Platelet Proteomes, Pathways, and Phenotypes as Informants of Vascular Wellness and Disease

Abstract

Platelets rapidly undergo responsive transitions in form and function to repair vascular endothelium and mediate hemostasis. In contrast, heterogeneous platelet subpopulations with a range of primed or refractory phenotypes gradually arise in chronic inflammatory and other conditions in a manner that may indicate or support disease. Qualitatively distinguishable platelet phenotypes are increasingly associated with a variety of physiological and pathological circumstances; however, the origins and significance of platelet phenotypic variation remain unclear and conceptually vague. As changes in platelet function in disease exhibit many similarities to platelets following the activation of platelet agonist receptors, the intracellular responses of platelets common to hemostasis and inflammation may provide insights to the molecular basis of platelet phenotype. Here, we review concepts around how protein-level relations-from platelet receptors through intracellular signaling events-may help to define platelet phenotypes in inflammation, immune responses, aging, and other conditions. We further discuss how representing systems-wide platelet proteomics data profiles as circuit-like networks of causally related intracellular events, or, pathway maps, may inform molecular definitions of platelet phenotype. In addition to offering insights into platelets as druggable targets, maps of causally arranged intracellular relations underlying platelet function can also advance precision and interceptive medicine efforts by leveraging platelets as accessible, dynamic, endogenous, circulating biomarkers of vascular wellness and disease. Graphic Abstract: A graphic abstract is available for this article.

Keywords: hemostasis; immunity; inflammation; proteomics; vascular endothelium.

Figure 1.. Platelet phenotype and function in endothelial injury (i.e., hemostasis) and dysfunction (i.e., inflammation).

Upper. Upon encountering cues of endothelial injury (i.e., VWF, subendothelial collagen), circulating platelets rapidly respond through adhesion, secretion (i.e., ADP, granules), thromboxane generation (TXA2) and aggregation to mediate hemostasis and prevent vessel leakage. Lower. Platelets can also more transiently interact with dysfunctional endothelium, where factors associated with chronic conditions may give rise to more heterogeneous “inflamed” platelet subpopulations (shaded in red) to progress vascular inflammation, further fueling platelet secretion (i.e., cytokines, granules), platelet-leukocyte aggregate formation and other responses driving disease.

Figure 2.. Platelet ligands, receptors and intracellular signaling pathways determine platelet function and phenotype.

A variety of hemostatic (i.e., VWF, collagen) and inflammatory (i.e., IL-6, lipopolysaccharide) agonists and other ligands (i.e., thrombopoietin TPO) engage a number of different receptors expressed on platelets to result in different platelet responses and platelet phenotypes. Following ligand binding and initial receptor responses, diverging signaling pathways globally reorganize platelet intracellular protein modifications and relations (“platelet proteotype”) to solicit specific platelet responses in physiological context (i.e., shape change, secretion). Specific signaling events downstream of platelet receptors are mediated by tyrosine kinases, phospholipases (PLC), protein kinase C (PKC), adenylyl cyclase, protein kinase A (PKA), phosphodiesterases (PDE), phosphoinositide 3-kinase (PI3K), Akt, mitogen activated protein kinases (MAPKs), Jak/STAT, IKK/NF-κB, and, potentially thousands of other distinct effectors. How these signals and their systemic, intracellular consequences globally determine platelet phenotype and function remains to be elucidated.

Figure 3.. From platelet phenotype to (phospho)proteome and pathway map.

Example workflow highlighting steps from blood collection and platelet preparation, through quantitative mass spectrometry (MS) and sample analysis and pathway map generation. First, blood is drawn from a set of control (i.e., “healthy”) and/or experimental subjects to prepare platelets. After isolation from whole blood, purified platelets may be kept in resting state, or stimulated with specific agonists (i.e., −/+ CRP-XL to activate GPVI). Following sample lysis and tryptic digestion to prepare peptides, phosphopeptides are enriched with reagents such as TiO₂. Next, each sample of enriched phosphopeptides is labeled with a specific tandem mass tag (TMT) label that serves as a “bar code” to inform the mass spectrometer which sample each peptide corresponds to. Finally, all TMT-labeled samples are mixed together for multiplexed, quantitative mass spectrometry analysis. Quantitative data and statistics of thousands of phosphopeptide reporter ion intensity measurements between samples are analyzed with CausalPath to note causal relations in each dataset (“phosphorylation of protein A at site X causes phosphorylation of protein B at site Y…”). Example #1 shows a subset of causal relations associated with platelet GPVI activation in vitro, where specific changes downstream of PKA (PRKACA) activity are highlighted. Example #2 illustrates some PI3K/Akt pathways that may be associated with disease when platelets are analyzed ex vivo from obese, elderly or other subjects with upregulated vascular inflammation relative to healthy control subjects.