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.

Neutrophils and platelets are among the most abundant cell types in peripheral blood and characterized by high plasticity and a readily available reservoir of surface proteins and secretable granule contents. Receptor-mediated activation and granule release predispose both cell types for rapid responses to various stimuli. While neutrophils provide the first line of defense to microbial infections and platelets are known for their aggregatory functions in hemostasis and thrombosis, research of the past decade has highlighted that both cell types jointly shape local and systemic immune responses and clot formation alike. Concomitant activation of neutrophils and platelets has been observed in a variety of cardiovascular diseases, including arterial and venous thrombosis, atherosclerosis as well as myocardial infarction and ischemia-reperfusion injury. In this review, we describe the mechanisms by which neutrophils and platelets interact physically, how release of granule contents and soluble molecules by either cell type affects the other and how this mutual activation supports the efficacy of immune responses. We go on to describe how activated platelets contribute to host defense by triggering neutrophil extracellular trap (NET) formation in a process termed immunothrombosis, which in turn promotes local platelet activation and coagulation. Further, we review current evidence of hazardous overactivation of either cell type and their respective role in cardiovascular disease, with a focus on thrombosis, myocardial infarction and ischemia-reperfusion injury, and describe how neutrophils and platelets shape thromboinflammation in COVID-19. Finally, we provide an overview of therapeutic approaches targeting neutrophil-platelet interactions as novel treatment strategy in cardiovascular disease.

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 HLADR^low CD9^low monocyte population expands in severe COVID‐19, which releases neutrophil chemokines in the lung, and might in turn explain neutrophil expansion and pulmonary recruitment in the late stages of severe COVID‐19. Conclusions In summary, our data underline an innate immune cell axis causing vascular inflammation and immunothrombosis in severe SARS‐CoV‐2 infection.

Platelets are key players in cardiovascular disease, and platelet aggregation represents a central pharmacologic target, particularly in secondary prevention. However, inhibition of adenosine diphosphate and thromboxane signaling has low efficacy in preventing venous thromboembolism, necessitating the inhibition of the plasmatic coagulation cascade in this disease entity. Anticoagulation carries a significantly higher risk of bleeding complications, highlighting the need of alternative therapeutic approaches. We hypothesized that procoagulant activation (PA) of platelets promotes venous thrombus formation and that targeting PA could alleviate venous thrombosis. Here, we found elevated levels of procoagulant platelets in the circulation and in thrombi of patients with deep vein thrombosis (DVT) and pulmonary embolism, and in mice developing DVT following inferior vena cava stenosis. Furthermore, we detected PA of recruited platelets within murine venous thrombi and human pulmonary emboli. Mice with platelet-specific deficiency in central pathways of PA—cyclophilin D and transmembrane protein 16F—were more resistant toward low flow–induced venous thrombosis. Finally, we found that a clinically approved carbonic anhydrase inhibitor, methazolamide, reduced platelet procoagulant activity and alleviated murine thrombus formation without affecting trauma-associated hemostasis. These findings identify an essential role of platelet procoagulant function in venous thrombosis and delineate novel pharmacologic strategies targeting platelets in the prevention of venous thromboembolism.

Acute and chronic coronary syndromes (ACS and CCS) are leading causes of mortality. Inflammation is considered a key pathogenic driver of these diseases, but the underlying immune states and their clinical implications remain poorly understood. Multiomic factor analysis (MOFA) allows unsupervised data exploration across multiple data types, identifying major axes of variation and associating these with underlying molecular processes. We hypothesized that applying MOFA to multiomic data obtained from blood might uncover hidden sources of variance and provide pathophysiological insights linked to clinical needs. Here we compile a longitudinal multiomic dataset of the systemic immune landscape in both ACS and CCS (n = 62 patients in total, n = 15 women and n = 47 men) and validate this in an external cohort (n = 55 patients in total, n = 11 women and n = 44 men). MOFA reveals multicellular immune signatures characterized by distinct monocyte, natural killer and T cell substates and immune-communication pathways that explain a large proportion of inter-patient variance. We also identify specific factors that reflect disease state or associate with treatment outcome in ACS as measured using left ventricular ejection fraction. Hence, this study provides proof-of-concept evidence for the ability of MOFA to uncover multicellular immune programs in cardiovascular disease, opening new directions for mechanistic, biomarker and therapeutic studies.

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 αIIbβ3 (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.