Procoagulant platelet sentinels prevent inflammatory bleeding through GPIIBIIIA and GPVI.

Impairment of vascular integrity is a hallmark of inflammatory diseases. We recently reported that single immune-responsive platelets migrate and reposition themselves to sites of vascular injury to prevent bleeding. However, it remains unclear how single platelets preserve vascular integrity once encountering endothelial breaches. Here we demonstrate by intravital microscopy combined with genetic mouse models that procoagulant activation (PA) of single platelets and subsequent recruitment of the coagulation cascade are crucial for the prevention of inflammatory bleeding. Using a novel lactadherin-based compound, we detect phosphatidylserine (PS)-positive procoagulant platelets in the inflamed vasculature. We identify exposed collagen as the central trigger arresting platelets and initiating subsequent PA in a CypD- and TMEM16F-dependent manner both in vivo and in vitro. Platelet PA promotes binding of the prothrombinase complex to the platelet membrane, greatly enhancing thrombin activity and resulting in fibrin formation. PA of migrating platelets is initiated by costimulation via integrin αIIbß3 (GPIIBIIIA)/Gα13-mediated outside-in signaling and glycoprotein VI signaling, leading to an above-threshold intracellular calcium release. This effectively targets the coagulation cascade to breaches of vascular integrity identified by patrolling platelets. Platelet-specific genetic loss of either CypD or TMEM16F as well as combined blockade of platelet GPIIBIIIA and glycoprotein VI reduce platelet PA in vivo and aggravate pulmonary inflammatory hemorrhage. Our findings illustrate a novel role of procoagulant platelets in the prevention of inflammatory bleeding and provide evidence that PA of patrolling platelet sentinels effectively targets and confines activation of coagulation to breaches of vascular integrity.

Thrombocytopenia and splenic platelet-directed immune responses after IV ChAdOx1 nCov-19 administration.

Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are based on a range of novel platforms, with adenovirus-based approaches (like ChAdOx1 nCov-19) being one of them. Recently, a novel complication of SARS-CoV-2-targeted adenovirus vaccines has emerged: immune thrombocytopenia, either isolated, or accompanied by thrombosis (then termed VITT). This complication is characterized by low platelet counts, and in the case of VITT, also by platelet-activating platelet factor 4 antibodies reminiscent of heparin-induced thrombocytopenia, leading to a prothrombotic state with clot formation at unusual anatomic sites. Here, we detected antiplatelet antibodies targeting platelet glycoprotein receptors in 30% of patients with proven VITT (n = 27) and 42% of patients with isolated thrombocytopenia after ChAdOx1 nCov-19 vaccination (n = 26), indicating broad antiplatelet autoimmunity in these clinical entities. We use in vitro and in vivo models to characterize possible mechanisms of these platelet-targeted autoimmune responses leading to thrombocytopenia. We show that IV but not intramuscular injection of ChAdOx1 nCov-19 triggers platelet-adenovirus aggregate formation and platelet activation in mice. After IV injection, these aggregates are phagocytosed by macrophages in the spleen, and platelet remnants are found in the marginal zone and follicles. This is followed by a pronounced B-cell response with the emergence of circulating antibodies binding to platelets. Our work contributes to the understanding of platelet-associated complications after ChAdOx1 nCov-19 administration and highlights accidental IV injection as a potential mechanism of platelet-targeted autoimmunity. Hence, preventing IV injection when administering adenovirus-based vaccines could be a potential measure against platelet-associated pathologies after vaccination.

Single platelet and megakaryocyte morpho-dynamics uncovered by multicolor reporter mouse strains in vitro and in vivo.

