The present study demonstrates a selective role for PKCε in GPVI signalling in mouse platelets with impaired aggregation and secretion to collagen and CRP. In comparison, aggregation and secretion induced through the PAR4 receptor are only marginally inhibited, consistent with a minor role for this PKC isoform in supporting activation alongside other PKC isoforms. The selective defect in GPVI signalling in PKCε-deficient platelets contrasts sharply with that of PKCδ, as platelets from mice deficient in the latter show increased filopodia formation on CRP and increased aggregation to collagen in suspension 
. This demonstrates unique functions for the two novel PKC isoforms downstream of GPVI in mouse platelets. The defect in GPVI signalling seen in the PKCε-null mouse platelets is not due to a change in the level of expression of GPVI or α2β1. The observation that aggregation to ADP was not altered in mouse platelets argues against a role for a change in expression of integrin αIIbβ3, a result confirmed by flow cytometry.
The reduction in response to GPVI can be accounted for by a decrease in tyrosine phosphorylation at a proximal stage of its signalling cascade, as phosphorylation of the FcRγ-chain and Syk are reduced. A mechanism to explain this has been described in mast cells, where, in response to activation of the FcεRI receptor, PKC phosphorylates the FcRγ-chain on threonine-60, two residues downstream of the conserved C-terminal tyrosine residue which is phosphorylated in the ITAM, leading to Syk association 
. Expression of a mutated form of the FcRγ-chain with threonine-60 replaced by alanine led to reduced association and activation of Syk and subsequent inhibition of degranulation in response to IgE 
. The authors speculate that phosphorylation of the ITAM by a novel PKC isoform increases binding of Syk to the ITAM and enhances activation, illustrated by the increase in tyrosine phosphorylation of Syk and downstream proteins. We speculate that this increase in binding of Syk to the FcRγ-chain protects the ITAM moiety from dephosphorylation leading to a net increase in tyrosine phosphorylation. In mast cells, PKCδ is responsible for the phosphorylation. However, although PKCδ is expressed robustly in human platelets, it is at low levels in mouse platelets. PKCε is present in mouse platelets and, as for PKCδ, its recombinant form does mediate phosphorylation of the FcRγ-chain ITAM in vitro 
. Tyrosine phosphorylation of the FcRγ-chain, Syk and downstream proteins is also reduced in the presence of a pan-PKC inhibitor in mouse platelets, confirming that the reduction in tyrosine phosphorylation of FcRγ-chain is mediated by the loss of a PKC isoform. Interestingly, a similar result is not seen in human platelets, which, although consistent with the low levels of PKCε, is surprising given the presence of PKCδ. This may reflect a further difference between mouse and human platelets in the role of novel PKC isoforms.
Our inability to detect expression of PKCε in human platelets was not expected as there are several reports describing its presence. However, Buensuceso et al.
were also unable to detect expression of PKCε in human platelets although both positive and negative studies used the same source of antibodies as used in the present one 
. Our attempt to address this through concentration of PKCε by immunoprecipitation was unsuccessful. Whilst the explanation for this discrepancy is not known, and may reflect differences in batches of antibodies, the cumulative reports illustrate that PKCε is either absent in human platelets or present at a very low level.
The present study demonstrates that the tyrosine phosphorylation of PKCδ seen in human platelets is down-regulated through a PKC-dependent pathway. The molecular basis of this is not known, although it could be dependent upon the direct or indirect regulation of a protein tyrosine phosphatase. For example the SHP-1 tyrosine phosphatase interacts with PKCα and is phosphorylated on a consensus PKC phosphorylation site following human platelet activation 
. Phosphorylation of SHP-1 negatively regulates its activity in vitro
, but the in vivo
consequences of phosphorylation may be more complex, with the potential to regulate substrate specificity and localisation. Interestingly, in mouse platelets tyrosine phosphorylation of PKCδ is sustained, further emphasising the species-specific functions and regulation of PKC isoforms. PAR4 has been reported to stimulate sustained tyrosine phosphorylation of PKCδ in human platelets 
, and this is the major signalling receptor for thrombin in mouse platelets. Thus, the difference in kinetics may reflect differential expression and roles of PAR1 and PAR4 in human and mouse platelets.
Although frequently used as a marker of PKCδ activation, the functional significance of its tyrosine phosphorylation is unclear. It has been reported to increase the kinase activity of PKCδ in human platelets but is not required for membrane association 
. This study shows that blockade of Src kinases, and hence phosphorylation of PKCδ, does not alter pleckstrin phosphorylation in thrombin-stimulated platelets, arguing against direct regulation of enzymatic activity, with the caveat that PKCδ may not play a major role in regulating pleckstrin phosphorylation. The lack of Src kinase blockade on pleckstrin phosphorylation is consistent with the fact that Src kinases play a minimal role in thrombin-induced platelet aggregation 
, again questioning the significance of PKCδ tyrosine phosphoryation in this signalling pathway. The significance of tyrosine phosphorylation of PKCδ downstream of GPVI remains to be established.
In conclusion, the present study has provided further evidence for specific regulation and functions of PKC isoforms by focussing on the two novel PKCs, PKCδ and PKCε, and in particular has shown that PKCε plays a critical role in potentiating the proximal events in the GPVI signalling pathway. Further, it has also demonstrated differences in expression and phosphorylation of the two isoforms in mouse and human platelets further revealing species-specific functions of individual isoforms.