Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.

Platelet homeostasis is essential for vascular integrity and immune defense. While the process of platelet formation by fragmenting megakaryocytes (thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of megakaryocytes by their progenitor cells (megakaryopoiesis) remains unclear. Here we use intravital 2 photon microscopy to track individual megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as crucial bone marrow niche cells that regulate megakaryopoiesis. pDCs monitor the bone marrow for platelet-producing megakaryocytes and deliver IFN-α to the megakaryocytic niche to trigger local on-demand proliferation of megakaryocyte progenitors. This fine-tuned coordination between thrombopoiesis and megakaryopoiesis is crucial for megakaryocyte and platelet homeostasis in steady state and stress. However, uncontrolled pDC function within the megakaryocytic niche is detrimental. Accordingly, we show that pDCs activated by SARS-CoV2 drive inappropriate megakaryopoiesis associated with thrombotic complications. Together, we uncover a hitherto unknown megakaryocytic bone marrow niche maintained by the constitutive delivery of pDC-derived IFN-α.

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.

Patients frequently visit the emergency department with conditions that place them at risk of worse outcomes when accompanied by coagulopathy. Routine tests of coagulation—prothrombin time, partial thromboplastin time, platelets, and fibrinogen—have shortcomings that limit their use in providing emergency care. One alternative is to investigate coagulation disturbance with viscoelastic monitoring (VEM), a coagulation test that measures the timing and strength of blood clot development in real time. VEM is widely used and studied in cardiac surgery, liver transplant surgery, anesthesia, and trauma. In this article, we review the technique of VEM and the biologic rationale of using it in addition to routine tests of coagulation in emergency clinical situations. Then, we review the evidence (or lack thereof) for using VEM in the diagnosis and treatment of specific conditions. Finally, we describe the limitations of the test and future directions for clinical use and research in emergency medicine. Patients frequently visit the emergency department with conditions that place them at risk of worse outcomes when accompanied by coagulopathy. Routine tests of coagulation—prothrombin time, partial thromboplastin time, platelets, and fibrinogen—have shortcomings that limit their use in providing emergency care. One alternative is to investigate coagulation disturbance with viscoelastic monitoring (VEM), a coagulation test that measures the timing and strength of blood clot development in real time. VEM is widely used and studied in cardiac surgery, liver transplant surgery, anesthesia, and trauma. In this article, we review the technique of VEM and the biologic rationale of using it in addition to routine tests of coagulation in emergency clinical situations. Then, we review the evidence (or lack thereof) for using VEM in the diagnosis and treatment of specific conditions. Finally, we describe the limitations of the test and future directions for clinical use and research in emergency medicine.

Patients frequently visit the emergency department with conditions that place them at risk of worse outcomes when accompanied by coagulopathy. Routine tests of coagulation-prothrombin time, partial thromboplastin time, platelets, and fibrinogen-have shortcomings that limit their use in providing emergency care. One alternative is to investigate coagulation disturbance with viscoelastic monitoring (VEM), a coagulation test that measures the timing and strength of blood clot development in real time. VEM is widely used and studied in cardiac surgery, liver transplant surgery, anesthesia, and trauma. In this article, we review the technique of VEM and the biologic rationale of using it in addition to routine tests of coagulation in emergency clinical situations. Then, we review the evidence (or lack thereof) for using VEM in the diagnosis and treatment of specific conditions. Finally, we describe the limitations of the test and future directions for clinical use and research in emergency medicine.

Abstract Coronavirus disease 2019 (COVID-19) is associated with a prothrombotic state with a high incidence of thrombotic events during hospitalization; however, data examining rates of thrombosis after discharge are limited. We conducted a retrospective observational cohort study of discharged patients with confirmed COVID-19 not receiving anticoagulation. The cohort included 163 patients with median time from discharge to last recorded follow-up of 30 days (interquartile range [IQR], 17-46 days). The median duration of index hospitalization was 6 days (IQR, 3-12 days) and 26% required intensive care. The cumulative incidence of thrombosis (including arterial and venous events) at day 30 following discharge was 2.5% (95% confidence interval [CI], 0.8-7.6); the cumulative incidence of venous thromboembolism alone at day 30 postdischarge was 0.6% (95% CI, 0.1-4.6). The 30-day cumulative incidence of major hemorrhage was 0.7% (95% CI, 0.1-5.1) and of clinically relevant nonmajor bleeds was 2.9% (95% CI, 1.0-9.1). We conclude that the rates of thrombosis and hemorrhage appear to be similar following hospital discharge for COVID-19, emphasizing the need for randomized data to inform recommendations for universal postdischarge thromboprophylaxis.

