Response:Role of PKCθ in collagen-related peptide-induced platelet activation

Response:

Harper and Poole report that collagen-related peptide (CRP) caused potentiation of dense granule secretion in murine platelets deficient in Protein kinase C (PKC) θ isoform.¹  They also show that pretreatment of human platelets with PKCθ isoform selective RACK peptide results in enhanced dense granule secretion upon stimulation with CRP.¹  They argue that these results do not agree with our recent work²  demonstrating a positive role for PKCθ in platelet activation.

Our experiments²  were done with higher concentrations of CRP (10 and 20 μg/mL). It is evident from Figure 1 of the work by Harper and Poole¹  that the potentiating effects of CRP on dense granule secretion are more apparent at lower agonist concentrations and there is no potentiation when 10 μg/mL CRP was used. Hence, we decided to perform additional experiments with lower concentrations of CRP (2 and 5 μg/mL), which were not used in our previous study.²  The lower concentrations of CRP (2 μg/mL) caused a potentiating effect on dense granule secretion from PKCθ^−/− platelets (0.320 ± 0.0064 nmoles/3.7 × 10⁸ platelets; P < .05), compared with wild-type (WT) littermates (0.238 ± 0.004 nmoles/3.7 × 10⁸ platelets). Slightly higher concentrations (5 μg/mL) showed no significant differences (0.685 ± 0.044 vs 0.512 ± 0.044 nmoles/3.7 × 10⁸ platelets; P > .1). Hence, we agree with Harper and Poole¹  that very low concentrations of CRP cause potentiation of dense granule release in platelets lacking functional PKCθ.

Harper and Poole¹  claimed that they reported that CRP-induced secretion is enhanced in their previous study.³  In their previous study³  they actually stated that “Interestingly, however, no difference in adenosine triphosphate (ATP) secretion was seen between PKCθ^−/− and WT platelets in response to CRP (Figure 3B) or collagen (Figure 3C).¹  It is not clear to us how, using the same concentrations of CRP used in their previous study (1 and 5 μg/mL),³  they could demonstrate significant potentiation in PKCθ null mouse platelets (P < .001) in Figure 1A of their study.¹  Hence their current results¹  are in conflict with their own previous work.³  It is also important to note that in their previous study,³  the ATP release numbers were nearly 10-fold higher than in the current work (Figure 1A¹ ).

Consistent with our study,²  Cohen et al⁴  recently reported that PKCθ plays a positive regulatory role in platelet activation and secretion. Consistent with our in vivo thrombosis results,²  they noticed that the bleeding times were increased in PKCθ^−/− mice.⁴ 

Harper and Poole¹  argue that phosphorylated syntaxin 4 would dissociate from SNAP23 and thereby reduce secretion, thus syntaxin 4 phosphorylation would negatively regulate release. Previous studies^5,6  reported dissociation of syntaxin 4 and SNAP23 upon platelet activation though no time course for this process was given. Dissociation could occur after fusion, during which the components of the SNARE complex might be recycled, though it is not clear whether this is significant in an activated platelet. The evidence that panPKC inhibitors abolish phosphorylation of syntaxin as well as secretion is indicative that syntaxin phosphorylation could be important for secretion.⁵ 

In conclusion, we emphasize that our data²  are supported by a recent independent study⁴  that confirms a positive regulatory role for PKCθ in platelets.