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.

Thrombin receptor PAR4 drives canonical NLRP3 inflammasome signaling in the heart.

The deleterious effects of diabetes in the heart are increasingly attributed to inflammatory signaling through the NLRP3 (NOD, LRR and PYD domains-containing protein 3) inflammasome. Thrombin antagonists reduce cardiac remodeling and dysfunction in diabetic mice, in part by suppressing fibrin-driven inflammation. The role of cellular thrombin receptor subtypes in this context is not known. We sought to determine the causal involvement of protease-activated receptors (PAR) in inflammatory signaling of the diabetic heart. Mice with diet-induced diabetes showed increased abundance of pro-caspase-1 and pro-interleukin (IL)-1ß in the left ventricle (LV), indicating transcriptional NLRP3 inflammasome priming, and augmented cleavage of active caspase-1 and IL-1ß, pointing to canonical NLRP3 inflammasome activation. Caspase-11 activation, which mediates non-canonical NLRP3 inflammasome signaling, was not augmented. Formation of the plasma membrane pore-forming protein N-terminal gasdermin D (GDSMD), a prerequisite for IL-1ß secretion, was also higher in diabetic vs. control mouse LV. NLRP3, ASC and IL-18 expression did not differ between the groups, nor did expression of PAR1 or PAR2. PAR3 was nearly undetectable. LV abundance of PAR4 by contrast increased with diabetes and correlated positively with active caspase-1. Genetic deletion of PAR4 in mice prevented the diet-induced cleavage of caspase-1, IL-1ß and GDSMD. Right atrial appendages from patients with type 2 diabetes also showed higher levels of PAR4, but not of PAR1 or PAR2, than non-diabetic atrial tissue, along with increased abundance of cleaved caspase-1, IL-1ß and GSDMD. Human cardiac fibroblasts maintained in high glucose conditions to mimic diabetes also upregulated PAR4 mRNA and protein, and increased PAR4-dependent IL-1ß transcription and secretion in response to thrombin, while PAR1 and PAR2 expressions were unaltered. In conclusion, PAR4 drives caspase-1-dependent IL-1ß production through the canonical NLRP3 inflammasome pathway in the diabetic heart, providing mechanistic insights into diabetes-associated cardiac thromboinflammation. The emerging PAR4-selective antagonists may provide a feasible approach to prevent cardiac inflammation in patients with diabetes.

Straight to the heart: Pleiotropic antiarrhythmic actions of oral anticoagulants.

Mechanistic understanding of atrial fibrillation (AF) pathophysiology and the complex bidirectional relationship with thromboembolic risk remains limited. Oral anticoagulation is a mainstay of AF management. An emerging concept is that anticoagulants may themselves have potential pleiotropic disease-modifying effects. We here review the available evidence for hemostasis-independent actions of the oral anticoagulants on electrical and structural remodeling, and the inflammatory component of the vulnerable substrate.

Components in Plasma-Derived Factor VIII, But Not in Recombinant Factor VIII Downregulate Anti-Inflammatory Surface Marker CD163 in Human Macrophages through Release of CXCL4 (Platelet Factor 4).

BACKGROUND: Hemarthrosis, or bleeding into the joints, is a hallmark of hemophilia. Heme triggers oxidative stress, inflammation, and destruction of cartilage and bone. The haptoglobin-CD163-heme oxygenase-1 (HO-1) pathway circumvents heme toxicity through enzymatic degradation of heme and transcription of antioxidant genes. Plasma-derived factor concentrates contain many proteins that might impact on cellular pathways in joints, blood, and vessels. METHODS: Activation of platelets from healthy volunteers was assessed by flow cytometry analysis of fibrinogen binding and CD62P expression. Platelet CXCL4 release was measured by ELISA. Human peripheral blood mononuclear cells were exposed to CXCL4 or platelet supernatants (untreated or pre-stimulated with factor VIII (FVIII) products) during their differentiation to macrophages and analyzed for CD163 expression. Some macrophage cultures were additionally incubated with autologous hemoglobin for 18 h for analysis of HO-1 expression. RESULTS: Platelet CXCL4 release was increased by all 8 tested plasma-derived FVIII products but not the 3 recombinant products. Macrophages exposed to supernatant from platelets treated with some plasma-derived FVIII products downregulated CD163 surface expression and failed to upregulate the athero- and joint protective enzyme HO-1 in response to hemoglobin. CONCLUSION: Plasma-derived FVIII products might promote bleeding-induced joint injury via generation of macrophages that are unable to counteract redox stress.

