CGX1037 is a novel PKC isoform delta selective inhibitor in platelets

Abstract

Platelets upon activation change their shape, aggregate and secrete alpha and dense granule contents among which ADP acts as a feedback activator. Different Protein Kinase C (PKC) isoforms have specific non-redundant roles in mediating platelet responses including secretion and thrombus formation. Murine platelets lacking specific PKC isoforms have been used to evaluate the isoform specific functions. Novel PKC isoform δ has been shown to play an important role in some pathological processes. Lack of specific inhibitors for PKCδ has restricted analysis of its role in various cells. The current study was carried out to evaluate a novel small molecule PKCδ inhibitor, CGX1037 in platelets. Platelet aggregation, dense granule secretion and western blotting experiments were performed to evaluate CGX1037. In human platelets, CGX1037 inhibited PAR4-mediated phosphorylation on PKD2, a PKCδ-specific substrate. Pre-treatment of human or murine platelets with CGX1037 inhibited PAR4-mediated dense granule secretion whereas it potentiated GPVI-mediated dense granule secretion similar to the responses observed in murine platelets lacking PKCδ· Furthermore, pre-treatment of platelets from PKCδ^−/− mice with CGX1037 had no significant additive effect on platelet responses suggesting the specificity of CGX1037. Hence, we show that CGX1037 is a selective small molecule inhibitor of PKCδ in platelets.

• Protein kinase Cδ
• CGX1037
• protein kinase D2
• protease activated receptor-4
• GPVI
• platelet dense granule secretion

Acknowledgements

The authors thank Monica Wright for maintenance and breeding of mice.

Declaration of interest

Dr. Swindle is the CEO of CompleGen, Inc. (Seattle, WA), which produces the new PKCδ inhibitor CGX1037. CompleGen, Inc. provided the product to Dr. Kunapuli’s laboratory at no charge. The planning, conduct, and reporting of the research were performed by Dr. Kunapuli and the members of his laboratory. Data analysis was performed by Dr. Kunapuli with no input or oversight from CompleGen, Inc. This work is supported by HL93231 and HL118593 from National Institutes of Health to SPK, HL111552 to LEK, and R416M103193 to JS and LEK. JCK is supported by NIH training grant in Thrombosis (HL07777). D. B. designed and performed experiments, analyzed data and wrote the article. J.C.K. analyzed data and edited the article. J.S. provided the inhibitor. L.E.K. analyzed data and edited the article. S.P.K. provided overall direction, designed experiments, analyzed data and edited the article. The authors declare no conflicts of interests. The authors alone are responsible for the content and writing of this article.