Individuals with type 1 diabetes are at increased cardiovascular risk, particularly in the presence of insulin resistance. A prothrombotic environment is believed to contribute to this risk but thrombotic pathways in type 1 diabetes are only partially understood and the role of platelets is incompletely studied. We hypothesised that platelets from individuals with type 1 diabetes exhibit platelet hyperactivity due to both increased propensity for activation and diminished sensitivity to inhibition, with an amplified maladaptive phenotype in those with insulin resistance.
Blood samples were obtained from individuals with type 1 diabetes enrolled on the 'Double diabEtes and adVErse cLinical Outcome: identification of mechanistic Pathways' (DEVELOP) study with insulin resistance assessed as estimated glucose disposal rate (eGDR), whereby eGDR >8 or <6 mg kg-1 min-1 indicates normal insulin sensitivity or advanced insulin resistance, respectively. Platelet function was analysed using whole blood multiparameter flow cytometry to simultaneously measure three distinct markers of activation, including integrin αIIbβ3 (PAC-1 binding), P-selectin (CD62P) and phosphatidylserine (PS) (Annexin V). Both activation and inhibition responses of the platelets were investigated, which were subjected to the machine learning tool Full Annotation Shape-constrained Trees (FAUST) to characterise platelet subpopulations.
A total of 32 individuals with type 1 diabetes were studied (median age [range] of 24 [18-34] years, 59% male, diabetes duration [mean ± SD] of 14.0 ± 6.3 years and HbA1c of 65.3 ± 14.0 mmol/mol [8.1%]). An increased basal expression, measured as mean fluorescence intensity, of all three platelet activation markers was detected in the type 1 diabetes group compared with healthy control participants (CD62P expression 521 ± 246 vs 335 ± 67; p<0.001, PAC-1 370 ± 165 vs 231 ± 88; p=0.011 and PS 869 ± 762 vs 294 ± 109; p=0.001). Following platelet stimulation, an enhanced activation of these markers was found in the type 1 diabetes group. Within the type 1 diabetes group, those with advanced insulin resistance (eGDR<6 mg kg-1 min-1) showed increased platelet activation compared with individuals with normal insulin sensitivity (eGDR>8 mg kg-1 min-1) with single agonist stimulation CD62P expression (29,167 ± 2177 vs 22,829 ± 2535, p<0.001 and PAC-1 19,339 ± 11,749 and 5187 ± 2872, p=0.02). Moreover, individuals with type 1 diabetes showed reduced sensitivity to platelet inhibition by prostacyclin (PGI2) compared with control participants. Stratification of individuals with type 1 diabetes by insulin resistance demonstrated that in the presence of PGI2, suppression of stimulated CD62P was 17 ± 11% and 33 ± 12% (p=0.02) for advanced insulin resistance and normal insulin sensitivity groups, respectively, with even larger differences demonstrated for PAC-1 (48 ± 17% and 75 ± 7%; p=0.006) and PS exposure (33 ± 12% and 84 ± 10%; p=0.001). Furthermore, FAUST analysis showed that, under basal conditions, there was a different distribution of the eight platelet subpopulations comparing advanced insulin resistance and normal insulin sensitivity groups, with differences also detected following PGI2 inhibition.
Our novel characterisation of platelets in type 1 diabetes shows a maladaptive phenotype with increased basal activity together with hyperactivation following stimulation and diminished responses to inhibition. Insulin resistance appears to further drive this adverse thrombotic phenotype, suggesting an enhanced platelet-driven cardiovascular risk in those with type 1 diabetes and reduced insulin sensitivity.
© 2025. The Author(s).

Platelets, known for maintaining blood balance, also participate in antimicrobial defense. Upon severeacute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, platelets become hyperactivated, releasing molecules such as cytokines, granule contents, and bioactive lipids. The key effector biolipids produced by platelets include 12-hydroxyeicosatetraenoic acid (12-HETE) and 12-hydroxyeicosatrienoic acid (12-HETrE), produced by 12-lipoxygenase (12-LOX), and prostaglandins and thromboxane, produced by cyclooxygenase-1. While prostaglandin E2 and thromboxane B2 were previously associated with lung inflammation in severe COVID-19, the role of platelet 12-LOX in SARS-CoV-2 infection remains unclear. Using mice deficient for platelets' 12-LOX, we report that SARS-CoV-2 infection resulted in higher lung inflammation characterized by histopathological tissue analysis, increased leukocyte infiltrates, and cytokine production relative to wild-type mice. In addition, distinct platelet and lung transcriptomic changes, including alterations in NOD-like receptor (NLR) family pyrin domain-containing 1 (NLRP1) inflammasome-related gene expression, were observed. Mass spectrometry lipidomic analysis in 12-LOX-deficient-infected mice revealed significant changes in bioactive lipid content, including reduced levels of 12-HETrE that inversely correlated with disease severity. Finally, platelet 12-LOX deficiency was associated with increased morbidity and lower survival rates relative to wild type (WT) mice. Overall, this study highlights the complex interplay between 12-LOX-related lipid metabolism and inflammatory responses during SARS-CoV-2 infection. The findings provide valuable insights into potential therapeutic targets aimed at mitigating severe outcomes, emphasizing the pivotal role of platelet enzymes in the host response to viral infections.