Thrombin receptor protease-activated receptor 4 is a key regulator of exaggerated intimal thickening in diabetes mellitus.

BACKGROUND: Diabetes mellitus predisposes to thrombotic and proliferative vascular remodeling, to which thrombin contributes via activation of protease-activated receptor (PAR) 1. However, the use of PAR-1 inhibitors to suppress remodeling may be limited by severe bleeding. We recently reported upregulation of an additional thrombin receptor, PAR-4, in human vascular smooth muscle cells exposed to high glucose and have now examined PAR-4 as a novel mediator linking hyperglycemia, hypercoagulation, and vascular remodeling in diabetes mellitus. METHODS AND RESULTS: PAR-4 expression was increased in carotid atherectomies and saphenous vein specimens from diabetic versus nondiabetic patients and in aorta and carotid arteries from streptozotocin-diabetic versus nondiabetic C57BL/6 mice. Vascular PAR-1 mRNA was not increased in diabetic mice. Ligated carotid arteries from diabetic mice developed more extensive neointimal hyperplasia and showed greater proliferation than arteries from nondiabetic mice. The augmented remodeling response was absent in diabetic mice deficient in PAR-4. At the cellular level, PAR-4 expression was controlled via the mRNA stabilizing actions of human antigen R, which accounted for the stimulatory actions of high glucose, angiotensin II, and H2O2 on PAR-4 expression, whereas cicaprost via protein kinase A activation counteracted this effect. CONCLUSIONS: PAR-4 appears to play a hitherto unsuspected role in diabetic vasculopathy. The development of PAR-4 inhibitors might serve to limit mainly proliferative processes in restenosis-prone diabetic patients, particularly those patients in whom severe bleeding attributed to selective PAR-1 blockade or complete thrombin inhibition must be avoided or those who do not require anticoagulation.

The thrombin receptor PAR4 supports visceral adipose tissue inflammation.

Thrombin inhibition suppresses adiposity, WAT inflammation and metabolic dysfunction in mice. Protease-activated receptor (PAR)1 does not account for thrombin-driven obesity, so we explored the culprit role of PAR4 in this context. Male WT and PAR-4-/- mice received a high fat diet (HFD) for 8 weeks, WT controls received standard chow. Body fat was quantified by NMR. Epididymal WAT was assessed by histology, immunohistochemistry, qPCR and lipase activity assay. 3T3-L1 preadipocytes were differentiated ± thrombin, acutely stimulated ± PAR4 activating peptide (AP) and assessed by immunoblot, qPCR and U937 monocyte adhesion. Epicardial adipose tissue (EAT) from obese and lean patients was assessed by immunoblot. PAR4 was upregulated in mouse WAT under HFD. PAR4-/- mice developed less visceral adiposity and glucose intolerance under HFD, featuring smaller adipocytes, fewer macrophages and lower expression of adipogenic (leptin, PPARγ) and pro-inflammatory genes (CCL2, IL-1ß) in WAT. HFD-modified activity and expression of lipases or perilipin were unaffected by PAR4 deletion. 3T3-L1 adipocytes differentiated with thrombin retained Ki67 expression, further upregulated IL-1ß and CCL2 and were more adhesive for monocytes. In mature adipocytes, PAR4-AP increased phosphorylated ERK1/2 and AKT, upregulated Ki67, CCl2, IL-ß and hyaluronan synthase 1 but not TNF-α mRNA, and augmented hyaluronidase-sensitive monocyte adhesion. Obese human EAT expressed more PAR4, CD68 and CD54 than lean EAT. PAR4 upregulated in obesity supports adipocyte hypertrophy, WAT expansion and thrombo-inflammation. The emerging PAR4 antagonists provide a therapeutic perspective in this context beyond their canonical antiplatelet action.

