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Heparin-induced thrombocytopenia/thrombosis (HIT) is a serious immune reaction to heparins, characterized by thrombocytopenia and often severe thrombosis with high morbidity and mortality. HIT is mediated by IgG antibodies against heparin/platelet factor 4 antigenic complexes. These complexes are thought to activate platelets leading to thrombocytopenia and thrombosis. Here we show that HIT immune complexes induce NETosis via interaction with FcγRIIa on neutrophils and through neutrophil-platelet association. HIT immune complexes induce formation of thrombi containing neutrophils, extracellular DNA, citrullinated histone H3 and platelets in a microfluidics system and in vivo, while neutrophil depletion abolishes thrombus formation. Absence of PAD4 or PAD4 inhibition with GSK484 abrogates thrombus formation but not thrombocytopenia, suggesting they are induced by separate mechanisms. NETs markers and neutrophils undergoing NETosis are present in HIT patients. Our findings demonstrating the involvement of NETosis in thrombosis will modify the current concept of HIT pathogenesis and may lead to new therapeutic strategies. The pathogenesis of heparin-induced thrombocytopenia and thrombosis (HIT) is mediated by heparin-reactive autoantibodies binding to platelets (thrombocytes). Here the authors show neutrophil activation and NETosis are elevated in patients with HIT, and are essential for thrombosis in HIT mouse models.

Platelets — blood cells continuously produced from megakaryocytes mainly in the bone marrow — are implicated not only in haemostasis and arterial thrombosis, but also in other physiological and pathophysiological processes. This Review describes current evidence for the heterogeneity in platelet structure, age, and activation properties, with consequences for a diversity of platelet functions. Signalling processes of platelet populations involved in thrombus formation with ongoing coagulation are well understood. Genetic approaches have provided information on multiple genes related to normal haemostasis, such as those encoding receptors and signalling or secretory proteins, that determine platelet count and/or responsiveness. As highly responsive and secretory cells, platelets can alter the environment through the release of growth factors, chemokines, coagulant factors, RNA species, and extracellular vesicles. Conversely, platelets will also adapt to their environment. In disease states, platelets can be positively primed to reach a pre-activated condition. At the inflamed vessel wall, platelets interact with leukocytes and the coagulation system, interactions mediating thromboinflammation. With current antiplatelet therapies invariably causing bleeding as an undesired adverse effect, novel therapies can be more beneficial if directed against specific platelet responses, populations, interactions, or priming conditions. On the basis of these novel concepts and processes, we discuss several initiatives to target platelets therapeutically.

There is a growing appreciation for the contribution of platelets to immunity; however, our knowledge mostly relies on platelet functions associated with vascular injury and the prevention of bleeding. Circulating immune complexes (ICs) contribute to both chronic and acute inflammation in a multitude of clinical conditions. Herein, we scrutinized platelet responses to systemic ICs in the absence of tissue and endothelial wall injury. Platelet activation by circulating ICs through a mechanism requiring expression of platelet Fcγ receptor IIA resulted in the induction of systemic shock. IC-driven shock was dependent on release of serotonin from platelet-dense granules secondary to platelet outside-in signaling by αIIbβ3 and its ligand fibrinogen. While activated platelets sequestered in the lungs and leaky vasculature of the blood–brain barrier, platelets also sequestered in the absence of shock in mice lacking peripheral serotonin. Unexpectedly, platelets returned to the blood circulation with emptied granules and were thereby ineffective at promoting subsequent systemic shock, although they still underwent sequestration. We propose that in response to circulating ICs, platelets are a crucial mediator of the inflammatory response highly relevant to sepsis, viremia, and anaphylaxis. In addition, platelets recirculate after degranulation and sequestration, demonstrating that in adaptive immunity implicating antibody responses, activated platelets are longer lived than anticipated and may explain platelet count fluctuations in IC-driven diseases.

