To the editor:
Protein kinase C (PKC) is a central regulator of platelet activation, and individual PKC isoforms are likely to have distinct roles.1 We and others had previously reported roles for the novel PKC isoform, PKCθ, in integrin signaling2 and platelet function.3 The recent paper by Nagy et al,4 which attempts to characterize further the importance of PKCθ, is valuable in this regard. However, their data conflict with other published data in several respects, and we are unable to repeat some of the findings of Nagy et al despite preparing platelets in the manner they describe.
While Nagy et al4 report that granule secretion in response to the glycoprotein VI collagen-related peptide (GPVI) agonist CRP is reduced in PKCθ−/− platelets, we have previously reported that CRP-induced secretion is enhanced in these cells.3 Similarly, we found increased CRP-induced integrin αIIbβ3 activation and thrombus formation under flow in vitro,3 whereas Nagy et al reported a decrease in integrin activation. Nagy et al4 indicate that their effects are independent of their proposed role for PKCθ in thromboxane synthesis, as they report that decreased aggregation and secretion also occurred in indomethacin-treated platelets. To address whether the differences between our studies reflect differences in platelet preparation, we have repeated our experiments using the platelet preparation method described by Nagy et al,4 and treated the platelets with indomethacin. However, under these conditions we still find enhanced CRP-induced dense granule secretion in PKCθ platelets (Figure 1A).
Nagy et al4 supported their data from mouse platelets with experiments using a peptide (Vθ1-1; CGLSNFD) predicted to inhibit PKCθ's interaction with its RACK adaptor protein, coupled to a TAT peptide (CYGRKKRRQRRR) to allow its entry into cells. To the best of our knowledge, this is the first published report of Vθ1-1-TAT. Mochly-Rosen and colleagues (Stanford University) have kindly provided us with the same peptide. We found that CRP-induced ATP secretion was enhanced by Vθ1-1-TAT pretreatment (1 μM) in wild-type (WT) platelets (Figure 1B), similar to the enhanced secretion seen in PKCθ−/− platelets, and Vθ1-1-TAT had no additional effect in PKCθ−/− platelets. TAT alone had no effect. Moreover, we found that Vθ1-1-TAT significantly enhanced CRP-induced granule secretion in human platelets (Figure 1C), prepared in the manner described by Nagy et al.4
Together, our data suggest that PKCθ negatively regulates CRP-induced ATP secretion in human and mouse platelets, consistent with our reported increased thrombus formation under flow over collagen in vitro.3 It is difficult to explain the differences between our data and those of Nagy et al4 However, we note that Vθ1-1-TAT inhibited CRP-induced syntaxin 4 phosphorylation in human platelets, as shown in Figure 4 of Nagy et al.4 Syntaxin 4 phosphorylation is expected to reduce its association with SNAP-23.5 However, interaction between SNAP and syntaxin is necessary for granule secretion.6-8 We would therefore expect that reduced syntaxin phosphorylation would lead to enhanced syntaxin-SNAP interaction and so enhanced secretion.
In conclusion, contrary to Nagy et al, we suggest that PKCθ negatively regulates CRP-induced dense granule secretion, although clearly there remains much to discover about the roles of PKC isoforms in platelet activation and thrombus formation.
Contribution: M.T.H. designed and performed experiments, analyzed results and cowrote the manuscript. A.W.P. designed experiments and cowrote the manuscript.
Acknowledgments: Vθ1-1-TAT was a kind gift from Drs Mochly-Rosen and Grant Budas (Stanford University, CA). M.T.H. is supported by the British Heart Foundation.
Conflict-of-interest disclosure: The authors declare no competing financial interests.