TY - JOUR
T1 - Phosphoproteomic quantitation and causal analysis reveal pathways in GPVI/ITAM-mediated platelet activation programs
AU - Babur, Özgün
AU - Melrose, Alexander R.
AU - Cunliffe, Jennifer M.
AU - Klimek, John
AU - Pang, Jiaqing
AU - Sepp, Anna Liisa I.
AU - Zilberman-Rudenko, Jevgenia
AU - Yunga, Samuel Tassi
AU - Zheng, Tony
AU - Parra-Izquierdo, Iván
AU - Minnier, Jessica
AU - McCarty, Owen J.T.
AU - Demir, Emek
AU - Reddy, Ashok P.
AU - Wilmarth, Phillip A.
AU - David, Larry L.
AU - Aslan, Joseph E.
N1 - Funding Information:
This work was supported by the Medical Research Foundation of Oregon, the American Heart Association (17SDG33350075 to J.E.A.), a Scholar Award from the American Society of Hematology (to J.E.A.), and the National Institutes of Health (NIH), National Heart, Lung, and Blood Institute (R01HL146549 [J.E.A.]; R01HL101972 [O.J.T.M.]). Mass spectrometric analysis was partially supported by core grants from the NIH, National Eye Institute (P30 EY010572) and NIH, National Cancer Institute (P30 CA069533) and a shared instrument grant from the Office of the Director (S10OD-012246).
Publisher Copyright:
© 2020 by The American Society of Hematology
PY - 2020/11/12
Y1 - 2020/11/12
N2 - Platelets engage cues of pending vascular injury through coordinated adhesion, secretion, and aggregation responses. These rapid, progressive changes in platelet form and function are orchestrated downstream of specific receptors on the platelet surface and through intracellular signaling mechanisms that remain systematically undefined. This study brings together cell physiological and phosphoproteomics methods to profile signaling mechanisms downstream of the immunotyrosine activation motif (ITAM) platelet collagen receptor GPVI. Peptide tandem mass tag (TMT) labeling, sample multiplexing, synchronous precursor selection (SPS), and triple stage tandem mass spectrometry (MS3) detected >3000 significant (false discovery rate < 0.05) phosphorylation events on >1300 proteins over conditions initiating and progressing GPVI-mediated platelet activation. With literature-guided causal inference tools, >300 site-specific signaling relations were mapped from phosphoproteomics data among key and emerging GPVI effectors (ie, FcRg, Syk, PLCg2, PKCd, DAPP1). Through signaling validation studies and functional screening, other less-characterized targets were also considered within the context of GPVI/ITAM pathways, including Ras/MAPK axis proteins (ie, KSR1, SOS1, STAT1, Hsp27). Highly regulated GPVI/ITAM targets out of context of curated knowledge were also illuminated, including a system of >40 Rab GTPases and associated regulatory proteins, where GPVI-mediated Rab7 S72 phosphorylation and endolysosomal maturation were blocked by TAK1 inhibition. In addition to serving as a model for generating and testing hypotheses from omics datasets, this study puts forth a means to identify hemostatic effectors, biomarkers, and therapeutic targets relevant to thrombosis, vascular inflammation, and other platelet-associated disease states.
AB - Platelets engage cues of pending vascular injury through coordinated adhesion, secretion, and aggregation responses. These rapid, progressive changes in platelet form and function are orchestrated downstream of specific receptors on the platelet surface and through intracellular signaling mechanisms that remain systematically undefined. This study brings together cell physiological and phosphoproteomics methods to profile signaling mechanisms downstream of the immunotyrosine activation motif (ITAM) platelet collagen receptor GPVI. Peptide tandem mass tag (TMT) labeling, sample multiplexing, synchronous precursor selection (SPS), and triple stage tandem mass spectrometry (MS3) detected >3000 significant (false discovery rate < 0.05) phosphorylation events on >1300 proteins over conditions initiating and progressing GPVI-mediated platelet activation. With literature-guided causal inference tools, >300 site-specific signaling relations were mapped from phosphoproteomics data among key and emerging GPVI effectors (ie, FcRg, Syk, PLCg2, PKCd, DAPP1). Through signaling validation studies and functional screening, other less-characterized targets were also considered within the context of GPVI/ITAM pathways, including Ras/MAPK axis proteins (ie, KSR1, SOS1, STAT1, Hsp27). Highly regulated GPVI/ITAM targets out of context of curated knowledge were also illuminated, including a system of >40 Rab GTPases and associated regulatory proteins, where GPVI-mediated Rab7 S72 phosphorylation and endolysosomal maturation were blocked by TAK1 inhibition. In addition to serving as a model for generating and testing hypotheses from omics datasets, this study puts forth a means to identify hemostatic effectors, biomarkers, and therapeutic targets relevant to thrombosis, vascular inflammation, and other platelet-associated disease states.
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U2 - 10.1182/BLOOD.2020005496
DO - 10.1182/BLOOD.2020005496
M3 - Article
C2 - 32640021
AN - SCOPUS:85092452802
SN - 0006-4971
VL - 136
SP - 2346
EP - 2358
JO - Blood
JF - Blood
IS - 20
ER -