Submaximal Inhibition of Protein Kinase C Restores ADP-induced Dense Granule Secretion in Platelets in the Presence of Ca2+

Amanda J Unsworth,Holly Smith,Paul Gissen,Steve P Watson,Catherine J Pears

doi:10.1074/jbc.m110.187138

Amanda J Unsworth, Holly Smith + Show 3 more

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https://doi.org/10.1074/jbc.m110.187138

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Abstract

Protein kinase C (PKC) is a family of serine/threonine kinases that play isoform-specific inhibitory and stimulatory roles in platelet activation. We show here that the pan-PKC inhibitor Ro31-8220 can be used to dissect these events following platelet activation by ADP. Submaximal concentrations of Ro31-8220 potentiated aggregation and dense granule secretion to ADP in plasma anticoagulated with citrate, in d-Phe-Pro-Arg-chloromethyl ketone-anticoagulated plasma, which has physiological levels of Ca2+, and in washed platelets. Potentiation was retained on inhibition of cyclooxygenase and was associated with an increase in intracellular Ca2+. Potentiation of aggregation and secretion was abolished by a maximally effective concentration of Ro31-8220, consistent with a critical role of PKC in secretion. ADP-induced secretion was potentiated in the presence of an inhibitor of PKCβ but not in the presence of available inhibitors of other PKC isoforms in human and mouse platelets. ADP-induced secretion was also potentiated in mouse platelets deficient in PKCϵ but not PKCθ. These results demonstrate that partial blockade of PKC potentiates aggregation and dense granule secretion by ADP in association with increased Ca2+. This provides a molecular explanation for the inability of ADP to induce secretion in plasma in the presence of physiological Ca2+ concentrations, and it reveals a novel role for PKC in inhibiting platelet activation by ADP in vivo. These results also demonstrate isoform-specific inhibitory effects of PKC in platelets.

Highlights

Platelets play a vital role in the generation of a thrombus or vascular plug, preventing excessive blood loss following vascular injury
In an attempt to dissect this balancing role of protein kinase C (PKC) in human platelets, we monitored the effect of a range of concentrations of the pan-PKC inhibitor Ro31-8220 on platelet activation in platelet-rich plasma (PRP) and washed platelets
The effect of different concentrations of Ro31-8220 on platelet aggregation and dense granule secretion was compared in PRP and washed platelets downstream of stimulation of the G protein-coupled PAR-1 receptor using the PAR-1 peptide TRAP, as both of these responses are known to be PKC-dependent (Fig. 1)

Summary

Introduction

Platelets play a vital role in the generation of a thrombus or vascular plug, preventing excessive blood loss following vascular injury. We observe a marked potentiation in dense granule secretion and aggregation to ADP in citrated platelet-rich plasma (PRP) by a submaximal but not maximal concentration of the PKC inhibitor.

Results

Conclusion