Philadelphia-negative myeloproliferative neoplasms (MPN), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (MF), are hematological malignancies driven by gain-of-function mutations in the JAK2, CALR, and MPL genes. Chronic inflammation significantly contributes to MPN pathogenesis and cardiovascular disorders such as thrombosis, stroke, and bleeding, which remain the main cause of patients’ morbidity and mortality. Although thrombocytosis is a common feature of MPN, platelet count alone does not fully explain or predict thrombotic burden in MPN. Several evidence suggest that altered surface markers expression and metabolic reprogramming may exert a more impactful role in MPN-associated thromboinflammation. Therefore, we hypothesized that immunophenotypic, transcriptomic, and metabolic alterations in platelets may contribute to a thromboinflammatory state in MPN.

To characterize platelet immunophenotype, gene signature, and bioactive lipids profile in patients with PV, ET, and MF.

Peripheral blood was collected from PV (n = 25), ET (n = 15), MF (n = 20) patients, and controls (n = 25). Platelet-rich plasma (PRP) was isolated and supplemented with prostaglandin E1 to prevent autonomous activation. Platelet activation markers (CD41, CD62P, CD36, CD63, CD154) were analyzed by flow cytometry at baseline and after stimulation with 0.5 µM calcium ionophore (A23187) or 50 U/mL thrombin, and control platelets were incubated with 10% MPN plasma to assess inflammatory activation. Gene Set Enrichment Analysis (GSEA) was applied to a public dataset (GSE26049) to assess platelet transcriptome. For metabolomics, purified platelets were stimulated with A23187 for eicosanoids detection or left at baseline for sphingolipids quantification, then submitted to high-resolution liquid chromatography–mass spectrometry (HRLC-MS).

MPN platelets showed increased CD62P, CD36, CD63, CD154 and reduced CD41 expression at baseline, indicating a pre-activated state. Thrombin responses were reduced in MPN platelets, and exposure to MPN plasma increased platelet activation. GSEA revealed downregulation of genes involved in cytoskeletal regulation, integrin signaling, and vesicular trafficking (eg.: ZYX, FLNA, ACTN4, GNAI2, HGS, PFN1). The downregulated genes were more prominent in MF and less in PV. In eicosanoid analysis, PV platelets showed increased levels of prostaglandins and oxidized lipids (PGE₁, 15-HETE); ET presented moderate increase in 12-HHTrE and PGF₂α; and MF presented dysregulation in TXB₂, long-chain fatty acids, polyamines, and amino acid–related metabolites. Sphingolipid analysis revealed reduced glucosylceramides (GlcCer C18:0, C24:1) and ceramide-1-phosphate (Cer1P) in PV; increased dihydroceramides (DHCer C18:0) and reduced long-chain sphingomyelins (SM C24:1, C22:1) in MF, and moderate alterations in long-chain sphingomyelins (SM C24:0) in ET.

Platelets are intrinsically activated, transcriptionally altered, and metabolically dysregulated in MPN, fueling a sustained thromboinflammatory state in PV, ET, and MF. This profile persists despite the standard treatments and suggests that immunometabolic alterations in platelets directly contribute to the paradox of thrombosis and bleeding in MPN. Altogether, these observations reveal that platelets can offer new potential therapeutic targets to effectively treat MPN-associated thromboinflammatory complications.

The study was financed by FAPESP (2022/13366-2) and CNPq.