Phosphoproteomic quantitation and causal analysis reveal pathways in GPVI/ITAM-mediated platelet activation programs

Abstract

<jats:p>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 cell surface, and through intracellular signaling mechanisms that remain systematically undefined. This study brings together cell physiological and phosphoproteomics methods incorporating peptide tandem mass tag (TMT) labeling, sample multiplexing, synchronous precursor selection (SPS) and triple stage tandem mass spectrometry (MS3) to profile signaling mechanisms downstream of the immunotyrosine activation motif (ITAM) platelet collagen receptor GPVI. Altogether, &amp;gt;3,000 significant (FDR&amp;lt;0.05) phosphorylation events on &amp;gt;1,300 proteins were detected in initial and progressing conditions of GPVI-mediated platelet activation. With literature-guided causal inference tools, &amp;gt;300 site-specific signaling relations were mapped from phosphoproteomics data among key and emerging GPVI effectors (i.e., 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 (i.e., KSR1, SOS1, STAT1, Hsp27). Networks highly regulated in GPVI/ITAM signaling out of context of curated knowledge were also illuminated, including a system of &amp;gt;40 Rab GTPases and associated regulatory proteins - where TAK1-mediated Rab7 S72 phosphorylation associated with endolysosomal maturation and GPVI-mediated platelet function. 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.</jats:p>

Fine-Tuning of Platelet Responses by Serine/Threonine Protein Kinases and Phosphatases—Just the Beginning

“…However, their targeted pathways need to be understood and assessed with valid assays. Such emerging strategies 65,98,99 are promising for the individualized screening of platelet-related diseases and drugs. Human platelets can serve here as prominent cellular model, but also as therapeutic and diagnostic target.…”

Bleeding diathesis in mice lacking JAK2 in platelets

“…Phosphoproteomics-based studies have identified JAK2 tyrosine phosphorylation downstream of both GPVI and CLEC-2. 44,45 In addition, Mpl/JAK2 signaling may also contribute to the regulation of platelet hemostatic function in vivo, as TPO potentiates GPVI signaling through a phosphatidylinositol 3-kinase-dependent pathway in human platelets. 46,47 Potentiation of platelet GPVI signaling by TPO is lost in platelets of JAK2 V617F patients.…”

Infrastructure‐scale sustainable energy planning in the cityscape: Transforming urban energy metabolism in East Asia

“…A PV system of this scale could support 30% of annual and about 67% of daylight electricity needs in the city (Byrne et al, 2015). Closer evaluation of the Gangnam district in Seoul finds that the area could be home to almost 5 million m 2 of suitable rooftop space across its 27,000 buildings (Hong et al, 2017;Lee, Hong, Jeong, et al, 2018a;Lee, Hong, Jeong, et al, 2018b). An evaluation of 710 projects of the Korean Local Energy Savings Program (2012Program ( -2014 likewise found that cities in Korea could expect energy savings ranging from 18 to 45% with an average 30.5% savings through cost-effective implementation of various energy efficiency interventions (Yoon et al, 2017).…”