OC 35.5 - Quantitative Phosphoproteomics And Causal Analysis Reveal Distinct And Combinatorial Signaling Mechanisms In Protease-activated Receptor PAR1 And PAR4 Platelet Activation Programs

Monday, July 11, 2022

11:45 AM – 12:00 PM

Location: ExCel Center, ICC Capital Suite Room 8&9

Presentation Format: In-Person

Presenting Author(s)

SR

Stephanie Reitsma

PhD student
Oregon Health & Science University
Portland, Oregon, United States

Co-Author(s)

Ivan Parra-Izquierdo, PhD

Postdoctoral Scholar
Oregon Health & Science University
Portland, Oregon, United States
JP

Jiaqing Pang

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

Alexander Melrose

Research assisant
Oregon Health & Science University
portland, Oregon, United States
Tia C.L. Kohs

PhD Candidate
Oregon Health & Science University
Portland, Oregon, United States
AR

Ashok Reddy

Director Proteomics Shared Resource
Oregon Health & Science University
Portland, Oregon, United States
PW

Phillip Wilmarth

Research Assistant Professor Proteomics Shared Resource
Oregon health & Science University
Portland, Oregon, United States
LD

Larry David

Professor Chemical Physiology & Biochemistry department
Oregon Health & Science University
Portland, Oregon, United States
OM

Owen J.T. McCarty, PhD, FAHA

Chair and Professor of Biomedical Engineering
Oregon Health & Science University
Portland, Oregon, United States
Joseph E. Aslan, PhD

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

Background: Intracellular signaling pathways downstream of platelet protease-activated receptors (PARs) mediate hemostasis and, also contribute to thrombosis in vascular diseases through mechanisms that remain unspecified.

Aims: Here, we assess the hypothesis that platelet PAR1 and PAR4 each activate specific, as well as overlapping signaling systems to drive platelet responses underlying hemostasis and thrombosis. Our systems biology approach incorporates state-of-the-art mass spectrometry, computational and cell physiological tools to measure and map phosphorylation events in platelet PAR responses.

Methods: Following platelet isolation from n=4 healthy human donors (in accordance with Helsinki guidelines), washed platelets were stimulated with PAR1 agonist (TRAP6), PAR4 agonist (AYPGFK), thrombin, or vehicle, prior to lysis, digestion, phosphopeptide enrichment and 16plex tandem-mass-tag (TMT) labeling.

Results: Relative to resting platelets, we measured >1,000 significant phosphorylation events in response to PAR agonists (fold-change >1.5; false discovery rate < 0.01), including >600 phosphorylation events common to TRAP6, AYPGFK and thrombin stimulation. These included phosphorylation of well-established mediators (GSK3α, PAK2) and more novel and emerging effectors in platelet activation pathways (BIN2, NKX3-2). Specific PAR1 and PAR4 agonist responses of mechanistic and translational interest were also noted, including phosphorylation of PAR1 T410 or PAR4 S369; thrombin uniquely activated tyrosine kinase Fer Y714 phosphorylation, in a manner that may integrate PAR1 and PAR4 signaling. Functional flow cytometry assays reveal a role of Fer Y714 in platelet function when inhibited with E260 Fer inhibitor. Furthermore, CausalPath analysis identified >100 signaling relations among site-specific phosphorylation changes downstream of PARs, around MAPK, PI3K/Akt, mTOR/S6K and other pathways.

Conclusion(s): In conclusion, we provide a quantitative omics study and causal analysis of platelet PAR signaling, including specific PAR1 and PAR4 agonist responses. Ultimately, this work will help to specify essential effectors, as well as biomarkers and therapeutic targets in platelet dysregulation, hyperactivity and thrombotic diseases.