Background Streptococcus oralis is the most prevalent viridans group streptococcus associated with infective endocarditis (IE), yet the platelet activation mechanisms that underpin its pathogenicity remain poorly defined. Platelet–bacterium interactions are central to vegetation formation, but the relative contributions of direct adhesion and antibody-dependent signaling in S. oralis-induced platelet aggregation are unclear.Objectives To define the molecular mechanisms by which S. oralis induces platelet activation and aggregation, and to determine how bacterial adhesins and host factors cooperate to drive this response.Methods Platelet aggregation and dense granule secretion were assessed using light transmission aggregometry and ATP release assays in platelet-rich plasma and washed platelets. An IE isolate of S. oralis and isogenic mutants lacking functional Fap1 were used alongside pharmacological and antibody-based inhibition of platelet receptors and signaling pathways.Results S. oralis ATCC 10557 induced robust platelet aggregation with a characteristic lag phase and strong dense granule release. Aggregation required integrin αIIbβ3 signaling and was abolished by inhibition of glycoprotein Ibα (GPIbα) or FcγRIIA. Loss of the serine-rich repeat protein Fap1, or mutation of its conserved sialic acid-binding motif, abrogated platelet aggregation. While most donors required a plasma component for aggregation, supplementation of washed platelets with human IgG fully restored aggregation in a dose-dependent manner. Importantly, IgG was unable to bypass the requirement for Fap1–GPIbα interaction, indicating that direct bacterial adhesion is an essential initiating step that enables antibody-dependent FcγRIIA signaling. Substantial inter-individual variability was observed in aggregation kinetics and IgG dependence.Conclusions S. oralis induces platelet aggregation through a dual, non-redundant mechanism requiring Fap1-mediated engagement of GPIbα and IgG-dependent FcγRIIA signaling. This cooperative pathway drives potent platelet activation and exhibits marked host-dependent variability. These findings provide new mechanistic insight into early events in IE pathogenesis and highlight platelet–bacterium interactions as potential targets for risk stratification and therapeutic intervention.
Hemolysis is a metric of hemocompatibility in total artificial hearts (TAHs) but provides limited insight into platelet biology and thrombogenicity. We demonstrated reduced hemolysis with Realheart® TAH (RH) compared to FDA-approved SynCardia TAH (Sync). Here, we evaluated platelet function following exposure to both devices in a whole-blood in vitro system using a standardized ASTM F1830 protocol. Human blood (n = 5) was circulated for 360 min under controlled conditions (5 L/min, 100 mmHg, 37 °C) in RH, Sync, while matched static controls were maintained under identical temperature conditions. Platelets were assessed using metabolic (ATP, mitochondrial potential), phenotypic (surface markers, microparticles), and functional (agonist response, thromboelastometry) assays. Differences were observed between circulating and static conditions, including higher pH, increased lactate, and reduced platelet surface receptor expression, consistent with the non-physiological nature of static controls. Despite these changes, no evidence of increased platelet activation (CD62P), thrombogenicity (CD61+ microparticles), or mitochondrial damage (ATP, JC-1) was observed, and platelet function remained preserved by agonist stimulation and thromboelastometry. SynCardia increased mean platelet volume and reduced ATP levels, suggesting altered particle composition rather than intrinsic platelet dysfunction. These findings indicate preserved platelet integrity under clinically relevant TAH conditions and highlight that hemolysis and platelet responses represent distinct aspects of device hemocompatibility.
