14 (peak height above basal was 77.8 ± 7.8 % of control; n = 3; p = 0.018). NED-14 also potently inhibited platelet granule secretion. CRP-induced dense granule secretion, was reduced to 27.1 ± 7.6 % of control (n = 3; p = 0.011) and α-granule secretion, measured as surface P-selectin expression, was reduced to 45.2 ± 3.4 % (n = 3, p = 0.035). Platelet aggregation in response to CRP and ADP was also inhibited. ADP-induced aggregation was completely abolished (n = 3), and CRP-induced aggregation was reduced to 44.8 ± 4.1 % of control (n = 4; p < 0.001). This was not wholly due to inhibition of ADP secretion from dense granules and subsequent stimulation of P2Y receptors, since CRP-induced aggregation was also inhibited by NED-14 in the presence of P2Y receptor antagonists. Consistent with this, CRP-induced Rap1b activation (required for integrin activation and so for aggregation), was inhibited by both NED-14 and by P2Y receptor antagonists, and completely abolished by a combination of these treatments (n = 3). These data suggest that NAADP is an important second messenger in platelet activation, and that NAADP-induced Ca2+ release is a key regulator, both of platelet granule secretion and also of secretion-independent aggregation. Inhibition of NAADP signalling may provide may offer an alternative therapeutic target in treatment of thrombosis.

Where applicable, experiments conform with Society ethical requirements