title: NAADP regulates human platelet function.
abstract: Platelets play a vital role in maintaining haemostasis. Human platelet activation depends on Ca2+ release, leading to cell activation, granule secretion and aggregation. NAADP (nicotinic acid-adenine dinucleotide phosphate) is a Ca2+-releasing second messenger that acts on acidic Ca2+ stores and is used by a number of mammalian systems. In human platelets, NAADP has been shown to release Ca2+ in permeabilized human platelets and contribute to thrombin-mediated platelet activation. In the present study, we have further characterized NAADP-mediated Ca2+ release in human platelets in response to both thrombin and the GPVI (glycoprotein VI)-specific agonist CRP (collagen-related peptide). Using a radioligand-binding assay, we reveal an NAADP-binding site in human platelets, indicative of a platelet NAADP receptor. We also found that NAADP releases loaded 45Ca2+ from intracellular stores and that total platelet Ca2+ release is inhibited by the proton ionophore nigericin. Ned-19, a novel cell-permeant NAADP receptor antagonist, competes for the NAADP-binding site in platelets and can inhibit both thrombin- and CRP-induced Ca2+ release in human platelets. Ned-19 has an inhibitory effect on platelet aggregation, secretion and spreading. In addition, Ned-19 extends the clotting time in whole-blood samples. We conclude that NAADP plays an important role in human platelet function. Furthermore, the development of Ned-19 as an NAADP receptor antagonist provides a potential avenue for platelet-targeted therapy and the regulation of thrombosis.
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Le Gros Clark Building, South Parks Road, Oxford OX1 3QX, U.K. carmen.coxon@dpag.ox.ac.uk
authors.forename: Carmen H
authors.initials: CH
authors.lastname: Coxon
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Lewis
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Sadler
authors.forename: Sridhar R
authors.initials: SR
authors.lastname: Vasudevan
authors.forename: Andrew
authors.initials: A
authors.lastname: Thomas
authors.forename: Kirsten A
authors.initials: KA
authors.lastname: Dundas
authors.forename: Lewis
authors.initials: L
authors.lastname: Taylor
authors.forename: R Duncan
authors.initials: RD
authors.lastname: Campbell
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Gibbins
authors.forename: Grant C
authors.initials: GC
authors.lastname: Churchill
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Tucker
mesh_terms: D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D002243:Carbolines / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D006801:Humans; D009249:NADP / Q000031:analogs & derivatives* / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology; D015539:Platelet Activation / Q000502:physiology*; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
chemical_list: C539827:1-(3-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyrido(3,4-b)indole-3-carboxylic acid; D002243:Carbolines; D002352:Carrier Proteins; D010455:Peptides; D010879:Piperazines; D043562:Receptors, G-Protein-Coupled; C435908:collagen-related peptide; D009249:NADP; C024376:NAADP; D020794:Receptor Protein-Tyrosine Kinases; D013917:Thrombin; D002118:Calcium