MASP2 inhibition by narsoplimab suppresses endotheliopathies characteristic of transplant-associated thrombotic microangiopathy: in vitro and ex vivo evidence.

Transplant-associated thrombotic microangiopathy (TA-TMA) is an endotheliopathy complicating up to 30% of allogeneic hematopoietic stem cell transplants (alloHSCT). Positive feedback loops among complement, pro-inflammatory, pro-apoptotic, and coagulation cascade likely assume dominant roles at different disease stages. We hypothesized that mannose-binding lectin-associated serine protease 2 (MASP2), principal activator of the lectin complement system, is involved in the microvascular endothelial cell (MVEC) injury characteristic of TA-TMA through pathways that are susceptible to suppression by anti-MASP2 monoclonal antibody narsoplimab. Pre-treatment plasmas from 8 of 9 TA-TMA patients achieving a complete TMA response in a narsoplimab clinical trial activated caspase 8, the initial step in apoptotic injury, in human MVEC. This was reduced to control levels following narsoplimab treatment in 7 of the 8 subjects. Plasmas from 8 individuals in an observational TA-TMA study, but not 8 alloHSCT subjects without TMA, similarly activated caspase 8, which was blocked in vitro by narsoplimab. mRNA sequencing of MVEC exposed to TA-TMA or control plasmas with and without narsoplimab suggested potential mechanisms of action. The top 40 narsoplimab-affected transcripts included upregulation of SerpinB2, which blocks apoptosis by inactivating procaspase 3; CHAC1, which inhibits apoptosis in association with mitigation of oxidative stress responses; and pro-angiogenesis proteins TM4SF18, ASPM, and ESM1. Narsoplimab also suppressed transcripts encoding pro-apoptotic and pro-inflammatory proteins ZNF521, IL1R1, Fibulin-5, aggrecan, SLC14A1, and LOX1, and TMEM204, which disrupts vascular integrity. Our data suggest benefits to narsoplimab use in high-risk TA-TMA and provide a potential mechanistic basis for the clinical efficacy of narsoplimab in this disorder.

Premortem Skin Biopsy Assessing Microthrombi, Interferon Type I Antiviral and Regulatory Proteins, and Complement Deposition Correlates with Coronavirus Disease 2019 Clinical Stage.

Apart from autopsy, tissue correlates of coronavirus disease 2019 (COVID-19) clinical stage are lacking. In the current study, cutaneous punch biopsy specimens of 15 individuals with severe/critical COVID-19 and six with mild/moderate COVID-19 were examined. Evidence for arterial and venous microthrombi, deposition of C5b-9 and MASP2 (representative of alternative and lectin complement pathways, respectively), and differential expression of interferon type I-driven antiviral protein MxA (myxovirus resistance A) versus SIN3A, a promoter of interferon type I-based proinflammatory signaling, were assessed. Control subjects included nine patients with sepsis-related acute respiratory distress syndrome (ARDS) and/or acute kidney injury (AKI) pre-COVID-19. Microthrombi were detected in 13 (87%) of 15 patients with severe/critical COVID-19 versus zero of six patients with mild/moderate COVID-19 (P < 0.001) and none of the nine patients with pre-COVID-19 ARDS/AKI (P < 0.001). Cells lining the microvasculature staining for spike protein of severe acute respiratory syndrome coronavirus 2, the etiologic agent of COVID-19, also expressed tissue factor. C5b-9 deposition occurred in 13 (87%) of 15 patients with severe/critical COVID-19 versus zero of six patients with mild/moderate COVID-19 (P < 0.001) and none of the nine patients with pre-COVID-19 ARDS/AKI (P < 0.001). MASP2 deposition was also restricted to severe/critical COVID-19 cases. MxA expression occurred in all six mild/moderate versus two (15%) of 13 severe/critical cases (P < 0.001) of COVID-19. In contrast, SIN3A was restricted to severe/critical COVID-19 cases co-localizing with severe acute respiratory syndrome coronavirus 2 spike protein. SIN3A was also elevated in plasma of patients with severe/critical COVID-19 versus control subjects (P ≤ 0.02). In conclusion, the study identified premortem tissue correlates of COVID-19 clinical stage using skin. If validated in a longitudinal cohort, this approach could identify individuals at risk for disease progression and enable targeted interventions.

