Protein Kinase C Isoform ε Negatively Regulates ADP-Induced Calcium Mobilization and Thromboxane Generation in Platelets

Abstract

Objective— Members of the protein kinase C (PKC) family are shown to positively and negatively regulate platelet activation. Although positive regulatory roles are extensively studied, negative regulatory roles of PKCs are poorly understood. We investigated the mechanism and specific isoforms involved in PKC-mediated negative regulation of ADP-induced functional responses. Methods and Results— A pan-PKC inhibitor, GF109203X, potentiated ADP-induced cPLA 2 phosphorylation and thromboxane generation as well as ERK activation and intracellular calcium (Ca 2+ i ) mobilization, 2 signaling molecules, upstream of cPLA 2 activation. Thus, PKCs inhibit cPLA 2 activation by inhibiting ERK and Ca 2+ i mobilization. Because the inhibitor of classic PKC isoforms, GO-6976, did not affect ADP-mediated thromboxane generation, we investigated the role of novel class of PKC isoforms. ADP-induced thromboxane generation, calcium mobilization, and ERK phosphorylation were potentiated in PKCε null murine platelets compared with platelets from wild-type littermates. Interestingly, when thromboxane release is blocked, ADP-induced aggregation in PKCε knockout and wild-type was similar, suggesting that PKCε does not affect ADP-induced aggregation directly. PKCε knockout mice exhibited shorter times to occlusion in an FeCl 3 -induced arterial injury model and shorter bleeding times in tail-bleeding experiments. Conclusion— We conclude that PKCε negatively regulates ADP-induced thromboxane generation in platelets and offers protection against thrombosis.

Protein Kinase C ε Expression in Platelets from Patients with Acute Myocardial Infarction

“…In platelets from PKCε null mouse, Pears et al showed a marked inhibition of aggregation and dense granule secretion in response to GPVI agonists but no significant functional change in response to ADP [24]. At the opposite, recently Bynagari-Settipalli et al [26] showed an increase in ADP-induced aggregation and secretion in platelets from PKCε null mice. Indeed, signaling through GPVI suggests a role for PKCε in the initial steps of thrombus formation in mouse platelets.…”

Protein kinase Cε and protein kinase Cθ double‐deficient mice have a bleeding diathesis

“…S1). The level of PKCε in human platelets, however, is unclear as we were not able to detect expression using commercially available antibodies, although a recent study using an in‐house antibody has reported robust expression [8].…”

“…Robust expression of several isoforms has been reported in human (α, β, δ, θ) and mouse (α, β, ε, δ, θ) platelets, with evidence of expression of additional isoforms. In contrast, there are conflicting reports on expression of PKCε in human platelets [4–7], although a recent study using an in‐house antibody has demonstrated robust expression [8].…”

Effect of tetramethylpyrazine (TMP) on Ca2+ signal transduction and cell viability in a model of renal tubular cells

“…Ca 2+ oscillations, Ca 2+ sensitization, and contraction were shown to be activated by PKC in small airway smooth muscle . Conversely, PKC was shown to negatively regulate ADP‐induced Ca 2+ mobilization and thromboxane generation in platelets . Because 30% of TMP‐induced [Ca 2+ ] i rises was via Ca 2+ influx, this influx appears to be contributed by PKC‐regulated store‐operated Ca 2+ entry.…”