The novel Syk inhibitor R406 reveals mechanistic differences in the initiation of GPVI and CLEC-2 signaling in platelets.
BACKGROUND
Syk is a key mediator of signaling pathways downstream of several platelet surface receptors including GPVI/FcRgamma collagen receptor, the C-type lectin receptor CLEC-2, and integrin alphaIIbbeta3. A recent study identified the novel small molecule R406 as a selective inhibitor of Syk.


OBJECTIVE
The present study evaluates the role of Syk in human platelets using the novel inhibitor R406.


METHODS
Agonist-induced GPVI and CLEC-2 signaling were assessed using aggregometry, immunoprecipitation and western blotting to determine the effects of R406 on platelet activation.


RESULTS
We demonstrate R406 to be a powerful inhibitor of Syk in human platelets. R406 abrogated shape change and aggregation induced by activation of GPVI and CLEC-2, and reduced platelet spreading on fibrinogen. The inhibitory effect of R406 was associated with inhibition of tyrosine phosphorylation of signaling proteins that lay downstream of Syk for all three receptors, including PLCgamma2. Strikingly, R406 markedly inhibited tyrosine phosphorylation of CLEC-2 and Syk downstream of CLEC-2 activation, whereas phosphorylation of Syk downstream of GPVI and integrin alphaIIbbeta3 was unaffected.


CONCLUSIONS
The inhibitory effect of R406 provides direct evidence of a role for Syk in GPVI, CLEC-2 and integrin alphaIIbbeta3 signaling in human platelets. Further, the results demonstrate a critical role for Syk in mediating tyrosine phosphorylation of CLEC-2, suggesting a novel model in which both Src and Syk kinases regulate tyrosine phosphorylation of the C-type lectin receptor leading to platelet activation.
Centre for Cardiovascular Sciences, Institute for Biomedical Research, The College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK.
J C
JC
Spalton
J
J
Mori
A Y
AY
Pollitt
C E
CE
Hughes
J A
JA
Eble
S P
SP
Watson
D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology; D015153:Blotting, Western; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors*; D037181:Lectins, C-Type / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D010078:Oxazines / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011725:Pyridines / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D000072377:Syk Kinase
C104778:CLEC2B protein, human; D047908:Intracellular Signaling Peptides and Proteins; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; C510788:N4-(2,2-dimethyl-3-oxo-4H-pyrid(1,4)oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine; D010078:Oxazines; D010980:Platelet Membrane Glycoproteins; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C098341:platelet membrane glycoprotein VI; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase