Key result
PKCβ inhibition triggers Syk hyperphosphorylation in human platelets, identifying this isoform as the negative regulator.
Why the study?
The role of protein kinase C isoforms in regulating tyrosine phosphorylation and activity of Syk in platelets was unknown.
PKCβ is identified as the isoform responsible for Syk negative regulation in human platelets, highlighting a species-specific pathway relevant for anti-platelet therapy development.
Supports PKCβ as platelet signaling target; leaves open translation to antithrombotic strategies.
Background: PKC regulating Syk activity has been demonstrated in other cells but is unknown in platelets. Results: PKCs regulate tyrosine phosphorylation and activity of Syk. Conclusion: PKCβ-dependent differential regulation of Syk activity is seen in human but not in murine platelets. Significance: Understanding this human pathway of platelet regulation might aid in development of anti-platelet therapy. Protein kinase C (PKC) isoforms differentially regulate platelet functional responses downstream of glycoprotein VI (GPVI) signaling, but the role of PKCs regulating upstream effectors such as Syk is not known. We investigated the role of PKC on Syk tyrosine phosphorylation using the pan-PKC inhibitor GF109203X (GFX). GPVI-mediated phosphorylation on Syk Tyr-323, Tyr-352, and Tyr-525/526 was rapidly dephosphorylated, but GFX treatment inhibited this dephosphorylation on Tyr-525/526 in human platelets but not in wild type murine platelets. GFX treatment did not affect tyrosine phosphorylation on FcRγ chain or Src family kinases. Phosphorylation of Lat Tyr-191 and PLCγ2 Tyr-759 was also increased upon treatment with GFX. We evaluated whether secreted ADP is required for such dephosphorylation. Exogenous addition of ADP to GFX-treated platelets did not affect tyrosine phosphorylation on Syk. FcγRIIA- or CLEC-2-mediated Syk tyrosine phosphorylation was also potentiated with GFX in human platelets. Because potentiation of Syk phosphorylation is not observed in murine platelets, PKC-deficient mice cannot be used to identify the PKC isoform regulating Syk phosphorylation. We therefore used selective inhibitors of PKC isoforms. Only PKCβ inhibition resulted in Syk hyperphosphorylation similar to that in platelets treated with GFX. This result indicates that PKCβ is the isoform responsible for Syk negative regulation in human platelets. In conclusion, we have elucidated a novel pathway of Syk regulation by PKCβ in human platelets.
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Buitrago et al. (2013) studied this question. Pan-PKC inhibitor GF109203X (GFX) and selective PKC inhibitors vs. Untreated/wild type platelets was evaluated on Syk tyrosine phosphorylation. PKCβ inhibition resulted in Syk hyperphosphorylation in human platelets, indicating that PKCβ is the isoform responsible for Syk negative regulation.
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