Background Alloantibodies against human platelet antigens (HPAs) and human leukocyte antigen (HLA) are implicated in several immune-mediated platelet disorders. Detection of these antibodies is crucial in the diagnosis and management of these disorders. The aim of this study was to establish a novel method to simultaneously detect HPA-1, HPA-2, HPA-3, HPA-5 and HLA antibodies with Luminex microbeads technology. Methods Monoclonal antibodies specific for platelet glycoproteins and HLA class I molecules were separately coupled to the Luminex microbeads. We validated specificity of the Luminex platform using the following antibodies: anti-HPA-1a, anti-HPA-2b, anti-HPA-3a, anti-HPA-5a, and anti-HLA positive samples. Sensitivity was evaluated by a serial dilution (from neat to 1/1024) using the following antibodies: anti-HPA-1a, anti-HPA-3a standard sera, and anti-HPA-5a positive serum. Serum samples were collected from 36 neonatal alloimmune thrombocytopenia (NAIT) patients suspected of having HPA or HLA antibodies and 8 samples from ISBT platelet workshop were tested using the Luminex assay. Results The Luminex assay detected all antibodies tested from the known samples. The sensitivities of the Luminex assay detecting anti-HPA-1a, anti-HPA-3a, and anti-HPA-5a were 1:512, 1:64, and 1:128, respectively. The sensitivity of Luminex assay was higher than monoclonal antibody immobilization of platelet antigen method (MAIPA). No cross-reactivity was observed in the samples containing multi-platelet antibodies or mixture antibodies against HPA and HLA. The results of 44 samples with platelet disorders were consistent with those of the same samples processed with the MAIPA assay. Conclusion Luminex microbeads coupled with monoclonal antibodies could be successfully used to detect HPA and HLA antibodies simultaneously, especially with high sensitivity in detecting HPA antibodies.

Coronavirus disease 2019 (COVID-19) is a clinical syndrome caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Patients with severe disease show hyperactivation of the immune system, which can affect multiple organs besides the lungs. Here, we propose that SARS-CoV-2 infection induces a process known as immunothrombosis, in which activated neutrophils and monocytes interact with platelets and the coagulation cascade, leading to intravascular clot formation in small and larger vessels. Microthrombotic complications may contribute to acute respiratory distress syndrome (ARDS) and other organ dysfunctions. Therapeutic strategies aimed at reducing immunothrombosis may therefore be useful. Several antithrombotic and immunomodulating drugs have been proposed as candidates to treat patients with SARS-CoV-2 infection. The growing understanding of SARS-CoV-2 infection pathogenesis and how it contributes to critical illness and its complications may help to improve risk stratification and develop targeted therapies to reduce the acute and long-term consequences of this disease.Here, the authors propose that SARS-CoV-2 induces a prothrombotic state, with dysregulated immunothrombosis in lung microvessels and endothelial injury, which drive the clinical manifestations of severe COVID-19. They discuss potential antithrombotic and immunomodulating drugs that are being considered in the treatment of patients with COVID-19.

Immune and inflammatory responses require leukocytes to migrate within and through the vasculature, a process that is facilitated by their capacity to switch to a polarized morphology with an asymmetric distribution of receptors. We report that neutrophil polarization within activated venules served to organize a protruding domain that engaged activated platelets present in the bloodstream.The selectin ligand PSGL-1 transduced signals emanating from these interactions, resulting in the redistribution of receptors that drive neutrophil migration. Consequently, neutrophils unable to polarize or to transduce signals through PSGL-1 displayed aberrant crawling, and blockade of this domain protected mice against thromboinflammatory injury. These results reveal that recruited neutrophils scan for activated platelets, and they suggest that the neutrophils' bipolarity allows the integration of signals present at both the endothelium and the circulation before inflammation proceeds.

Key Points Platelets have a plethora of soluble and cell-associated immunomodulatory molecules that can enhance and in some instances inhibit immune responses in a variety of diverse settings. Platelets are naturally adhesive and can interact with various cells of the innate and adaptive immune system. Platelets store preformed and synthesized immunomodulatory molecules that can markedly affect immune responses. These molecules include transforming growth factor-β, interleukin-1, platelet-derived growth factor and CC-chemokine ligand 5 (also known as RANTES). Platelets express and secrete CD40 and CD154 and are the largest source of soluble CD154, which can affect both dendritic cell maturation and T cell activation. Platelets express Toll-like receptors and actively bind to microorganisms, and in some instances can be directly cytopathic against bacteria. This, together with their large numbers within the circulation, makes them the ultimate bacterial sentinels in the bloodstream. Platelets have been shown to have a central role in the pathogenesis of a diverse array of immune-mediated and infectious diseases, such as atherosclerosis, sepsis-induced lung damage, rheumatoid arthritis and malaria. Platelets are well known as mediators of haemostasis, but their roles in immunity are less well appreciated. Here, the authors relate the diverse immune functions of platelets and discuss how these anucleate cells can promote both innate and adaptive immune responses. Platelets are anucleate cells that are crucial mediators of haemostasis. Most immunologists probably don't think about platelets every day, and may even consider these cells to be 'nuisances' in certain in vitro studies. However, it is becoming increasingly clear that platelets have inflammatory functions and can influence both innate and adaptive immune responses. Here, we discuss the mechanisms by which platelets contribute to immunity: these small cells are more immunologically savvy than we once thought.

