[Abstract unavailable

Summary Background Increasing evidence implicates both platelets and neutrophils in the formation, stabilization, and growth of peripheral and coronary thrombi. Neutrophil extracellular traps (NETs) play a key role. The early events in the deregulated cross‐talk between platelets and neutrophils are poorly characterized. Objectives To identify at the molecular level the mechanism through which platelets induce the generation of NETs in sterile conditions. Patients/Methods The presence of NETs was determined in 26 thrombi from patients with acute myocardial infarction by immunohistochemistry and immunofluorescence and markers of NETs assessed in the plasma. In vitro NET generation was studied in static and in physiological flow conditions. Results Coronary thrombi mainly consist of activated platelets, neutrophils, and NETs in close proximity of platelets. Activated platelets commit neutrophils to NET generation. The event abates in the presence of competitive antagonists of the high mobility group box 1 (HMGB1) protein. Hmgb1−/− platelets fail to elicit NETs, whereas the HMGB1 alone commits neutrophils to NET generation. Integrity of the HMGB1 receptor, Receptor for Advanced Glycation End products (RAGE), is required for NET formation, as assessed using pharmacologic and genetic tools. Exposure to HMGB1 prevents depletion of mitochondrial potential, induces autophagosome formation, and prolongs neutrophil survival. These metabolic effects are caused by the activation of autophagy. Blockade of the autophagic flux reverts platelet HMGB1‐elicited NET generation. Conclusions Activated platelets present HMGB1 to neutrophils and commit them to autophagy and NET generation. This chain of events may be responsible for some types of thromboinflammatory lesions and indicates novel paths for molecular intervention.

Since the discovery and definition of neutrophil extracellular traps (NETs) 14 years ago, numerous characteristics and physiological functions of NETs have been uncovered. Nowadays, the field continues to expand and novel mechanisms that orchestrate formation of NETs, their previously unknown properties, and novel implications in disease continue to emerge. The abundance of available data has also led to some confusion in the NET research community due to contradictory results and divergent scientific concepts, such as pro-and anti-inflammatory roles in pathologic conditions, demarcation from other forms of cell death, or the origin of the DNA that forms the NET scaffold. Here, we present prevailing concepts and state of the science in NET-related research and elaborate on open questions and areas of dispute.

Human type 1 diabetes (T1D) is an autoimmune disease associated with major histocompatibility complex polymorphisms, β-cell autoantibodies, and autoreactive T cells. However, there is increasing evidence that innate cells may also play critical roles in T1D. We aimed to monitor peripheral immune cells in early stages of T1D (i.e., in healthy autoantibody-positive subjects) and in more advanced phases of the disease (i.e., at disease onset and years after diagnosis). We found a mild but significant and reproducible peripheral neutropenia that both precedes and accompanies the onset of T1D. This reduction was not due to peripheral neutrophil cell death, impaired differentiation, or the presence of anti-neutrophil antibodies. Neutrophils were observed by electron microscopy and immunohistochemical analysis in the exocrine pancreas of multiorgan donors with T1D (both at onset and at later stages of the disease) and not in that of multiorgan donors with type 2 diabetes or nondiabetic donors. These pancreas-infiltrating neutrophils mainly localized at the level of very small blood vessels. Our findings suggest the existence of a hitherto unrecognized clinical phenotype that might reflect unexplored pathogenic pathways underlying T1D.

SARS-CoV-2 infection poses a major threat to the lungs and multiple other organs, occasionally causing death. Until effective vaccines are developed to curb the pandemic, it is paramount to define the mechanisms and develop protective therapies to prevent organ dysfunction in patients with COVID-19. Individuals that develop severe manifestations have signs of dysregulated innate and adaptive immune responses. Emerging evidence implicates neutrophils and the disbalance between neutrophil extracellular trap (NET) formation and degradation plays a central role in the pathophysiology of inflammation, coagulopathy, organ damage, and immunothrombosis that characterize severe cases of COVID-19. Here, we discuss the evidence supporting a role for NETs in COVID-19 manifestations and present putative mechanisms, by which NETs promote tissue injury and immunothrombosis. We present therapeutic strategies, which have been successful in the treatment of immunο-inflammatory disorders and which target dysregulated NET formation or degradation, as potential approaches that may benefit patients with severe COVID-19.

