Reticulated platelets: unveiling molecular drivers of coronary syndromes

Abstract Background Cardiovascular disease (CVD) spans a continuum from chronic coronary syndrome (CCS) to acute coronary syndrome (ACS). Platelets play distinct roles across this spectrum, contributing to vascular inflammation in CCS and driving occlusive thrombosis in ACS. Reticulated platelets (RPs), chracterised by high RNA content and enhanced reactivity, have emerged as key determinants of thrombogenic potential. Elevated RP levels correlate with impaired antiplatelet response and increased risk of thrombosis, yet their molecular phenotype across CCS and ACS remains incompletely characterised. Purpose To compare transcriptomic and proteomic profiles of RPs and mature platelets (MPs) in healthy, CCS, and ACS patients, focusing on disease-specific alterations in platelet subpopulations. Methods Peripheral blood was collected from healthy, CCS, and ACS patients. Platelet subpopulations were analysed using bulk RNA sequencing and mass cytometry (CyTOF). Differential gene expression, gene set enrichment analysis (GSEA), and protein profiling were performed. Reactivity assay and measurement of S100A8 expression were done using CyTOF and flow cytometry, respectively. Platelet-leukocyte aggregates (PLAs) were evaluated by CyTOF. Results Although GP6 was consistently higher in RPs than in MPs across all patient groups, transcriptomic analysis showed no significant difference in GP6 expression between healthy, ACS, and CCS patients. (Fig. 1A). On the contrary, we observed disease-specific upregulation of S100A8 and TLR4 in ACS patients compared with CCS and healthy (Fig. 1B). GSEA analysis indicated enhanced MHC I protein complex assembly pathways in ACS compared with CCS group (Fig. 1C). Proteomic profiling demonstrated increased GPVI and CD62P expression in ACS RPs compared with CCS RPs, with concomitant decreased activation of αIIbβ3 (recognized by PAC1 antibody, Fig. 1D). RPs exhibited higher S100A8 levels than MPs across CCS and ACS patients on protein level (Fig. 1E). Following TRAP stimulation, RPs from CCS patients showed activation levels comparable to baseline ACS RPs, as evidenced by CD62P expression. However, RPs from ACS patients did not significantly respond to TRAP-induced activation, probably due to platelets exhaustion as a result of chronic activation during ACS (Fig. 1F). PLA analysis revealed reduced platelet-CD8+ T cell aggregates in ACS (Fig. 1G), which may suggest an immune-escaping mechanism through increased MHC I expression on platelets against cytotoxicity by CD8+ T cells in ACS patients. Conclusion RPs display distinct molecular signatures across CCS and ACS, reflecting their role in thrombo-inflammatory processes. ACS is marked by inflammatory mediator upregulation and immune-modulatory adaptations, while CCS RPs maintain activation potential (Fig. 1H). These findings highlight RPs profiling as a biomarker for platelet activation and antiplatelet resistance, offering insights into therapeutic targets for coronary syndromes.For image description, please refer to the figure legend and surrounding text.