Coronavirus disease 2019 (Covid-19) is associated with venous and arterial thrombotic complications, especially among those hospitalized with more severe disease. Disturbances in coagulation indices are commonly observed in severe Covid-19 including marked elevations of plasma D-dimer that portend a poor prognosis. Emerging evidence points to the inadequacy of standard thromboprophylaxis. And, in an attempt to prevent thrombotic events and potentially limit microvasculature fibrin deposition, more aggressive thromboprophylaxis anticoagulation regimens are considered, including therapeutic dosing of unfractionated heparin (UFH).1 Heparin induced thrombocytopenia (HIT) is an uncommon but potentially life-threatening complication of heparin exposure. Autoimmune hematologic phenomena have increasingly been recognized during the course of Covid-19.2, 3 Accordingly, we evaluated the incidence of HIT among hospitalized patients with Covid-19 treated with intravenous, therapeutic-dose heparin. Institutional Review Board approval was obtained. A retrospective cohort analysis was performed for patients hospitalized at Beth Israel Deaconess Medical Center with Covid-19 confirmed by PCR between February 1, 2020 to May 7, 2020 and received intravenous UFH for ≥5 days. A cut off of 5 days was selected based on the known natural history of HIT. The HIT antibody testing was ordered by the treating physicians and performed by latex immunoassay for anti-platelet factor 4/heparin antibodies (HemosIL HIT-Ab).4 Confirmatory testing performed by serotonin release assay (Versiti, Wisconsin). To account for death as a competing risk, estimation of the cumulative incidence of HIT was calculated by competing risk analyses (stcompet, Stata Corp, TX) along with 95% confidence intervals at 25 days from start of heparin infusion. Among 439 hospitalized patients with Covid-19, 88 patients who received at least 5 days of UFH were included in the analyses. The target partial thromboplastin time was above 60 seconds in 84 patients and the remaining four patients the target was above 50 seconds. Median duration of heparin exposure was 11 days (IQR 7-15 days). The most common indications for therapeutic heparin were Covid-19 coagulopathy (N = 58), confirmed or presumed venous thromboembolism (N = 15), and atrial fibrillation (N = 15). Among eight patients where there was suspicion for HIT, five patients tested positive for HIT by latex immunoassay (Table S1). The cumulative incidence of positive HIT immunoassay assay was 12% at 25 days (95% CI, 4% to 26%, Figure 1). Direct thrombin inhibitors were administered following HIT diagnosis (argatroban N = 4, bivalirudin N = 1). One patient was identified with thrombotic complications in the setting of HIT, this included evidence of acute cerebrovascular infarct as well as extensive areas of splenic infarction. Three patients developed major hemorrhagic events. Three patients died during hospitalization of progressive respiratory failure, one was discharged, and one remains hospitalized. Therapeutic UFH is commonly administered in order to mitigate the prothrombotic state in severe Covid-19. We report a cumulative incidence of detectable HIT antibodies of 12% at 25 days. In non-Covid-19 cohort analyses conducted in hospitalized medical populations, the rate of diagnosed HIT by antibody testing alone (ie, without serotonin release confirmation) is typically less than 3%.5, 6 For instance, in a cohort of 5415 patients receiving therapeutic heparin, the overall incidence was 0.76%.5 These results raise several important issues. First, while immunologic detection of auto-antibodies such as lupus anticoagulant appear to be prevalent among patients with Covid-19, whether or not these are pathologic is unclear. In our cohort, the majority of the positive HIT antibody tests were not definitively confirmed by serotonin release assay, which reinforces the importance of confirmatory testing. Second, while the benefit of empiric therapeutic heparin to mitigate the hypercoagulable state of Covid-19 is not established, the diagnosis of HIT necessitates the initiation of non-heparin anticoagulants. Three of the five patients diagnosed with HIT developed major hemorrhages after switching to direct thrombin inhibitors. Bleeding risks must be balanced in prothrombotic disorders and critically ill patients may have multiple risk factors, a recent paper showed that the rate of major bleeding in critically ill patients with Covid-19 was considerable.7 Lastly, the incidence of HIT commonly correlates with duration and intensity of heparin exposure. Even in the absence of immune hyperstimulation, prolonged hospitalizations are common with severe Covid-19 and thus an increased incidence of HIT would be expected. This study is the first to investigate the incidence of HIT in hospitalized patients with Covid-19 being treated with therapeutic unfractionated heparin, however we acknowledge limitations including the limited sample size given the single center design. Given the retrospective design of the study, HIT testing was only performed when considered indicated by the treating providers. However cytopenias and thrombosis are common in critically ill patients and given the widespread use of heparin in hospitalized Covid-19 patients, it is possible that screening serologies would detect higher rates of HIT antibodies. This would lead to an underestimate of the true prevalence of HIT antibodies by detecting unsuspected patients. The latex immune turbidimetric assay used in our center is semi-quantitative with operating characteristics superior to the more common ELISA.4 The serotonin release assay, which is the confirmatory test, was performed in all four of the patients with low positive values, and only one was negative, belying that the antibodies detected in a majority of the cases were not false positives. In this single center retrospective cohort study, we found a high incidence of HIT antibody positivity in critically ill Covid-19 patients being treated with therapeutic unfractionated heparin. Despite the limitations of the study design, our findings suggest that HIT may be an unrecognized clinical entity at least in a subpopulation of patients and merits further study. Considering the frequency of HIT following prolonged UFH exposure in the intensive care setting, we urge caution in the empiric use of intravenous UFH for the management of Covid-19 coagulopathy. JIZ reports research funding from Incyte and Quercegen; consultancy: Sanofi, CSL, Parexel; honoraria/advisory boards: Pfizer/BMS, Portola, Daiichi. KAB reports consultancy with Janssen. WCA is employed by DynaMed. Table S1 Characteristics of patients with COVID-19 receiving UFH who tested positive for HIT antibodies by latex immunoassay. 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Homogeneous populations of mature differentiated primary cell types can display variable responsiveness to extracellular stimuli, although little is known about the underlying mechanisms that govern such heterogeneity at the level of gene expression. In this article, we show that morphologically homogenous human endothelial cells exhibit heterogeneous expression of VCAM1 after TNF-α stimulation. Variability in VCAM1 expression was not due to stochasticity of intracellular signal transduction but rather to preexisting established heterogeneous states of promoter DNA methylation that were generationally conserved through mitosis. Variability in DNA methylation of the VCAM1 promoter resulted in graded RelA/p65 and RNA polymerase II binding that gave rise to a distribution of VCAM1 transcription in the population after TNF-α stimulation. Microarray analysis and single-cell RNA sequencing revealed that a number of cytokine-inducible genes shared this heterogeneous response pattern. These results show that heritable epigenetic heterogeneity is fundamental in inflammatory signaling and highlight VCAM1 as a metastable epiallele.