Glycoprotein Ibα (GPIbα) is a platelet-specific receptor that is subjected to proteolytic shedding by ADAM17, releasing its ectodomain glycocalicin. GPIbα proteolysis is induced by strong platelet activation in a process too slow to be crucial in primary hemostasis. The spatiotemporal fate of glycocalicin was assessed during thrombosis using human and murine samples, integrating in vitro approaches and an inferior vena cava (IVC) ligation model. We found that the glycocalicin concentration inside human thrombi was 4-fold higher than in initial plasma (4.0 ± 0.8 µg/mL vs 0.9 ± 0.0 µg/mL, respectively). This was confirmed in vitro starting from human platelet-rich plasma or murine whole blood, with 6-fold and 3-fold increases, respectively. In vivo, containment of glycocalicin inside thrombi following IVC ligation was even more pronounced with glycocalicin levels reaching 135.1 ± 44.3 µg/mL in thrombi compared to 8.2 ± 1.0 µg/mL in plasma. In the presence of ADAM17 inhibitors, the glycocalicin concentration inside human thrombi was 3-fold lower than in thrombi with competent ADAM17. In contrast, inhibition of ADAM17 in mice, either pharmacologically or genetically, had no limiting effect on intrathrombic GPIbα shedding. In conclusion, GPIbα shedding occurs inside thrombi and results in maximal retention of glycocalicin. Intrathrombic GPIbα shedding is conserved between species, but is regulated differently.KEY POINTS Glycocalicin is contained within the thrombus microenvironmentRegulation of GPIbα ectodomain shedding is species-dependent and not universally mediated by ADAM17
Background In aspirin-treated unstable angina (UA), heterogeneity in on-treatment arachidonic acid (AA)–induced platelet aggregation and cholesterol reduction contributes to residual ischemic risk. Insomnia is prevalent in coronary disease, yet its association with specific aspirin-sensitive platelet aggregation kinetics and lipid parameters remains undefined.Methods In a cross-sectional cohort of 198 aspirin-treated UA patients, 38 routine hematologic or biochemical variables including AA-induced platelet aggregation indices were analyzed using principal component analysis (PCA) and unsupervised phenotyping (k-means and hierarchical clustering). Elastic-net logistic regression and machine learning classifiers were used for multivariable modeling, and performance was estimated by cross-validation. Correlation-network analysis and Shapley additive explanations (SHAP) supported interpretability.Results Unsupervised analyses consistently identified two phenotypes. Insomnia was enriched in a platelet–lipid phenotype characterized by lower platelet aggregation indices and a lower lipid marker profile. The K-nearest neighbors (KNN) model demonstrated strong cross-validated discrimination (AUC = 0.934; average precision = 0.923), while all four classifiers converged with AUC > 0.925. After age matching, elastic-net logistic regression retained only AA-induced maximal platelet aggregation (PmaxAggR) and total cholesterol (CHOL) as independent predictors, with odds ratios of 0.927 and 0.340, respectively (both p < .001). Inflammatory markers, including neutrophil count and monocyte count, were no longer selected. Network analysis placed insomnia within the platelet–lipid module linked to PMaxAggR, CHOL, and some other platelet or lipid indices.Conclusions In age-matched, aspirin-treated UA patients, insomnia is independently associated with reduced PMaxAggR and lower CHOL, with inflammatory markers no longer significant after age-balancing. These findings delineate an age-independent platelet–cholesterol axis linked to insomnia, potentially reflecting shared autonomic and neurobiological dysregulation, and motivate sleep evaluation as part of platelet-reactivity phenotyping in this population.
Neonatal and adult platelets are functionally different, with neonatal platelets being comparatively hypo-reactive to multiple platelet agonists. When transfused, neonates receive platelets from adult donors, resulting in a cellular developmental mismatch. Platelets are key regulators of the monocyte inflammatory response, yet how the developmental stage of the platelet affects this response is unknown. In this study, we modeled this developmental mismatch in vitro to investigate the neonatal (cord blood) monocyte inflammatory response and migratory phenotype after exposure to either neonatal (cord blood) or adult platelets. Adult platelets expressed more P-selectin on the surface and released more beta-2-microglobulin (B2M) after activation compared to neonatal platelets. While exposure to platelets of either developmental stage increased the release of IL-8 and MCP-1 from neonatal monocytes to a similar degree, exposure to adult platelets induced significantly greater monocyte CCR2 and CCR5 surface expression, consistent with a pro-migratory monocyte phenotype. Blocking P-selectin/PSGL-1 interactions had no effect on platelet induced monocyte cytokine release but completely abrogated monocyte CCR2 and CCR5 surface upregulation. These findings suggest that the monocyte pro-migratory phenotype is regulated by P-selectin exposure, which is influenced by the platelet developmental stage, while monocyte cytokine release is independent of platelet P-selectin expression or developmental stage.