Angiopoietin 2 Is Associated with Vascular Necroptosis Induction in Coronavirus Disease 2019 Acute Respiratory Distress Syndrome.

Vascular injury is a well-established, disease-modifying factor in acute respiratory distress syndrome (ARDS) pathogenesis. Recently, coronavirus disease 2019 (COVID-19)-induced injury to the vascular compartment has been linked to complement activation, microvascular thrombosis, and dysregulated immune responses. This study sought to assess whether aberrant vascular activation in this prothrombotic context was associated with the induction of necroptotic vascular cell death. To achieve this, proteomic analysis was performed on blood samples from COVID-19 subjects at distinct time points during ARDS pathogenesis (hospitalized at risk, N = 59; ARDS, N = 31; and recovery, N = 12). Assessment of circulating vascular markers in the at-risk cohort revealed a signature of low vascular protein abundance that tracked with low platelet levels and increased mortality. This signature was replicated in the ARDS cohort and correlated with increased plasma angiopoietin 2 levels. COVID-19 ARDS lung autopsy immunostaining confirmed a link between vascular injury (angiopoietin 2) and platelet-rich microthrombi (CD61) and induction of necrotic cell death [phosphorylated mixed lineage kinase domain-like (pMLKL)]. Among recovery subjects, the vascular signature identified patients with poor functional outcomes. Taken together, this vascular injury signature was associated with low platelet levels and increased mortality and can be used to identify ARDS patients most likely to benefit from vascular targeted therapies.

Severe COVID-19: A multifaceted viral vasculopathy syndrome.

The objective of this study was to elucidate the pathophysiology that underlies severe COVID-19 by assessing the histopathology and the in situ detection of infectious SARS-CoV-2 and viral capsid proteins along with the cellular target(s) and host response from twelve autopsies. There were three key findings: 1) high copy infectious virus was limited mostly to the alveolar macrophages and endothelial cells of the septal capillaries; 2) viral spike protein without viral RNA localized to ACE2+ endothelial cells in microvessels that were most abundant in the subcutaneous fat and brain; 3) although both infectious virus and docked viral spike protein was associated with complement activation, only the endocytosed pseudovirions induced a marked up-regulation of the key COVID-19 associated proteins IL6, TNF alpha, IL1 beta, p38, IL8, and caspase 3. Importantly, this microvasculitis was associated with characteristic findings on hematoxylin and eosin examination that included endothelial degeneration and resultant basement membrane zone disruption and reduplication. It is concluded that serious COVID-19 infection has two distinct mechanisms: 1) a microangiopathy of pulmonary capillaries associated with a high infectious viral load where endothelial cell death releases pseudovirions into the circulation, and 2) the pseudovirions dock on ACE2+ endothelial cells most prevalent in the skin/subcutaneous fat and brain that activates the complement pathway/coagulation cascade resulting in a systemic procoagulant state as well as the expression of cytokines that produce the cytokine storm. The data predicts a favorable response to therapies based on either removal of circulating viral proteins and/or blunting of the endothelial-induced response.

Anti-complement C5 therapy with eculizumab in three cases of critical COVID-19.

Respiratory failure and acute kidney injury (AKI) are associated with high mortality in SARS-CoV-2-associated Coronavirus disease 2019 (COVID-19). These manifestations are linked to a hypercoaguable, pro-inflammatory state with persistent, systemic complement activation. Three critical COVID-19 patients recalcitrant to multiple interventions had skin biopsies documenting deposition of the terminal complement component C5b-9, the lectin complement pathway enzyme MASP2, and C4d in microvascular endothelium. Administration of anti-C5 monoclonal antibody eculizumab led to a marked decline in D-dimers and neutrophil counts in all three cases, and normalization of liver functions and creatinine in two. One patient with severe heart failure and AKI had a complete remission. The other two individuals had partial remissions, one with resolution of his AKI but ultimately succumbing to respiratory failure, and another with a significant decline in FiO2 requirements, but persistent renal failure. In conclusion, anti-complement therapy may be beneficial in at least some patients with critical COVID-19.