Platelet-cancer cell interactions play a significant role in metastasis. Indeed, they interact via a plethora of receptors, including integrins (e.g. ⍺IIbβ3 and ⍺vβ3), and calcium is essential for both their stability and function. Additionally, calcium plays a significant role in the coagulation cascade, and the implication of calcium level changes on metastatic dissemination and cancer-associated thrombosis are not fully understood. A significant proportion of cancer patients suffer from hypercalcemia and have a worse prognosis. We hypothesized that calcium levels are important for platelet-cancer cell interactions that are mediated via integrins, thus this can be leveraged to disrupt platelet support to the metastatic process. In this study, we assessed the detection of integrins ⍺IIbβ3 and ⍺vβ3 on platelets and cancer cells, platelet function, and the respective receptors implicated in platelet function, while modulating calcium levels. The effect of calcium levels on platelet-cancer cell interactions and cancer cell invasion in vitro was also assessed. Our data demonstrates that calcium levels affect surface integrins, and receptors involved in platelet-cancer cell interactions. In addition, calcium levels significantly affect platelet activation and aggregation. In our experimental scenarios, calcium depletion modulates platelet-cancer cell interaction with MDA-MB-231 breast cancer cells, while hypercalcemic environments did not affect interaction. Meanwhile, hypercalcemia leads to enhanced cancer cell invasion for both MDA-MB-231 and A549 cells in the presence of platelets. Thus, this study provides a greater understanding of the dynamics associated with the effects of calcium and platelet-cancer cell interactions mediated by integrins.
© 2025. The Author(s).

Iloprost, a prostacyclin (PGI2) analogue, stimulates the IP receptor (PTGIR) to interact with the Gsα β/γ complex, leading to the activation of adenylate cyclase, which enzyme produces the second messenger cAMP. Elevation in cAMP triggers intracellular signalling events and regulates a wide variety of cellular activities. Thus, we evaluated the effects of Iloprost on platelet function and apoptosis and in vivo haemostasis and thrombosis, as well as the underlying mechanisms. Firstly, we showed that Iloprost concentration-dependently inhibited agonist-induced P-selectin exposure, integrin αIIbβ3 activation, platelet aggregation, ATP release, platelet spreading, and clot retraction. Moreover, Iloprost dose-dependently inhibited FeCl3-induced mouse mesenteric arteriole thrombosis and markedly prolonged the tail bleeding time. Iloprost also concentration-dependently inhibited mitochondrial membrane potential (ΔΨm) depolarisation and phosphatidylserine (PS) externalisation in platelets, thereby inhibiting platelet apoptosis, and Iloprost at concentrations lower than 2 nM inhibited only platelet apoptosis but not platelet function. Importantly, Iloprost at low doses markedly elevated peripheral platelet counts in GPIbα antibody-induced immune thrombocytopenia (ITP). Mechanistic studies showed that Iloprost concentration-dependently antagonised agonist-induced decline of protein kinase A (PKA) activity and elevation of cytoplasmic Ca2+ in platelets, thereby attenuating platelet activation and aggregation. Elevation in PKA activity inhibited dephosphorylation of proapoptotic protein BAD and reduced caspase-3 activity, thus retarding platelet apoptosis. These data demonstrate that Iloprost dose-dependently inhibits platelet function and apoptosis by elevating PKA activity. Moderate-dose Iloprost impairs haemostasis and thrombosis via suppression of platelet function, and low-dose Iloprost elevates peripheral platelet levels by inhibiting platelet apoptosis while having no effects on platelet function.
© 2025 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.

Genome-wide platelet transcriptomics is increasingly used to uncover new aspects of platelet biology and as a diagnostic and prognostic tool. Nevertheless, platelet isolation methods for transcriptomic studies are not standardized, introducing challenges for cross-study comparisons, data integration, and replication. In this prospective multicenter study, called "Standardizing Platelet Transcriptomics for Discovery, Diagnostics, and Therapeutics in the Thrombosis and Hemostasis Community (STRIDE)" by the International Society on Thrombosis and Haemostasis Scientific and Standardization Committees, we assessed how 3 of the most commonly used platelet isolation protocols influence metrics from next-generation bulk RNA sequencing and functional assays. Compared with washing alone, more stringent removal of leukocytes by anti-CD45 beads or PALL filters resulted in a sufficient quantity of RNA for next-generation sequencing and similar quality of RNA sequencing metrics. Importantly, stringent removal of leukocytes resulted in the lower relative expression of known leukocyte-specific genes and the higher relative expression of known platelet-specific genes. The results were consistent across enrolling sites, suggesting that the techniques are transferrable and reproducible. Moreover, all 3 isolation techniques did not influence basal platelet reactivity, but agonist-induced integrin αIIbβ3 activation is reduced by anti-CD45 bead isolation compared with washing alone. In conclusion, the isolation technique chosen influences genome-wide transcriptional and functional assays in platelets. These results should help the research community make informed choices about platelet isolation techniques in their own platelet studies.
Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.