The silent saboteurs: reticulated platelets in coronary artery disease

This editorial refers to ‘Reticulated platelets in coronary artery disease: a multidimensional approach unveils prothrombotic signalling and novel therapeutic targets’, by K. Kirmes et al., https://doi.org/10.1093/eurheartj/ehaf694.

Chronic coronary artery disease (CAD) is a leading cause of cardiovascular morbidity and mortality worldwide. While antiplatelet therapy has improved outcomes, recurrent thrombotic events persist in a subset of patients in spite of antiplatelet therapy. An underexplored area of investigation is understanding and potentially targeting reticulated platelets (RPs), which are immature platelets enriched in RNA and believed to have a lower threshold for activation and precipitation of thrombosis in vivo. In this issue of the European Heart Journal, Kirmes et al. provide functional and molecular characterization of RPs in patients with CAD.¹ The findings suggest that RPs may represent a high-risk subpopulation of platelets insensitive to conventional antiplatelet therapy, thereby contributing to adverse thrombotic events. This investigation, for the first time, highlights RPs as a potential antithrombotic target in the treatment of CAD, especially for patients who display high residual platelet reactivity despite guideline-recommended therapy.

Using a multidimensional approach combining transcriptomics, high-dimensional proteomics (mass cytometry), flow-cytometry-based assays to determine platelet reactivity, phospho-proteomics, and in vitro models of thrombus formation, the authors demonstrate that RPs are distinct prothrombotic entities beyond our understanding of adult platelet biology. Notably, RPs have persistent activation of the surface glycoprotein VI (GPVI) receptor which is activated in response to collagen as well as external mechanical forces (mechano-transduction). In this study, GPVI is shown to promote activation of the phosphoinositide 3-kinase (PI3K) signal transduction pathway in RPs, even in patients prescribed dual antiplatelet therapy (DAPT) consisting of aspirin and a P2Y₁₂ receptor antagonist.

Methodological rigour and novel insights

Rreticulated platelets are not as abundant as mature platelets and are therefore more challenging to study. A strength of the study is the integration of ‘omics and RP function in the same donor population, enabling intra-individual comparisons between RPs and mature platelets (MPs) which avoid heterogeneity common to investigations in platelet biology. The authors employ several strategies to determine the thrombotic potential of RPs and engagement of RPs in inflammatory pathways through a rigorous RP selection pipeline involving leucocyte exclusion using CD41⁺/DNA⁻ gating for transcriptomics, and CD45⁻ selection by cytometry by time-of-flight (CyTOF) to ensure platelet integrity, consistent with acceptable research practice recommended by the International Society on Thrombosis and Haemostasis (ISTH).² Their analyses reveal up-regulation of signalling through the RP GPVI receptor, the thromboxane A₂ receptor (TBxA2R), the protease activated receptor 1 (PAR1), and the discovery of novel non-coding RNAs (including circular RNAs) in RPs. CyTOF was used to demonstrate phosphorylation of PI3K, Akt, and SYK.

The authors revealed that RPs are more reactive than mature platelets using a comprehensive constellation of assays. Beyond conventional surface activation markers, they employ a fluorescence-activated cell sorting (FACS)-based system that capitalizes on the sensitivity of this technique to characterize low abundance RPs and assess interactions between RPs and mature platelets that would occur during thrombus formation. This study represents a meaningful advancement over traditional FACS characterization of platelet reactivity that relies only on alpha granule and dense granule exocytosis as an indirect surrogate of platelet reactivity. By enabling the visualization of interactions between two distinct platelet populations, the method offers a more physiologically relevant perspective. While light transmission aggregometry (LTA) remains the most dependable ex vivo assay to determine platelet behaviour expected in an artery during acute thrombosis, this approach nonetheless provides complementary mechanistic insight at the cellular level. Moreover, targeted inhibition with Glenzocimab, an anti-GPVI Fab (a monoclonal antibody fragment targeting GPVI), and LY294002 (a PI3K inhibitor), significantly attenuates RP reactivity, providing pharmacological validation in a separate cohort of patients (Graphical Abstract).

Graphical Abstract.

Reticulated platelets (RPs) are prothrombotic in coronary artery disease, distinct drivers of thromboinflammation, and may be targets for precision antiplatelet therapy. RPs exhibit elevated GPVI-PI3K signaling that are attenuated by Glenzocimab and LY294002. Experimental techniques to evaluate platelet function include Fluorescence-Assisted Flow Sorting (FACS) and will require murine models in future for mechanistic insight.

Critical considerations and translational gaps

This study defines the presence of RPs based solely on RNA content using established protocols. However, since mature platelets are also enriched in RNA, additional markers are necessary to confirm the identity of RPs and distinguish them from mature platelets. The mechanistic connection between GPVI–PI3K signaling in CAD deserves special attention. GPVI serves as a mechanosensor in platelets and can be activated under conditions of shear stress which is common in obstructive CAD in which disturbed blood flow (d-flow) occurs.³ Supporting the current findings, PI3K activation was recently shown be important for d-flow-mediated activation of human platelets.⁴ D-flow is also observed in arterial aneurysmal disease and during blood passage through stenotic heart valves. The GPVI–PI3K module therefore represents a relevant mechanism linking vessel wall pathology to platelet hyper-reactivity. The broader physiological impact of RP heterogeneity on thrombosis is less clear given that platelets act as a population. This question is difficult to address in ex vivo assays alone in which platelets are deprived access to interactions with erythrocytes and leucocytes as well circulating mediators and mechanical forces in blood that also impact thrombosis. Future studies in flow chambers that model mechanical forces in coronary arteries or murine models of atherothrombosis are needed to progress therapeutics.