Ischemia does not provoke the full immune training repertoire in human cardiac fibroblasts.

Trained immunity of monocytes, endothelial, and smooth muscle cells augments the cytokine response to secondary stimuli. Immune training is characterized by stabilization of hypoxia-inducible factor (HIF)-1α, mTOR activation, and aerobic glycolysis. Cardiac fibroblast (CF)-myofibroblast transition upon myocardial ischemia/reperfusion (I/R) features epigenetic and metabolic adaptations reminiscent of trained immunity. We assessed the impact of I/R on characteristics of immune training in human CF and mouse myocardium. I/R was simulated in vitro with transient metabolic inhibition. CF primed with simulated I/R or control buffer were 5 days later re-stimulated with Pam3CSK for 24 h. Mice underwent transient left anterior descending artery occlusion or sham operation with reperfusion for up to 5 days. HIF-regulated metabolic targets and cytokines were assessed by qPCR, immunoblot, and ELISA and glucose consumption, lactate release, and lactate dehydrogenase (LDH) by chromogenic assay. Simulated I/R increased HIF-1α stabilization, mTOR phosphorylation, glucose consumption, lactate production, and transcription of PFKB3 and F2RL3, a HIF-regulated target gene, in human CF. PGK1 and LDH mRNAs were suppressed. Intracellular LDH transiently increased after simulated I/R, and extracellular LDH showed sustained elevation. I/R priming increased abundance of pro-caspase-1, auto-cleaved active caspase-1, and the expression and secretion of interleukin (IL)-1ß, but did not augment Pam3CSK-stimulated cytokine transcription or secretion. Myocardial I/R in vivo increased abundance of HIF-1 and the precursor and cleaved forms of caspase-1, caspase-11, and caspase-8, but not of LDH-A or phospho-mTOR. I/R partially reproduces features of immune training in human CF, specifically HIF-1α stabilization, aerobic glycolysis, mTOR phosphorylation, and PFKB3 transcription. I/R does not augment PGK1 or LDH expression or the cytokine response to Pam3CSK. Regulation of PAR4 and inflammasome caspases likely occurs independently of an immune training repertoire.

Direct neuronal protection by the protease-activated receptor PAR4 antagonist ML354 after experimental stroke in mice.

BACKGROUND AND PURPOSE: Thrombo-inflammation is a key feature of stroke pathophysiology and provides multiple candidate drug targets. Thrombin exerts coagulation-independent actions via protease-activated receptors (PAR), of which PAR1 has been implicated in stroke-associated neuroinflammation. The role of PAR4 in this context is less clear. This study examined if the selective PAR4 antagonist ML354 provides neuroprotection in experimental stroke and explored the underlying mechanisms. EXPERIMENTAL APPROACH: Mouse primary cortical neurons were exposed to oxygen-glucose deprivation (OGD) and simulated reperfusion ± ML354. For comparison, functional Ca2+-imaging was performed upon acute stimulation with a PAR4 activating peptide or glutamate. Male mice underwent sham operation or transient middle cerebral artery occlusion (tMCAO), with ML354 or vehicle treatment beginning at recanalization. A subset of mice received a platelet-depleting antibody. Stroke size and functional outcomes were assessed. Abundance of target genes, proteins, and cell markers was determined in cultured cells and tissues by qPCR, immunoblotting, and immunofluorescence. KEY RESULTS: Stroke up-regulated PAR4 expression in cortical neurons in vitro and in vivo. OGD augments spontaneous and PAR4-mediated neuronal activity; ML354 suppresses OGD-induced neuronal excitotoxicity and apoptosis. ML354 applied in vivo after tMCAO reduced infarct size, apoptotic markers, macrophage accumulation, and interleukin-1ß expression. Platelet depletion did not affect infarct size in mice with tMCAO ± ML354. CONCLUSIONS AND IMPLICATIONS: Selective PAR4 inhibition during reperfusion improves infarct size and neurological function after experimental stroke by blunting neuronal excitability, apoptosis, and local inflammation. PAR4 antagonists may provide additional neuroprotective benefits in patients with acute stroke beyond their canonical antiplatelet action.

