The PKC superfamily is a key regulator of platelet activation downstream of a range of platelet agonists, including ADP as shown in this study. Studies of the individual isoforms of PKC have highlighted both positive and negative regulatory roles for the kinase in several processes required for platelet activation and thrombus formation (15
). In an attempt to differentiate the net effect of the PKC superfamily on ADP activation, we investigated the effect of a range of concentrations of the pan-PKC inhibitors Ro31-8220 and Ro31-8425 on platelet activation by ADP. The relative affinities of Ro31-8220 and Ro31-8425 for the classical PKC isoforms have been reported (50
) and both are believed to be pan-PKC inhibitors that do not discriminate between isoforms at the concentrations required to inhibit PKC in intact cells.
Many pharmacological reagents have a reduced bioavailability in plasma (31
). Comparison of PAR-1-dependent responses in PRP and washed platelets confirmed that Ro31-8220 had reduced bioavailability in PRP in comparison with washed platelets. A 100-fold higher concentration was required to inhibit dense granule secretion in PRP, yet this concentration was not sufficient to achieve the same level of inhibition of aggregation as seen in washed platelets.
We have made the novel observation that submaximal PKC inhibition causes a marked potentiation in the extent of dense granule secretion and converts reversible to sustained aggregation to a low concentration of ADP (3 μm). At high ADP concentrations, a partial blockade of PKC increases the rate of onset of secretion, although this is followed by a diminished overall secretory response, consistent with both inhibitory and stimulatory actions of the PKC superfamily. This potentiation effect was not seen downstream of PAR-1 receptor stimulation possibly because it initiates activation through a Gq-dependent pathway. Consistent with this, potentiation was also absent in platelets solely activated through the P2Y1 ADP receptor, which also signals through a Gq-dependent pathway. Our results suggest the PKC superfamily has opposing regulatory roles in platelet activation downstream of ADP, an inhibitory role sensitive to low concentrations of Ro31-8220 and an activatory role sensitive to high concentrations. This is the first indication that pan-PKC inhibitors can be used to selectively block one of these opposing roles.
These results raise the question of why these broad spectrum PKC inhibitors have differential and opposing effects at different concentrations. It is possible that this is due to differential sensitivities of the different PKC isoforms to Ro31-8220 and Ro31-8425 or differential dose-response relationships for substrate phosphorylation. It has been reported that PKCϵ is one of the most highly expressed novel isoforms of PKC in mouse platelets even though it is absent in human platelets. In contrast, mice platelets express relatively low levels of PKCδ, which is expressed at a high level in human platelets (18
). Using PRP from mice deficient in the novel isoforms, we have highlighted a role for PKCϵ in the potentiation. Treatment of PKCϵ-deficient mice with Ro31-8220 prior to ADP stimulation partially reduced dense granule secretion demonstrating that the PKC component that activates secretion is mediated by other PKC isoforms. Because PKCϵ is not expressed in human platelets, potentiation must be mediated by another PKC isoform. We therefore extended our study to the use of various PKC isoform-selective inhibitors that are currently available. The range of concentrations of the inhibitors used included concentrations that were lower than previously described in human platelets (15
) in an attempt to ensure that observations made were a result of isoform selectivity of the inhibitor rather than nonspecific inhibition of the PKC superfamily. Treatment of human washed platelets with the PKCβ inhibitor potentiated aggregation and dense granule secretion. This potentiation was also observed following treatment with Gö6983, which primarily inhibits the classical isoforms PKCα and PKCβ, further implicating inhibition of PKCβ in the potentiation.
Treatment of platelets with indomethacin confirmed a critical role for TxA2
in the ability of ADP to stimulate sustained aggregation and marked dense granule secretion in citrated plasma. However, submaximal Ro31-8220 partially rescued this, indicating that, although it may contribute, enhanced TxA2
formation is not essential for potentiation. Use of inhibitors of the two ADP G protein-coupled receptors, ARC that inhibits P2Y12
and MRS that inhibits P2Y1
, indicates that signaling through P2Y12
but not P2Y1
is essential for the potentiation, although this may be mediated through potentiation of the synergy of the two receptors as the synergy is associated with an increase in intracellular Ca2+
. Initially, it seems that these results are at odds with previous reports that P2Y12
potentiates thrombin-induced calcium mobilization and that inhibition of the P2Y12
receptor inhibits ADP-induced calcium mobilization (54
). However, these observations refer to two different mechanisms of calcium regulation. There is synergy between P2Y12
-coupled receptors upstream of phospholipase C activation, and therefore calcium responses are increased as a result of increased inositol 1,4,5-trisphosphate production. The PKC superfamily, however, has both inhibitory and stimulatory effects on platelet Ca2+
mobilization. For example, the novel isoform PKCθ negatively regulates intracellular Ca2+
following GPVI signaling (47
). We observed an elevation in intracellular Ca2+
following treatment with both submaximal and maximal concentrations of Ro31-8220 supporting published data that PKC suppresses agonist-induced Ca2+
). This increase in Ca2+
has been attributed to inhibition of the plasma membrane Ca2+
-ATPase pump (56
), which is inhibited by PKC, and this mechanism to increase Ca2+
potentially underlies the increase in platelet activation observed here following submaximal PKC inhibition.
The inhibition of dense granule secretion by high Ro31-8220 has been reported downstream of multiple platelet agonists suggesting a common target (21
). Multiple proteins, including components of the soluble NSF attachment protein receptors complex and the vesicular trafficking machinery, which are essential for secretion, are phosphorylated in a PKC-dependent manner, including SNAP-23, syntaxin 4, and Munc18c (59
). Inhibition of one or more of these events may underlie the positive role for PKC.
The data presented here identify both positive and negative regulatory roles for the PKC superfamily in the regulation of activation by ADP in human and mouse platelets. The data also support previous reports that suggest differential regulatory roles for the individual isoforms of PKC, indicating a role for the classical isoform PKCβ in human platelets and PKCβ and the novel isoform PKCϵ in the regulation of ADP-induced platelet activation in mouse platelets. This is the first report of an inhibitory role for a classical PKC isoform in platelets.
It is essential that powerful inhibitory pathways exist to prevent unwanted platelet activation within the intact circulation. ADP is a key feedback mediator in platelet activation and thrombus formation. The release of low levels of ADP from damaged cells is therefore potentially very dangerous as this could give rise to unwanted thrombus formation. The work described here demonstrates a previously unrecognized role for members of the PKC superfamily in inhibiting platelet activation by low concentrations of ADP and therefore presents a new pathway of prevention of unwanted platelet activation in plasma. PKC activity is a key regulator of many signal transduction pathways in a variety of cell types, and it is therefore considered as a possible target for therapy for treatment of cancer and various other diseases in addition to being a putative target for antithrombotic therapies (64
). The potentiation of platelet aggregation downstream of low concentrations of ADP therefore has implications for the use of PKC-targeted antithrombotic and anti-cancer therapies.