Clinical implications and therapeutic potential

From a clinical perspective, the study reinforces the notion that RPs are not merely immature platelets but represent a functionally primed platelet subpopulation with distinct signalling behaviour and potential for targeted intervention. Recent data, including pre-specified analyses of the ISAR-REACT 5 trial, have associated elevated RPs with increased risk in patients for the composite primary endpoint death, myocardial infarction, or stroke at 1 year despite optimal antiplatelet therapy.⁵ Kirmes et al. now provide mechanistic explanations for this association. While the current study demonstrates promising ex vivo results with the GPVI antagonist Glenzocimab, previous clinical trials of GPVI antagonists have shown mixed results. The ISAR-PLASTER phase II trial of Revacept, a competitive GPVI antagonist, failed to demonstrate clinical benefit in patients with obstructive CAD undergoing elective percutaneous coronary intervention.⁶ Notably, Revacept functions by binding to collagen and preventing its interaction with GPVI, rather than directly blocking GPVI, a mechanism that requires in vivo conditions that cannot be fully recapitulated ex vivo. This disconnect between pre-clinical promise and clinical need highlights the complexity of translating platelet biology into therapeutic wins. The downstream PI3K pathway may offer more promising therapeutic opportunities since the PI3K antagonist LY294002 inhibits RP reactivity across multiple functional assays. However, the challenge lies in achieving platelet-specific inhibition without compromising other cellular functions dependent on the mitogenic requirement of the PI3K pathway.

Considerations and future directions

The study introduces several methodological strengths that enhance the reliability and depth of the findings. The authors employed mass cytometry to perform single-cell phospho-protein profiling and developed a FACS-based platelet interaction assay. This approach addresses the inherent challenges of working with low-abundance platelet subpopulations such as RPs, and overcomes technical limitations posed by conventional methodologies. A key strength lies in the within-subject comparison design, enabling direct evaluation of reticulated platelets vs mature platelets from the same individual. This strategy minimizes the confounding effects of demographic or clinical variability, such as age, comorbidities, or medication use, all of which are common in coronary artery disease populations and known to influence platelet function. As a result, the differences observed probably reflect true biological distinctions between reticulated and mature platelet subsets. The use of FACS for phosphoprotein analysis and platelet reactivity assessment, while innovative, represents a departure from established gold-standard methodologies. Additionally, the study still relies on surface marker expression as surrogates for platelet activation and may not fully capture the complex functional behaviour observed in vivo.

Unaddressed limitations and future research needs

Several important limitations that may impact the interpretation of these findings warrant discussion. The study population and clinical characteristics stratified by reticulated vs mature platelet groups will account for intrinsic differences between platelets and RPs, but a very high population of patients enrolled in this study—peripheral artery disease (21%), diabetes (32%), obesity (22%)—are patients well-known to have non-standard responses to antiplatelet therapy and platelet surface agonists. The present study cannot separate the impact of these diseases on RP and platelet function in patients with CAD without these diseases. The potential for white blood cell contamination in RNA sequencing experiments, despite the authors’ gating strategies, remains a concern that could significantly impact transcriptomic results. Although the authors applied CD41+/CD45−/DNA− gating and included scatter plots to minimize contamination, the possibility of residual leucocyte RNA remains. ISTH consensus recommendations emphasize additional purification strategies such as magnetic depletion or leucocyte RNA quantification to improve transcriptomic specificity in platelet-rich preparations.²

Translational opportunities and precision medicine

Developing point-of-care assays capable of quantifying RPs could enhance risk stratification and enable personalized antiplatelet therapy in coronary artery disease—a field where biomarker-guided treatment remains a significant unmet need. The identification of novel GPVI splice variants and circular RNA signatures by the authors opens up promising avenues for biomarker development and mechanistic exploration. Since platelet behaviour in vivo represents a ‘population average’, understanding how hyper-reactive reticulated platelets influence overall thrombotic risk within the context of less reactive mature platelets will be essential for therapeutic development.

Conclusion

As platelet biology continues to evolve, integrating platelet heterogeneity into treatment paradigms will be essential. This impressive study lays the groundwork for future investigations that move from mechanistic insight to therapeutic intervention, while highlighting the need for biomarker-driven patient selection in the quest to reduce thrombotic events without increasing bleeding risk. The integration of transcriptomic, proteomic, and functional data in low-yield platelet subpopulations such as RPs demonstrates the feasibility and the challenge of mechanistically grounded platelet phenotyping in cardiovascular disease.

Declarations

Disclosure of Interest

All authors declare no disclosure of interest for this contribution.

Funding

National Heart, Blood, and Lung Institute grant no. R01HL158801 to S.J.C.