title: Toll-like receptor 2 stimulation of platelets is mediated by purinergic P2X1-dependent Ca2+ mobilisation, cyclooxygenase and purinergic P2Y1 and P2Y12 receptor activation.
abstract: Toll-like receptor 2 (TLR2), which recognise and respond to conserved microbial pathogen-associated molecular patterns, is expressed on the platelet surface. Furthermore, it has recently been shown that the TLR2/1 agonist Pam3CSK4 stimulates platelet activation. The aim of the present study was to clarify important signalling events in Pam3CSK4-induced platelet aggregation and secretion. Platelet interaction with Pam3CSK4 and the TLR2/6 agonist MALP-2 was studied by analysing aggregation, ATP-secretion, [Ca2+]i mobilisation and thromboxane B2 (TxB2) production. The results show that Pam3CSK4 but not MALP-2 induces [Ca2+]i increase, TxB2 production, dense granule secretion and platelet aggregation. Preincubation of platelets with MALP-2 inhibited the Pam3CSK4-induced responses. The ATP-secretion and aggregation in Pam3CSK4-stimulated platelets was impeded by the purinergic P2X1 inhibitor MRS 2159, the purinergic P2Y1 and P2Y12 antagonists MRS 2179 and cangrelor, the phospholipase C inhibitor U73122, the calcium chelator BAPT-AM and aspirin. The calcium mobilisation was lowered by MRS 2159, aspirin and U73122 whereas the TxB2 production was antagonised by MRS 2159, aspirin and BAPT-AM. When investigating the involvement of the myeloid differentiation factor-88 (MyD88) -dependent pathway, we found that platelets express MyD88 and interleukin 1 receptor-associated kinase (IRAK-1), which are proteins important in TLR signalling. However, Pam3CSK4 did not stimulate a rapid (within 10 minutes) phosphorylation of IRAK-1 in platelets. In conclusion, the results show that Pam3CSK4-induced platelet aggregation and secretion depends on a P2X1-mediated Ca2+ mobilisation, production of TxA2 and ADP receptor activation. The findings in this study further support a role for platelets in sensing bacterial components.
authors.affiliation: Division of Clinical Medicine, Department of Biomedicine, Orebro University, SE-701 82 Orebro, Sweden. hanna.kalvegren@oru.se
authors.forename: H
authors.initials: H
authors.lastname: Kälvegren
authors.forename: C
authors.initials: C
authors.lastname: Skoglund
authors.forename: C
authors.initials: C
authors.lastname: Helldahl
authors.forename: M
authors.initials: M
authors.lastname: Lerm
authors.forename: M
authors.initials: M
authors.lastname: Grenegård
authors.forename: T
authors.initials: T
authors.lastname: Bengtsson
mesh_terms: D020013:Calcium Signaling / Q000276:immunology*; D055666:Lipopeptides / Q000494:pharmacology; D015539:Platelet Activation / Q000276:immunology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058469:Receptors, Purinergic P2X; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D013928:Thromboxane A2 / Q000096:biosynthesis; D051195:Toll-Like Receptor 2 / Q000819:agonists / Q000502:physiology*
chemical_list: D055666:Lipopeptides; C508075:Pam(3)CSK(4) peptide; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D051195:Toll-Like Receptor 2; D013928:Thromboxane A2; D011451:Prostaglandin-Endoperoxide Synthases