Age-Dependent Control of Collagen-Dependent Platelet Responses by Thrombospondin-1—Comparative Analysis of Platelets from Neonates, Children, Adolescents, and Adults

Platelet function is developmentally regulated. Healthy neonates do not spontaneously bleed, but their platelets are hypo-reactive to several agonists. The mechanisms underlying immature platelet function in neonates are incompletely understood. This critical issue remains challenging for the establishment of age-specific reference ranges. In this study, we evaluated platelet reactivity of five pediatric age categories, ranging from healthy full-term neonates up to adolescents (11–18 years) in comparison to healthy adults (>18 years) by flow cytometry. We confirmed that platelet hypo-reactivity detected by fibrinogen binding, P-selectin, and CD63 surface expression was most pronounced in neonates compared to other pediatric age groups. However, maturation of platelet responsiveness varied with age, agonist, and activation marker. In contrast to TRAP and ADP, collagen-induced platelet activation was nearly absent in neonates. Granule secretion markedly remained impaired at least up to 10 years of age compared to adults. We show for the first time that neonatal platelets are deficient in thrombospondin-1, and exogenous platelet-derived thrombospondin-1 allows platelet responsiveness to collagen. Platelets from all pediatric age groups normally responded to the C-terminal thrombospondin-1 peptide RFYVVMWK. Thus, thrombospondin-1 deficiency of neonatal platelets might contribute to the relatively impaired response to collagen, and platelet-derived thrombospondin-1 may control distinct collagen-induced platelet responses

Impact of NKG2D Signaling on Natural Killer and T‐Cell Function in Cerebral Ischemia

Background Typically defined as a thromboinflammatory disease, ischemic stroke features early and delayed inflammatory responses, which determine the extent of ischemia‐related brain damage. T and natural killer cells have been implicated in neuronal cytotoxicity and inflammation, but the precise mechanisms of immune cell‐mediated stroke progression remain poorly understood. The activating immunoreceptor NKG2D is expressed on both natural killer and T cells and may be critically involved. Methods and Results An anti‐NKG2D blocking antibody alleviated stroke outcome in terms of infarct volume and functional deficits, coinciding with reduced immune cell infiltration into the brain and improved survival in the animal model of cerebral ischemia. Using transgenic knockout models devoid of certain immune cell types and immunodeficient mice supplemented with different immune cell subsets, we dissected the functional contribution of NKG2D signaling by different NKG2D‐expressing cells in stroke pathophysiology. The observed effect of NKG2D signaling in stroke progression was shown to be predominantly mediated by natural killer and CD8+ T cells. Transfer of T cells with monovariant T‐cell receptors into immunodeficient mice with and without pharmacological blockade of NKG2D revealed activation of CD8+ T cells irrespective of antigen specificity. Detection of the NKG2D receptor and its ligands in brain samples of patients with stroke strengthens the relevance of preclinical observations in human disease. Conclusions Our findings provide a mechanistic insight into NKG2D‐dependent natural killer– and T‐cell–mediated effects in stroke pathophysiology

NLRP3 inflammasome is a key driver of obesity-induced atrial arrhythmias

AIMS: Obesity, an established risk factor of atrial fibrillation (AF), is frequently associated with enhanced inflammatory response. However, whether inflammatory signaling is causally linked to AF pathogenesis in obesity remains elusive. We recently demonstrated that the constitutive activation of the ‘NACHT, LRR, and PYD Domains-containing Protein 3’ (NLRP3) inflammasome promotes AF susceptibility. In this study, we hypothesized that the NLRP3 inflammasome is a key driver of obesity-induced AF. METHODS AND RESULTS: Western blotting was performed to determine the level of NLRP3 inflammasome activation in atrial tissues of obese patients, sheep, and diet-induced obese (DIO) mice. The increased body weight in patients, sheep, and mice was associated with enhanced NLRP3-inflammasome activation. To determine whether NLRP3 contributes to the obesity-induced atrial arrhythmogenesis, wild-type (WT) and NLRP3 homozygous knockout (NLRP3(−/−)) mice were subjected to high-fat-diet (HFD) or normal chow (NC) for 10 weeks. Relative to NC-fed WT mice, HFD-fed WT mice were more susceptible to pacing-induced AF with longer AF duration. In contrast, HFD-fed NLRP3(−/−) mice were resistant to pacing-induced AF. Optical mapping in DIO mice revealed an arrhythmogenic substrate characterized by abbreviated refractoriness and action potential duration (APD), two key determinants of reentry-promoting electrical remodeling. Upregulation of ultra-rapid delayed-rectifier K(+)-channel (Kv1.5) contributed to the shortening of atrial refractoriness. Increased profibrotic signaling and fibrosis along with abnormal Ca(2+) release from sarcoplasmic reticulum (SR) accompanied atrial arrhythmogenesis in DIO mice. Conversely, genetic ablation of Nlrp3 (NLRP3(−/−)) in HFD-fed mice prevented the increases in Kv1.5 and the evolution of electrical remodeling, the upregulation of profibrotic genes, and abnormal SR Ca(2+) release in DIO mice. CONCLUSION: These results demonstrate that the atrial NLRP3 inflammasome is a key driver of obesity-induced atrial arrhythmogenesis and establishes a mechanistic link between obesity-induced AF and NLRP3-inflammasome activation