Phosphorylation of protein kinase Cδ Tyr311 positively regulates thromboxane generation in platelets

Figures

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  Figure 1. Phosphorylation of Y311 on PKCδ is agonist dependent.A, schematic showing the locations of several tyrosine phosphorylation sites on PKCδ. Mouse and human sites are listed. An “Hu” prior to the number denotes human. B, western blots showing Y311 phosphorylation of PKCδ following stimulation of platelets with 500 μM AYPGKF with or without 10 μM Indomethacin, 100 μM MRS2179 and 500 nM AR-C69931MX. The combination of indomethacin, MRS2179 and AR-C69931MX are referred to as feedback inhibitors (FBIs). Washed human platelets were stimulated with 500 mM AYPGKF for the indicated time and the resulting platelet proteins were separated via SDS-PAGE. C, quantification of fluorescent band intensity from “B” expressed a ratio of pY311 to total PKCδ. D, western blots showing Y311 phosphorylation of PKCδ following stimulation (3 min) of human platelets with 5 μg/ml CRP with or without 100 nM YM-254890 and 500 nM AR-C69931MX. YM-254890 and AR-C69931MX are FBIs in this experiment. E, quantification of “D” expressed as a ratio of pY311 to total PKCδ, which was used to assess loading. ∗p < 0.05, n = 5. CRP, collagen-related peptide; PKC, protein kinase C.

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  Figure 2. Production of PKCδ Y311F mice.A, agarose gel of PCR products following SSpI restriction enzyme digest to identify PKCδ Y311F mice. WT PCR products remain uncleaved and are 432 bp, while heterozygotes (het) have the full length 432 bp product and the cleaved 147 bp and 285 bp products. The homozygous samples contain only the cleaved 147 bp and 285 bp products. B, western blot following immunoprecipitation of PKCδ from Y311F and WT control platelets. Washed platelets were stimulated with 1.25 μg/ml CRP for 3 min. The resulting platelet proteins were subjected to immunoprecipitation targeting PKCδ. The immunoprecipitated protein was resolved via SDS-PAGE, and the membranes were probed using an antibody against Y311 of PKCδ. Total PKCδ was used to assess loading. CRP, collagen-related peptide; PKC, protein kinase C.

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  Figure 3. Hemostasis and thrombosis are altered in PKCδ Y311F mice.A, tail bleeding times from WT (+/+), heterozygous (+/−), and PKCδY311F KI (−/−) mice. The distal 3 mm of the tail was clipped from mice aged 4 to 6 weeks and allowed to bleed into warm saline. The time until bleeding ceased was recorded. ∗p < 0.05. B, representative blood flow tracings from WT control and PKCδY311F Ki mice subjected to FeCl3 injury of the carotid artery. C, time to occlusion following vessel injury was recorded. ∗p < 0.05, n = 7. PKC, protein kinase C.

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  Figure 4. PAR-4 induced activation is impaired in platelets from PKCδ Y311F mice.A, washed platelets from WT control and PKCδ Y311F mice were stimulated with the indicated concentration of AYPGKF, and aggregation and secretion were recorded. B, representative tracings that show aggregation was unaltered in PKCδ Y311F platelets stimulated with AYPGKF. C, ATP secretion from PKCδ Y311F platelets was diminished when stimulated with 100 μM and 200 μM AYPGKF compared with WT control platelets. ∗p < 0.05, n = 5. PAR, protease-activated receptor; PKC, protein kinase C.

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  Figure 5. Inhibition of thromboxane generation equalizes ATP secretion between Y311F and WT platelets.A, washed platelet from PKCδ Y311F or WT control mice were either pretreated with indomethacin to inhibit thromboxane production or dimethyl sulfoxide as a vehicle control. Platelets were then stimulated with 200 μg/ml AYPGKF, and aggregation and secretion were observed. Representative tracings are shown. B, aggregation was unaltered with indomethacin pretreatment. C, ATP secretion was reduced in Y311F platelets when vehicle control was added but was normalized in PKCδ Y311F platelets following indomethacin pretreatment. ∗p < 0.05 compared with WT, n = 5. PKC, protein kinase C.

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  Figure 6. Y311 on PKCδ regulates thromboxane generation.A, representative western blots of platelet proteins from WT and Y311F mice. Platelets were stimulated with the indicated concentration of AYPGKF, and the membranes were probed for phosphorylated ERK1/2 and total ERK1/2, which was used to assess loading. B, quantification from multiple experiments described in B, expressed as a ratio of phosphorylated ERK1/2 to total ERK1/2. C, washed platelets from WT control and PKCδ Y311F mice were stimulated with the indicated concentration of AYPGKF for 3 min under stirring conditions. Thromboxane generation was assessed using a kit from Enzo Scientific. ∗p < 0.05, n = 5. ERK1/2, extracellular signal-regulated protein kinase; PKC, protein kinase C.

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  Figure 7. Schematic representation of Y311 phosphorylated PKCδ signaling in the platelet. Inhibitors and antagonists used during the collection of our data, as well as their targets, are also listed. Activation of PAR4 via AYPGKF leads to PLCβ activation and subsequent IP3 and DAG production. PKCδ responds to DAG and its activation results in ERK1/2 phosphorylation and thromboxane generation. Our data, presented in this manuscript, demonstrate that phosphorylation of Y311 on PKCδ regulates ERK1/2 phosphorylation and thromboxane generation. DAG, diacylglycerol; ERK1/2, extracellular signal-regulated protein kinase; PAR, protease-activated receptor; PKC, protein kinase C.