PKCα negatively regulates in vitro proplatelet formation and in vivo platelet production in mice

Abstract

Proplatelet formation is a part of the intricate process by which platelets are generated by their precursor cell, the megakaryocyte. The processes that drive megakaryocyte maturation and platelet production are however still not well understood. The protein kinase C (PKC) family of serine/threonine kinases has been demonstrated as an important regulator of megakaryocyte maturation and proplatelet formation, but little investigation has been made on the individual isoforms. We have previously shown, in mouse models, that PKCα plays a vital role in regulating platelet function, so in this study we aimed to investigate the role of PKCα in megakaryocyte function using the same Prkca^−/− mice. We assessed the role of global PKC and specifically PKCα in proplatelet formation in vitro, analyzed polyploidy in Prkca^−/−-derived megakaryocytes and followed platelet recovery in platelet-depleted Prkca^−/− mice. We show reduced proplatelet formation in the presence of global PKC blockade. However, in the presence of a selective classical PKC isoform inhibitor, Go6976, proplatelet formation was conversely enhanced. PKCα null megakaryocytes also showed enhanced proplatelet formation, as well as a shift to greater polyploidy. In vivo, platelet production was enhanced in response to experimentally induced immune thrombocytopenia. In conclusion, our data indicate that classical PKC isoforms, and more specifically PKCα, are negative regulators of proplatelet formation. PKCα appears to negatively regulate endomitosis, with the enhanced polyploidy observed in Prkca^−/−-derived megakaryocytes. In vivo, these observations may culminate in the observed ability of Prkca^−/− mice to recover more rapidly from a thrombocytopenic insult.

• Megakaryocyte
• PKC
• proplatelet formation
• polyploidy

Acknowledgments

We thank Elizabeth Aitken for their expert technical assistance supporting this study. We gratefully acknowledge the assistance of the Wolfson BioImaging Facility. The work was supported by grants from the British Heart Foundation to Alastair W. Poole (grant nos RG/10/006/28299 & PG/08/049).

Authorship contributions

Christopher M. Williams designed experiments, performed experiments, analyzed data and wrote the manuscript; Matthew T. Harper designed experiments, performed experiments, and analyzed data and Alastair W. Poole designed experiments and wrote the manuscript.

Conflict of interest declaration

The authors have declared that no conflict of interest exists.