Toll-like receptor 2 stimulation of platelets is mediated by purinergic P2X1-dependent Ca2+ mobilisation, cyclooxygenase and purinergic P2Y1 and P2Y12 receptor activation

Thromb Haemost. 2010 Feb;103(2):398-407. doi: 10.1160/TH09-07-0442. Epub 2009 Dec 18.

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.

MeSH terms

  • Calcium Signaling / immunology*
  • Lipopeptides / pharmacology
  • Platelet Activation / immunology*
  • Prostaglandin-Endoperoxide Synthases / metabolism*
  • Receptors, Purinergic P2 / metabolism*
  • Receptors, Purinergic P2X
  • Receptors, Purinergic P2Y1
  • Receptors, Purinergic P2Y12
  • Thromboxane A2 / biosynthesis
  • Toll-Like Receptor 2 / agonists
  • Toll-Like Receptor 2 / physiology*

Substances

  • Lipopeptides
  • Pam(3)CSK(4) peptide
  • Receptors, Purinergic P2
  • Receptors, Purinergic P2X
  • Receptors, Purinergic P2Y1
  • Receptors, Purinergic P2Y12
  • Toll-Like Receptor 2
  • Thromboxane A2
  • Prostaglandin-Endoperoxide Synthases