Platelet proteomics emerges from the womb: mass spectrometry insights into neonatal platelet biology

Platelet function fluctuates over the human lifespan in concert with the hemostatic demands of the developing and aging vascular system [1]. Notably, in the earliest days of life, platelets are generally less reactive than in adulthood, and differences in neonatal and adult platelet physiology have many important clinical implications [2, 3]. For instance, thrombocytopenia is common in pre-term infants, where the transfusion of adult-derived platelets can lead to complications such as bleeding, inflammation, and clotting issues [4]. While several physiological differences between neonatal and adult platelets have been described, little is known about whether and how the molecular composition of platelets – namely the complete set of platelet proteins, or platelet proteome – determines or is reflective of platelet function. In this issue of the Journal of Thrombosis and Haemostasis, Thom et al. [5] use quantitative mass spectrometry methods to compare the proteomes of neonatal platelets from cord blood relative to platelets from adults. Ultimately, their study uncovers distinctive proteomic signatures that differentiate neonatal and adult platelets, shedding light on different platelet reactivities between these groups. Beyond providing mechanistic and translational insights, this research also represents a leap in the technical and theoretical realms of what platelet proteomics can offer to the hemostasis and thrombosis research community [6–8].

Over the past several decades, laboratory and clinical studies have delineated how platelet reactivities differ across various life stages – from neonates to the elderly [9]. Despite gross morphological similarities amongst platelets throughout these stages, biochemical and cell biological methods have uncovered significant differences. Techniques like flow cytometry have identified variations in the expression of platelet surface receptors and ligands such as P-selectin [3]. High-throughput studies of platelet RNA transcriptomes have also hinted at the molecular bases of these differences in neonatal platelet function [10, 11], but comprehensive proteomic analyses of platelets from infants have remained limited or elusive [12, 13].

To expand proteomic details for neonatal as well as adult platelets, the Sola-Visner team takes advantage of data independent acquisition (DIA) methods to analyze platelet proteomes with quantitative mass spectrometry (MS) [14, 15]. Consequently, their study not only offers new physiological and clinical insights, but also serves as an inaugural publication of a DIA-MS analysis of human platelet proteomes. Previous mass spectrometry studies of platelet proteomes have typically relied on data dependent acquisition (DDA) methods, which, while precise in identifying and quantifying peptides, can overlook numerous potential peptide and protein identifications [14, 15]. Accordingly, the platelet proteomics community has been eagerly anticipating DIA-MS studies of platelets to ascertain whether proteins, previously undetected by DDA yet hypothesized to be present in platelets, would be revealed through DIA [16, 17]. Indeed, this study’s application of DIA greatly expands the list of proteins now known to be present in neonatal platelets and opens the door to a more comprehensive understanding of platelet protein composition in general.

Ultimately, well over 4,000 different proteins are measured and compared across all samples in this study, shedding light on potential physiological differences between adult and neonatal platelets. Adult platelets were found to contain higher levels of several immunological and proinflammatory proteins, including CXCL2 (SDF1), PDGFB, β2M and TGFβ2, as well as complement systems proteins, suggesting that adult platelets may have the capacity to incite more inflammation than neonatal platelets. In general, neonatal platelets expressed higher levels of several metabolic enzymes and ribosomal protein components. Crucially, many of these findings from MS analysis are corroborated by Western blot or ELISA experiments verifying protein content. These results align with both classical and contemporary studies of neonatal transfusion, including recent research in animal models that indicates transfusions with adult platelets could increase inflammatory complications [18].

Additionally, by combining advanced phosphopeptide enrichment strategies together with DIA methodologies, this study also documents more than 13,000 unique phosphorylation sites in human platelets, likely setting a new record for the most phosphorylation sites ever identified in a single study of the platelet phosphoproteome. Earlier studies using peptide isobaric labeling (e.g., iTRAQ, TMT) and mass spectrometry have quantified between 500 and 3,000 phosphorylation sites in platelets under agonist-stimulated conditions [19, 20]. While these previous studies have their strengths, particularly in quantitative analysis, the sheer volume of phosphorylation sites discovered here with DIA is notable. Many of these newly identified phosphorylation sites are located on proteins that are already known to be phosphorylated on different residues, or on proteins within known signaling pathways that are subject to tight regulation by reversible protein phosphorylation (e.g., Rho GTPase regulatory proteins).

However, despite the substantial number of phosphorylation sites identified and quantified, certain key elements of platelet signaling pathways remain elusive in this DIA workflow. For instance, many tyrosine-phosphorylated residues with critical roles in platelet regulation (e.g., PLCG2 Y₁₂₁₇) do not appear to be readily captured by this method, and likely require specific enrichment strategies for detection under agonist-stimulated conditions. Furthermore, while the research provides evidence of increased mTOR signaling in neonatal platelets associated with growth factor signaling and protein translation, and heightened MAP kinase signaling in adult platelets, the physiological underpinnings of these signaling discrepancies are not yet fully understood. Factors such as the plasma environment [21], blood flow shear forces [22], hematopoiesis and developmental stages [23] are all potential influencers of neonatal and adult platelet phenotype and functionality.

Overall, this collaborative study led by Dr. Davenport and Dr. Thom delivers an extensive set of data that platelet biologists and neonatologists will find valuable when approaching platelet function from a systems biology standpoint. The insights provided in this study pave the way for future research, but specifics on actions and recommendations regarding platelet transfusion practices in neonates still call for further investigation. In terms of mechanism, the study sheds light on the inner workings of platelet homeostasis and activation, yet the pathways and mechanisms underlying remain to be explored. Looking forward, advances in isobaric labeling [20, 24], analysis of protein complexes [25, 26] , and high-throughput imaging [27, 28] are promising directions for forthcoming research into both neonatal and adult platelet phenotype and functionality.

As the platelet omics field progresses, more and more protein and other molecular features are continually identified in platelet samples, yet the translation of these findings into biological understanding and clinical application still remains a challenge [6–8]. Nonetheless, this study showcases how mass spectrometry tools can not only find more proteins and phosphorylation sites in platelets, but also verify and generate translationally relevant hypotheses through a combination of exploratory and high-throughput, replicated experimentation. Altogether, this work marks a significant milestone in the progression of proteomics and systems biology studies of platelets, highlighting the potential and practicality of these tools in understanding platelet biology.