Abstract 140: Exploring Protein Kinase C Theta (PKCθ) Signaling In The Generation Of Procoagulant Platelet: Insights From Phosphoproteomics And Systems Analysis

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Background: Platelet activation plays a pivotal role in physiological hemostasis and thrombosis. Protein Kinase C theta (PKCθ) isoforms exert significant regulatory influence on platelet activation. While specific PKC family members have well-defined roles in platelet function, the roles of PKCθ remain less explored. Objectives: To determine mechanisms of PKCθ signaling that orchestrate procoagulant platelet generation. Methods: Platelets were prepared from healthy human donors for ex vivo experiments. Platelet signaling and function were studied following stimulation with GPVI agonist crosslinked collagen-related peptide (CRP-XL). To investigate PKCθ inhibition mechanisms in platelets, small molecule inhibitors targeting the PKCθ (CC-90005 and C20) were employed. Results: From phosphoproteomics, we noted prominent phosphorylation on the C-terminus PKCθ at Ser685. Pretreatment of platelets with inhibitors specific PKC isoforms revealed that PKCα/β, PKCθ, and PKCΔ activities determine PKCθ Ser685 phosphorylation. From the PKC Inhibitors Activity Curves, we noted a robust and selective inhibitory impact of CC-90005 on PKCθ, with specificity surpassing that of C20. Parallel platelet function analyses revealed that inhibiting PKCθ had minimal impact on platelet spreading and aggregation. However, PKCθ inhibition in the presence of Ca 2+ upregulated platelet phosphatidylserine (PS) exposure, as measured by flow cytometry. This was accompanied by an increase in platelet procoagulant activity, where PKCθ inhibition reduced the lag time for fibrin formation. Furthermore, fluorescence microscopy also found that PKCθ inhibition significantly augmented the proportion of procoagulant platelets and microparticles. Additionally, compared to the control group, the use of cyclosporin A (CsA) reduced the production of procoagulant platelets, but the addition of CC-90005 or C20 rescued this phenomenon, with signal intensity surpassing that of the control group. Conclusions: Our study proposes a model wherein PKCΔ and PKCα/β activities induce the phosphorylation of PKCθ at S685, influencing mitochondrial calcium signaling and supporting procoagulant platelet generation.

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