Background Coronavirus disease-2019 (COVID-19) is a recognized prothrombotic state. Patients hospitalized with active cancer are predisposed to thrombosis but whether active cancer further amplifies thrombotic risk with COVID-19 is not known. Objectives To evaluate cumulative incidences of thrombotic and hemorrhagic events in hospitalized COVID-19 patients with and without active cancer at 28 days. Methods A retrospective cohort analysis of consecutive adults hospitalized with COVID-19 was performed. Active cancer required cancer-directed therapy within previous 6 months. The cumulative incidences of thrombosis or hemorrhage were estimated considering death as a competing risk. Results Patients without cancer (n = 353) and active cancer (n = 45) were comparable in terms of age, sex, antibiotics administered, length of hospitalization, and critical care. The most common malignancies were lymphoid (17.8%), gastrointestinal (15.6%), lung (13.3%), and genitourinary (13.3%). At day 28, the cumulative incidence of thrombotic events was 18.2% (95% confidence interval [CI], 10.2%-27.9%) in the non-cancer cohort and 14.2% (95% CI, 4.7%-28.7%) in the cancer cohort. The cumulative incidence of major and fatal bleeding at day 28 was 20.8% (95% CI, 12.1%-31.0%) in the non-cancer group and 19.5% (95% CI, 5.5%-39.8%) in the cancer cohort. Three patients experienced fatal bleeds, all of whom were in the non-cancer cohort. Survival was significantly shorter in the group with active cancer (P = .038). Conclusions We observed a similarly high incidence of thrombosis and bleeding among patients admitted with COVID-19 with or without active cancer.