Visualizing cell behavior and effector function on a single cell level has been crucial for understanding key aspects of mammalian biology. Due to their small size, large number and rapid recruitment into thrombi, there is a lack of data on fate and behavior of individual platelets in thrombosis and hemostasis. Here we report the use of platelet lineage restricted multi-color reporter mouse strains to delineate platelet function on a single cell level. We show that genetic labeling allows for single platelet and megakaryocyte (MK) tracking and morphological analysis in vivo and in vitro, while not affecting lineage functions. Using Cre-driven Confetti expression, we provide insights into temporal gene expression patterns as well as spatial clustering of MK in the bone marrow. In the vasculature, shape analysis of activated platelets recruited to thrombi identifies ubiquitous filopodia formation with no evidence of lamellipodia formation. Single cell tracking in complex thrombi reveals prominent myosin-dependent motility of platelets and highlights thrombus formation as a highly dynamic process amenable to modification and intervention of the acto-myosin cytoskeleton. Platelet function assays combining flow cytrometry, as well as in vivo, ex vivo and in vitro imaging show unaltered platelet functions of multicolor reporter mice compared to wild-type controls. In conclusion, platelet lineage multicolor reporter mice prove useful in furthering our understanding of platelet and MK biology on a single cell level.

Self-sustaining IL-8 loops drive a prothrombotic neutrophil phenotype in severe COVID-19.

Neutrophils provide a critical line of defense in immune responses to various pathogens, inflicting self-damage upon transition to a hyperactivated, procoagulant state. Recent work has highlighted proinflammatory neutrophil phenotypes contributing to lung injury and acute respiratory distress syndrome (ARDS) in patients with coronavirus disease 2019 (COVID-19). Here, we use state-of-the art mass spectrometry-based proteomics and transcriptomic and correlative analyses as well as functional in vitro and in vivo studies to dissect how neutrophils contribute to the progression to severe COVID-19. We identify a reinforcing loop of both systemic and neutrophil intrinsic IL-8 (CXCL8/IL-8) dysregulation, which initiates and perpetuates neutrophil-driven immunopathology. This positive feedback loop of systemic and neutrophil autocrine IL-8 production leads to an activated, prothrombotic neutrophil phenotype characterized by degranulation and neutrophil extracellular trap (NET) formation. In severe COVID-19, neutrophils directly initiate the coagulation and complement cascade, highlighting a link to the immunothrombotic state observed in these patients. Targeting the IL-8-CXCR-1/-2 axis interferes with this vicious cycle and attenuates neutrophil activation, degranulation, NETosis, and IL-8 release. Finally, we show that blocking IL-8-like signaling reduces severe acute respiratory distress syndrome of coronavirus 2 (SARS-CoV-2) spike protein-induced, human ACE2-dependent pulmonary microthrombosis in mice. In summary, our data provide comprehensive insights into the activation mechanisms of neutrophils in COVID-19 and uncover a self-sustaining neutrophil-IL-8 axis as a promising therapeutic target in severe SARS-CoV-2 infection.

Vascular neutrophilic inflammation and immunothrombosis distinguish severe COVID-19 from influenza pneumonia.

OBJECTIVE: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can lead to severe pneumonia, but also thrombotic complications and non-pulmonary organ failure. Recent studies suggest intravascular neutrophil activation and subsequent immune cell-triggered immunothrombosis as a central pathomechanism linking the heterogenous clinical picture of coronavirus disease 2019 (COVID-19). We sought to study whether immunothrombosis is a pathognomonic factor in COVID-19 or a general feature of (viral) pneumonia, as well as to better understand its upstream regulation. APPROACH AND RESULTS: By comparing histopathological specimens of SARS-CoV-2 with influenza-affected lungs, we show that vascular neutrophil recruitment, NETosis, and subsequent immunothrombosis are typical features of severe COVID-19, but less prominent in influenza pneumonia. Activated neutrophils were typically found in physical association with monocytes. To explore this further, we combined clinical data of COVID-19 cases with comprehensive immune cell phenotyping and bronchoalveolar lavage fluid scRNA-seq data. We show that a HLADRlow CD9low monocyte population expands in severe COVID-19, which releases neutrophil chemokines in the lungs, and might in turn explain neutrophil expansion and pulmonary recruitment in the late stages of severe COVID-19. CONCLUSIONS: Our data underline an innate immune cell axis causing vascular inflammation and immunothrombosis in severe SARS-CoV-2 infection.