CLEC-2 is required for the activation of mouseplatelets by bacterial DNA mimetics

Background: Short nuclease-resistant phosphorothioate synthetic CpG
motif-bearing oligonucleotides (CpG ODNs) mimicking bacterial
DNA display potent immunostimulatory activity and are therefore
being used in clinical trials as vaccine adjuvants. Cellular uptake and
activation depends on the interaction of CpG ODNs with the C-type
lectin receptor DEC-205 and subsequent stimulation of the Toll-like
receptor 9 (TLR9) and myeloid differentiation primary response 88
(MyD88) signaling cascade. Platelets express TLR9, MyD88, and the
C-type lectin-like receptor 2 (CLEC-2). However, the impacts of CpG
ODNs on platelet function have been elusive.
Aims: To evaluate whether CpG ODNs affect platelet activation and
thrombus formation via CLEC-2 and TLR9.
Methods: We incubated washed platelets or whole blood from TLR9-,
MyD88- or CLEC-2- deficient mice with CpG ODNs. We performed
platelet aggregometry, flow cytometric binding and platelet activation
assays as well as signal transduction analyses. Thrombus formation
and fibrin generation were also analyzed by intravital microscopy in
mouse microcirculation upon intravenous injection of CpG ODNs.
Results: We show that CpG ODNs bind on platelet surface and are
internalized. They activate platelets and induce their aggregation.
TLR9- or MyD88-deficient platelets aggregated normally in response
to CpG ODN. Interestingly, platelets deficient for the C-type lectin
receptor CLEC-2 were unable to capture and internalize CpG ODN.
CLEC-2 deficiencyabolished CpG ODN-induced platelet activation
and aggregation. CpG ODN stimulated CLEC-2 dependent tyrosine
kinase pathway and Syk phosphorylation. In vivo, intravenously
injected CpG ODN interacted with platelets adhered to laser injured
arteriolar endothelia and promoted fibrin generation and thrombus
growth.
Conclusion: CLEC-2 mediates CpG ODN uptake and subsequent
platelet activation, independently of TLR9, which may serve an
important role in the interplay between platelets and immunity.