Activated platelets express ligands, which are recognized by counterreceptors on neutrophils. Here, we show that the ensuing cell-to-cell interaction programs neutrophil phagocytic function, resulting in activated platelet clearance. Neutrophils that have internalized platelets circulate in the blood of patients with acute myocardial infarction, and the extent of platelet clearance correlates with expression of platelet activation, including P-selectin. Activated platelets injected intravenously in experimental animals are detectable in circulating neutrophils 60 minutes after, and within 3 hours, more than 70% circulating neutrophils have internalized platelets. Platelet clearance comprises 2 events: adhesion to neutrophils, which requires divalent cations and depends on P-selectin, on the P-selectin glycoprotein ligand-1 (PSGL-1), and on the CD11b/CD18 ␤2 integrin; and internalization, which is abrogated by the phosphatidylserine-binding protein annexin A5. Adhesion to platelets causes neutrophil degranulation and is blocked by antibodies specific for P-selectin and PSGL-1, either in a synthetic medium in vitro or in the whole blood, therefore in the presence of a physiologic array of plasma cofactors and opsonins. The data suggest that the interaction between circulating platelets and neutrophils influences innate immune functions, possibly contributing to regulate vascular inflammation. IntroductionNeutrophils are recruited to inflamed sites, where they are required for microbial clearance. Neutrophil recruitment is a multistep process, which comprises initial tethering and rolling along the vessel wall, firm adhesion to endothelial cells, and eventual extravasation. It involves consecutively various adhesion molecules, including selectins and ␤2 integrins. [1][2][3][4] Granules of endothelial cells (Weibel-Palade bodies) and of platelets (␣-granules) contain P-selectin. Inflammatory stimuli cause its translocation at the endothelial cell surface. The interaction with the counterreceptor, PSGL-1, prompts leukocyte tethering and rolling and initiates the second phase of the process, firm adhesion. During these events, integrins shift to an active conformation. [5][6][7] Integrin activation depends on the signaling cascade downstream the P-selectin/PSGL-1 interaction. [8][9][10] Platelets adhere and are activated at sites of vascular injury: there, they produce a releasate, some contents of which penetrate and/or interact biochemically with neutrophils. This includes unprocessed free arachidonic acid which can then be transformed into leukotriene A4 and B4. 11-14 Furthermore, activated platelets express P-selectin. Platelet P-selectin guarantees the access of leukocytes to perivascular tissues even when dying or severely damaged endothelial cells fail to sustain leukocyte rolling and adhesion. 15,16 Indeed, endothelial cells surrounding the lesion release signals that amplify the expression of P-selectin on adhering platelets. 17 The P-selectin-dependent interaction of neutrophils and platelets amplifies mut...

Endothelial cell damage and platelet activation contribute to sustained vasculopathy, which is a key clinical characteristic of systemic sclerosis (SSc), also known as scleroderma. Microparticles released from activated platelets in the blood of SSc patients (SSc-microparticles) are abundant and express the damage-associated molecular pattern (DAMP) HMGB1. SSc-microparticles interacted with neutrophils in vitro and in immunocompromised mice and promoted neutrophil autophagy, which was characterized by mobilization of their granule content, enhanced proteolytic activity, prolonged survival, and generation of neutrophil extracellular traps (NETs). Neutrophils migrated within the mouse lung, with collagen accumulation in the interstitial space and the release of soluble E-selectin by the vascular endothelium. Microparticle-neutrophil interaction, neutrophil autophagy and survival, and generation of NETs abated in the presence of BoxA, a competitive inhibitor of HMGB1. Consistent with these results, neutrophils in the blood of SSc patients were autophagic and NET by-products were abundant. Our findings implicate neutrophils in SSc vasculopathy and suggest that platelet-derived, microparticle-associated HMGB1 may be a potential indicator of disease and target for novel therapeutics.

Summary. Background: Blood-borne tissue factor (TF) plays a crucial role in thrombogenesis. Aim: To study whether polymorphonuclear leukocytes (PMN) are a source of TF. Methods and Results: Human PMN were carefully separated from other blood cells and stimulated for 3 min with purified P-selectin or the chemotactic peptide formyl-MetLeuPhe (fMLP): they expressed both TF procoagulant activity, as identified by specific TF MoAb and inactivated factor VIIa blockade; and TF:Ag (four to six times), as shown by flowcytometry and immunocytochemistry. About 40% of permeabilized PMN, both resting and stimulated, contained TF:Ag, indicating that stimulation only modifies the location of TF:Ag within PMN. By real time-polymerase chain reaction (RT-PCR), a very low amount of TF mRNA was detectable in resting PMN, but a 3-to 5-fold increase was observed after 1-h stimulation with P-selectin or fMLP, respectively. Conclusions: These findings suggest that TF is not constitutively expressed in peripheral PMN, but can be up-regulated and produced upon stimulation and specific gene transcription, as for instance during contact with activated platelets or endothelium. The stored TF is rapidly expressed in vitro as a functional molecule on the surface of activated PMN. The availability of PMN TF supports the relevance of inflammatory cells and their interaction with platelets for fibrin deposition and thrombus formation.