Key Points Thrombosis involves the pathological occlusion of blood vessels, which inhibits the blood supply to organs. It is the most frequent cause of mortality worldwide, as it directly causes myocardial infarction, stroke, pulmonary embolism, thrombotic microangiopathies and complications during sepsis as well as other diseases. Thrombosis is traditionally seen as a pathological form of blood vessel repair via haemostasis. Indeed, both thrombosis and haemostasis are induced by two core processes: blood coagulation, which leads to fibrin formation; and platelet activation. However, thrombosis is also supported by cellular mediators (for example, neutrophils) and molecular mediators (for example, intravascular tissue factor) that are largely irrelevant for haemostasis. Immune cells and thrombosis-specific molecular mediators induce a physiological type of thrombosis in microvessels (such as sinusoids in the liver and spleen). This form of thrombosis has been designated here as immunothrombosis. Immunothrombosis involves a local platform consisting of fibrin, monocytes, neutrophils and platelets, which together contribute to pathogen recognition. This process helps to suppress the tissue invasion and dissemination of pathogens and to reduce their survival. The local nature of immunothrombosis and its occurrence in only a restricted number of microvessels probably ensures that immunothrombosis does not seriously perturb overall organ perfusion. Together, these properties characterize immunothrombosis as an independent process of innate immunity that is specifically activated by blood-borne microorganisms and by circulating altered-self components. Pathological thrombosis in large veins and microvessels (such as venous thromboembolism and thrombotic macroangiopathies) shares similar triggers (namely, pathogens and altered-self components), the same evolutionary origin and identical molecular and cellular mediators with immunothrombosis. This suggests that together with haemostasis, immunothrombosis is the most relevant biological process underlying pathological thrombosis. Thrombosis is the most frequent cause of mortality worldwide. In this Review, the authors propose that thrombosis might also have a conserved physiological role in immune defence via a process termed immunothrombosis. However, if uncontrolled, immunothrombosis facilitates pathological clot formation. Thrombosis is the most frequent cause of mortality worldwide and is closely linked to haemostasis, which is the biological mechanism that stops bleeding after the injury of blood vessels. Indeed, both processes share the core pathways of blood coagulation and platelet activation. Here, we summarize recent work suggesting that thrombosis under certain circumstances has a major physiological role in immune defence, and we introduce the term immunothrombosis to describe this process. Immunothrombosis designates an innate immune response induced by the formation of thrombi inside blood vessels, in particular in microvessels. Immunothrombosis is supported by immune cells and by specific thrombosis-related molecules and generates an intravascular scaffold that facilitates the recognition, containment and destruction of pathogens, thereby protecting host integrity without inducing major collateral damage to the host. However, if uncontrolled, immunothrombosis is a major biological process fostering the pathologies associated with thrombosis.

Dengue virus (DENV) infection in the presence of reactive, non-neutralizing immunoglobulin G (IgG) (RNNIg) is the greatest risk factor for dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Progression to DHF/DSS is attributed to antibody-dependent enhancement (ADE); however, because only a fraction of infections occurring in the presence of RNNIg advance to DHF/DSS, the presence of RNNIg alone cannot account for disease severity. We discovered that DHF/DSS patients respond to infection by producing IgGs with enhanced affinity for the activating Fc receptor FcγRIIIA due to afucosylated Fc glycans and IgG1 subclass. RNNIg enriched for afucosylated IgG1 triggered platelet reduction in vivo and was a significant risk factor for thrombocytopenia. Thus, therapeutics and vaccines restricting production of afucosylated, IgG1 RNNIg during infection may prevent ADE of DENV disease.

Influenza infection increases the incidence of myocardial infarction but the reason is unknown. Platelets mediate vascular occlusion through thrombotic functions but are also recognized to have immunomodulatory activity. To determine if platelet processes are activated during influenza infection, we collected blood from 18 patients with acute influenza infection. Microscopy reveals activated platelets, many containing viral particles and extracellular-DNA associated with platelets. To understand the mechanism, we isolate human platelets and treat them with influenza A virus. Viral-engulfment leads to C3 release from platelets as a function of TLR7 and C3 leads to neutrophil-DNA release and aggregation. TLR7 specificity is confirmed in murine models lacking the receptor, and platelet depletion models support platelet-mediated C3 and neutrophil-DNA release post-influenza infection. These findings demonstrate that the initial intrinsic defense against influenza is mediated by platelet–neutrophil cross-communication that tightly regulates host immune and complement responses but can also lead to thrombotic vascular occlusion. Influenza viremia is rare in human blood and not well studied. Here, the authors show that influenza can be found in human platelets and that platelet engulfment of influenza A results in TLR7-dependent C3 release, which in turn promotes neutrophil-DNA release and formation of platelet-DNA aggregates.