Protein Disulfide Isomerase and Extracellular Adherence Protein Cooperatively Potentiate Staphylococcal Invasion into Endothelial Cells.

Invasion of host cells is an important feature of Staphylococcus aureus. The main internalization pathway involves binding of the bacteria to host cells, e.g., endothelial cells, via a fibronectin (Fn) bridge between S. aureus Fn binding proteins and α5ß1-integrin, followed by phagocytosis. The secreted extracellular adherence protein (Eap) has been shown to promote this cellular uptake pathway of not only S. aureus, but also of bacteria otherwise poorly taken up by host cells, such as Staphylococcus carnosus. The exact mechanisms are still unknown. Previously, we demonstrated that Eap induces platelet activation by stimulation of the protein disulfide isomerase (PDI), a catalyst of thiol-disulfide exchange reactions. Here, we show that Eap promotes PDI activity on the surface of endothelial cells, and that this contributes critically to Eap-driven staphylococcal invasion. PDI-stimulated ß1-integrin activation followed by increased Fn binding to host cells likely accounts for the Eap-enhanced uptake of S. aureus into non-professional phagocytes. Additionally, Eap supports the binding of S. carnosus to Fn-α5ß1 integrin, thereby allowing its uptake into endothelial cells. To our knowledge, this is the first demonstration that PDI is crucial for the uptake of bacteria into host cells. We describe a hitherto unknown function of Eap-the promotion of an enzymatic activity with subsequent enhancement of bacterial uptake-and thus broaden mechanistic insights into its importance as a driver of bacterial pathogenicity. IMPORTANCE Staphylococcus aureus can invade and persist in non-professional phagocytes, thereby escaping host defense mechanisms and antibiotic treatment. The intracellular lifestyle of S. aureus contributes to the development of infection, e.g., in infective endocarditis or chronic osteomyelitis. The extracellular adherence protein secreted by S. aureus promotes its own internalization as well as that of bacteria that are otherwise poorly taken up by host cells, such as Staphylococcus carnosus. In our study, we demonstrate that staphylococcal uptake by endothelial cells requires catalytic disulfide exchange activity by the cell-surface protein disulfide isomerase, and that this critical enzymatic function is enhanced by Eap. The therapeutic application of PDI inhibitors has previously been investigated in the context of thrombosis and hypercoagulability. Our results add another intriguing possibility: therapeutically targeting PDI, i.e., as a candidate approach to modulate the initiation and/or course of S. aureus infectious diseases.

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.

Platelet depletion does not alter long-term functional outcome after cerebral ischaemia in mice.

Platelets are key mediators of thrombus formation and inflammation during the acute phase of ischaemic stroke. Particularly, the platelet glycoprotein (GP) receptors GPIbα and GPVI have been shown to mediate platelet adhesion and activation in the ischaemic brain. GPIbα and GPVI blockade could reduce infarct volumes and improve functional outcome in mouse models of acute ischaemic stroke, without concomitantly increasing intracerebral haemorrhage. However, the functional role of platelets during long-term stroke recovery has not been elucidated so far. Thus, we here examined the impact of platelet depletion on post-stroke recovery after transient middle cerebral artery occlusion (tMCAO) in adult male mice. Platelet depleting antibodies or isotype control were applied from day 3-28 after tMCAO in mice matched for infarct size. Long-term functional recovery was assessed over the course of 28 days by behavioural testing encompassing motor and sensorimotorical functions, as well as anxiety-like or spontaneous behaviour. Whole brain flow cytometry and light sheet fluorescent microscopy were used to identify resident and infiltrated immune cell types, and to determine the effects of platelet depletion on the cerebral vascular architecture, respectively. We found that delayed platelet depletion does not improve long-term functional outcome in the tMCAO stroke model. Immune cell abundance, the extent of thrombosis and the organisation of the cerebral vasculature were also comparable between platelet-depleted and control mice. Our study demonstrates that, despite their critical role in the acute stroke setting, platelets appear to contribute only marginally to tissue reorganisation and functional recovery at later stroke stages.