Background and aims: We have previously demonstrated that in response to hypoxia, von Willebrand factor (VWF) expression is upregulated in lung and heart endothelial cells both in vitro and in vivo, but not in kidney endothelial cells. The aim of our current study was to determine whether endothelial cells of different organs employ distinct molecular mechanisms to mediate VWF response to hypoxia. Methods: We used cultured human primary lung, heart and kidney endothelial cells to determine the activation of endogenous VWF as well as exogenously expressed VWF promoter in response to hypoxia. Chromatin immunoprecipitation and siRNA knockdown analyses were used to determine the roles of VWF promoter associated transacting factors in mediating its hypoxia response. Platelet aggregates formations in vascular beds of mice were used as a marker for potential functional consequences of hypoxia-induced VWF upregulation in vivo. Results: Our analyses demonstrated that while Yin Yang 1 (YY1) and specificity protein 1 (Sp1) participate in the hypoxia-induced upregulation of VWF specifically in lung endothelial cells, GATA6 mediates this process specifically in heart endothelial cells. In both cell types, the response to hypoxia involves the decreased association of the NFIB repressor with the VWF promoter, and the increased acetylation of the promoter-associated histone H4. In mice exposed to hypoxia, the upregulation of VWF expression was concomitant with the presence of thrombi in heart and lung, but not kidney vascular beds. Conclusions: Heart and lung endothelial cells demonstrated VWF upregulation in response to hypoxia, using distinct mechanisms, while this response was lacking in kidney endothelial cells.

We are presenting a case of a middle-aged woman with history of remote kidney transplantation who had multiple admissions for septic shock-like picture, recurrent fever, and hypotension. Her shock manifestation would resolve after stress dose steroid administration and less than 24 hours of vasopressor administration. Initially, extensive workup was performed without revealing etiology. Eventually, a bone marrow biopsy was carried out leading to the diagnosis of hemophagocytic lymphohistiocytosis, most likely related to recent cytomegalovirus infection.

Endothelial cells (ECs) are highly specialized across vascular beds. However, given their interspersed anatomic distribution, comprehensive characterization of the molecular basis for this heterogeneity in vivo has been limited. By applying endothelial-specific translating ribosome affinity purification (EC-TRAP) combined with high-throughput RNA sequencing analysis, we identified pan EC-enriched genes and tissue-specific EC transcripts, which include both established markers and genes previously unappreciated for their presence in ECs. In addition, EC-TRAP limits changes in gene expression after EC isolation and in vitro expansion, as well as rapid vascular bed-specific shifts in EC gene expression profiles as a result of the enzymatic tissue dissociation required to generate single-cell suspensions for fluorescence-activated cell sorting or single-cell RNA sequencing analysis. Comparison of our EC-TRAP with published single-cell RNA sequencing data further demonstrates considerably greater sensitivity of EC-TRAP for the detection of low abundant transcripts. Application of EC-TRAP to examine the in vivo host response to lipopolysaccharide (LPS) revealed the induction of gene expression programs associated with a native defense response, with marked differences across vascular beds. Furthermore, comparative analysis of whole-tissue and TRAP-selected mRNAs identified LPS-induced differences that would not have been detected by whole-tissue analysis alone. Together, these data provide a resource for the analysis of EC-specific gene expression programs across heterogeneous vascular beds under both physiologic and pathologic conditions.

