PB0421 - Phosphoproteomics and systems analysis reveal protein kinase C theta (PKCθ) signaling events associated with the generation of procoagulant platelets

Sunday, June 25, 2023

18:30 – 19:30 ET

Room: Exhibition

Co-Author(s)

Yiheng Zhang, MD

Student
Oregon Health & Science University
Portland, Oregon, United States
Stéphanie E. Reitsma, PhD

Postdoc
Department of Pathology and Laboratory Medicine, UNC Blood Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Chapel Hill, North Carolina, United States
OB

Ozgun Babur

Assistant Professor
UMass Boston
Boston, Massachusetts, United States
AM

Alexander Melrose

Research assisant
Oregon Health & Science University
portland, Oregon, United States
JP

Jiaqing Pang

Research Associate
Oregon Health & Science University
Portland, Oregon, United States
SH

Stefanie Hodapp

Graduate Student
University of California, San Diego
San Diego, California, United States
AN

Alexandra Newton

Professor
University of California, San Diego
San Diego, California, United States
PW

Phillip Wilmarth

Senior Core Scientist
Oregon Health & Science University
Portland, Oregon, United States
AR

Ashok Reddy

Core Director
Oregon Health & Science University
Portland, Oregon, United States
Owen J.T McCarty, PhD, FAHA

Professor and Chair of Biomedical Engineering
Oregon Health & Science University
Portland, Oregon, United States

Presenting Author(s)

Joseph E. Aslan, PhD

Associate Professor
Oregon Health & Science University
Portland, Oregon, United States

Background: Protein kinase C (PKC) isoforms have integrated roles in platelet activation programs to support hemostasis, and also contribute to inflammation and thrombosis. While PKCδ and PKC⍺/β are known to regulate platelet function, roles for PKCθ are less specified.

Aims: Here, we aim to determine how mechanisms around PKCθ signaling orchestrate platelet function.

Methods: Platelets were prepared from healthy human donors for ex vivo biochemical, physiological and systems biology studies. Platelet signaling and platelet function were studied following stimulation with agonists, including the GPVI agonist crosslinked collagen-related peptide (CRP-XL), and PAR1 and PAR4 agonists, TRAP6 and AYPGKF-NH2. Platelets were treated with small molecule inhibitors of PKC family members to study PKC activation mechanisms by Western blot using antisera against specific phosphorylation sites on PKC isoforms and PKC substrates.

Results: Phosphoproteomics analyses of agonist-stimulated platelets quantified significant, site-specific changes in the phosphorylation of PKC isoforms as well as numerous PKC substrates. In addition to well established phosphorylation events (e.g., PKCδ Y311), we noted prominent phosphorylation on the C-terminus PKCθ at Ser685. After generating and characterizing antisera specifically reactive to PKCθ phospho-Ser685, Western blot analysis determined that PKCθ Ser685 phosphorylation occurred later following GPVI activation (>5 min) relative to PKC Y311 phosphorylation and the majority of PKC substrate phosphorylation driven by calcium-dependent PKCs. Pretreatment of platelets with inhibitors specific PKC isoforms revealed that PKC⍺/β, PKCθ, and, to a lesser extent, PKCδ activities determine PKCθ Ser685 phosphorylation. Parallel platelet function analyses found that inhibition of PKCθ had only minor effects on platelet secretion and aggregation; however, PKCθ inhibition significantly reduced platelet phosphatidylserine exposure in response to GPVI and PAR agonists.

Conclusion(s): Together, our results inform a model, where PKCδ and PKC⍺/β activities drive the phosphorylation of PKCθ S685 to support later stages of platelet activation programs, including procoagulant platelet generation.