Direct oral anticoagulation and severe obesity - One size fits all?

Oral anticoagulation is obligatory in patients with atrial fibrillation (AF) to prevent thromboembolic stroke. Direct direct oral anticoagulants (DOAC) exhibit improved safety over Vitamin K antagonists, but any interference in haemostasis can impact on bleeding. Optimal anticoagulation remains challenging particularly in patients with co-morbidities. International Society of Thrombosis and Haemostasis (ISTH) guidelines recommend avoiding DOAC in patients with severe obesity, and systematic data on individual DOAC drug concentrations, clinical efficacy and safety in relation to body weight are lacking. A new study now provides reassurance that DOAC are safe and effective in a real-world cohort of morbidly obese patients, going some way to fill the knowledge gap pertaining to optimal management of concomitant obesity and AF.

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 Ca2+ 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 Ca2+ 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.

Bound to bleed: How altered albumin binding may dictate warfarin treatment outcome.

Although non-vitamin-K anticoagulants are now the preferred option for stroke prevention in atrial fibrillation (AF), warfarin is still used in a significant number of patients. Warfarin dosing requirements are susceptible to drug interactions and genetic polymorphisms in metabolising enzymes. Human serum albumin (HSA) is a candidate modifier of warfarin pharmacokinetics, with hypoalbuminemia now shown to correlate with supratherapeutic INR levels and annual bleeding risk. Warfarin is highly bound to HSA, and a relatively small shift, resulting from displacement by other xenobiotics, hypoalbuminemia or reduced binding capacity, can potentially lead to marked alterations in the free warfarin fraction. Precisely how this relates to the actual concentration of free, pharmacodynamically active, warfarin, is not clear, since measurement of this critical moiety remains an unsolved caveat. Yet awareness how disease, nutrition and polypharmacy affect warfarin binding to HAS and how this may impact (or not) on bioavailability and outcome, is essential for optimal treatment.

Decreased M1 macrophage polarization in dabigatran-treated Ldlr-deficient mice: Implications for atherosclerosis and adipose tissue inflammation.

BACKGROUND AND AIMS: The non-vitamin K oral anticoagulant dabigatran etexilate (dabigatran) is increasingly prescribed to patients with non-valvular atrial fibrillation and venous thromboembolism. Adipose tissue (AT) inflammation during obesity plays a crucial role in the development of insulin resistance, type II diabetes and atherogenesis. The aim of the present study was to investigate the effects of thrombin inhibition by dabigatran in a combined model of diet-induced obesity and atherosclerosis. METHODS: Female Low density lipoprotein receptor knockout (Lldr-/-) mice were fed a high-fat diet containing 5 mg/g dabigatran or matching control for 20 weeks. RESULTS: Dabigatran-treated animals showed increased adipocyte hypertrophy, but reduced numbers of pro-inflammatory M1-polarized macrophages in the adipose tissue. Abundance of pro-inflammatory M1 macrophages was also decreased in the aortic wall of dabigatran-fed mice. Multiple circulating cytokines were reduced, indicating an effect in systemically relevant secretory compartments such as the AT. CONCLUSIONS: Dabigatran treatment reduces pro-inflammatory M1 macrophages in atherosclerotic lesions, thereby contributing to plaque stabilizing and atheroprotective effects of the thrombin inhibitor. This finding is not restricted to the vascular wall but is also present in AT where dabigatran treatment reduced the release of pro-inflammatory cytokines and accumulation of M1 macrophages.