Background: Previous studies have provided insight into the molecular basis of human tissue factor (TF) activation of activated factor VII (FVIIa). TF-induced allosteric networks of FVIIa activation have been rationalized through analysis of the dynamic changes and residue connectivities in the human soluble TF (sTF)/FVIIa complex structure during molecular dynamics (MD) simulation. Evolutionary conservation of the molecular mechanisms for TF-induced allosteric FVIIa activation between humans and extant vertebrate jawless fish (lampreys), where blood coagulation emerged more than 500 million years ago, is unknown and of considerable interest. Objective: To model the sTf/FVIIa complex from cloned Petromyzon marinus lamprey sequences, and with comparisons to human sTF/FVlla investigate conservation of allosteric mechanisms of FVIIa activity enhancement by soluble TF using MD simulations. Methods: Full-length cDNAs of lamprey tf and f7 were cloned and characterized. Comparative models of lamprey sTf/FVIIa complex and free FVIIa were determined based on constructed human sTF/FVIIa complex and free FVIIa models, used in full-atomic MD simulations, and characterized using dynamic network analysis approaches. Results: Allosteric paths of correlated motion from Tf contact points in lamprey sTf/FVIIa to the FVIIa active site were determined and quantified, and were found to encompass residue-residue interactions along significantly similar paths compared with human. Conclusions: Despite low conservation of residues between lamprey and human proteins, 30% TF and 39% FVII, the structural and protein dynamic effects of TF activation of FVIIa appear conserved and, moreover, present in extant vertebrate proteins from 500 million years ago when TF/FVIIa-initiated extrinsic pathway blood coagulation emerged.

We sought to determine the effects of alternative resuscitation strategies on microcirculatory perfusion and examine any association between microcirculatory perfusion and mortality in sepsis.

Roundabout guidance receptor 4 (Robo4) is an endothelial cell-specific receptor that stabilizes the vasculature in pathological angiogenesis. Although Robo4 has been shown to suppress vascular hyperpermeability induced by vascular endothelial growth factor (VEGF) in angiogenesis, the role of Robo4 in inflammation is poorly understood. In this study, we investigated the role of Robo4 in vascular hyperpermeability during inflammation. Endotoxemia models using Robo4-/- mice showed increased mortality and vascular leakage. In endothelial cells, Robo4 suppressed tumor necrosis factor α (TNFα)-induced hyperpermeability by stabilizing VE-cadherin at cell junctions, and deletion assays revealed that the C-terminus of Robo4 was involved in this suppression. Through binding and localization assays, we demonstrated that in endothelial cells, Robo4 binds to TNF receptor-associated factor 7 (TRAF7) through interaction with the C-terminus of Robo4. Gain- and loss-of-function studies of TRAF7 with or without Robo4 expression showed that TRAF7 is required for Robo4-mediated suppression of hyperpermeability. Taken together, our results demonstrate that the Robo4-TRAF7 complex is a novel negative regulator of inflammatory hyperpermeability. We propose this complex as a potential future target for protection against inflammatory diseases.

The use of in vivo videomicroscopy at the bedside has demonstrated microcirculatory flow disturbances in sepsis. The ability of in vivo videomicroscopy to detect changes in the prevalence of rolling and adhered leukocytes that occur in sepsis is not well-described in humans. We sought to (1) develop methodology for accessing and quantifying sublingual leukocyte rolling and adherence with sidestream dark field (SDF) imaging; (2) compare the number of rolling and adhered leukocytes between patients with septic shock and non-infected controls; and (3) compare the number of rolling and adhered leukocytes between survivors and non-survivors of septic shock.

Although transcription factors regulating endothelial cell (EC)-specific gene expression have been identified, it is not known how those factors induce EC-specificity. We previously reported that DNA hypomethylation of the proximal promoter elicits EC-specific expression of Roundabout4 (Robo4). However, the mechanisms establishing EC-specific hypomethylation of the Robo4 promoter remain unknown. In this study, we demonstrated that the hypermethylated Robo4 proximal promoter is demethylated as human iPS cells differentiate into endothelial cells. Reporter assays demonstrated that ETV2, an ETS family transcription factor, bound to ETS motifs in the proximal promoter and activated Robo4 expression. Immunoprecipitation demonstrated direct interaction between ETV2 and methylcytosine-converting enzymes TET1 and TET2. Adenoviral expression of ETV2-TET1/TET2 complexes demethylated the Robo4 promoter and induced Robo4 expression in non-ECs. In summary, we propose a novel regulatory model of EC-specific gene expression via promoter demethylation induced by ETV2-TET1/TET2 complexes during endothelial differentiation.