Defining treatment duration in atypical hemolytic uremic syndrome in adults: a clinical and pathological approach.

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy (TMA) that is driven by uncontrolled activation of the alternative complement pathway, classically in the context of a genetic or autoimmune complement abnormality. Initial guidelines suggested lifelong treatment with the C5 inhibitor eculizumab, which until recently was the only therapy approved by the US Food and Drug Administration and European Medicines Agency for aHUS. However, multicenter observational studies provide compelling evidence that discontinuation of eculizumab, with careful monitoring for recurrence of renal injury, is an option for some patients. Although relapse occurs in 20% to 35% of patients with aHUS after a median of 3 months (range, 1-30 months) following eculizumab cessation, ostensibly irrespective of initial treatment duration, successful rescue with reinstitution of drug has been described in small cohorts if relapse is promptly recognized and eculizumab is immediately re-started. Rates of off-treatment TMA are higher in children than in adults; they are also elevated in those with a personal or family history of aHUS, certain complement mutations or anti-complement factor H autoantibodies, a renal allograft, or extrarenal manifestations of aHUS. Given the complex and unpredictable nature of aHUS, prospective trials defining the optimal treatment duration in diverse settings are required. In the interim, this review-which excludes pediatric patients and hematopoietic stem cell transplant recipients-suggests that eculizumab may be discontinued in some groups of patients; discontinuation should be undertaken on a case-by-case basis and with careful monitoring, following 6 to 12 months of treatment for aHUS that encompasses at least 3 months of normalization of renal function or stabilization of chronic renal disease.

Death from Transfusion-Transmitted Anaplasmosis, New York, USA, 2017.

We report a death from transfusion-transmitted anaplasmosis in a 78-year-old man. The patient died of septic shock 2 weeks after a perioperative transfusion with erythrocytes harboring Anaplasma phagocytophilum. The patient's blood specimens were positive for A. phagocytophilum DNA beginning 7 days after transfusion; serologic testing remained negative until death.

The role of complement activation in thrombosis and hemolytic anemias.

OBJECTIVE: The objective of this study was to describe complement activation in hemostatic and pathologic states of coagulation and in the acquired and congenital hemolytic anemias. METHODS AND RESULTS: We review published and emerging data on the involvement of the classic, alternative and lectin-based complement pathways in coagulation and the hemolytic anemias. The alternative pathway in particular is always "on," at low levels, and is particularly sensitive to hyper-activation in a variety of physiologic and pathologic states including infection, autoimmune disorders, thrombosis and pregnancy, requiring tight control predicated on a variety of soluble and membrane bound regulatory proteins. In acquired hemolytic anemias such as paroxysmal nocturnal hemoglobinuria (PNH) and cold agglutinin disease (CAD), the complement system directly induces red blood cell injury, resulting in intravascular and extravascular hemolysis. In congenital hemolytic anemias such as sickle cell disease and ß-thalassemia, the complement system may also contribute to thrombosis and vascular disease. Complement activation may also lead to a storage lesion in red blood cells prior to transfusion. CONCLUSION: Complement pathways are activated in hemolytic anemias and are closely linked with thrombosis. In acquired disorders such as PNH and possibly CAD, inhibition of the alternative complement pathway improves clinical outcomes and reduces thrombosis risk. Whether complement inhibition has a similar role in congenital hemolytic anemias apart from the atypical hemolytic-uremic (aHUS)-type thrombotic microangiopathies remains to be determined.

Atypical hemolytic uremic syndrome (aHUS): essential aspects of an accurate diagnosis.

Atypical hemolytic uremic syndrome (aHUS), a thrombotic microangiopathy (TMA), is a rare, life-threatening, systemic disease. When unrecognized or inappropriately treated, aHUS has a high degree of morbidity and mortality. aHUS results from chronic, uncontrolled activity of the alternative complement pathway, which activates platelets and damages the endothelium. Two-thirds of aHUS cases are associated with an identifiable complement-activating condition. aHUS is clinically very similar to the other major TMAs: Shiga toxin-producing Escherichia coli (STEC)-HUS, thrombotic thrombocytopenic purpura (TTP), and disseminated intravascular coagulation (DIC). The signs and symptoms of all the TMAs overlap, complicating the differential diagnosis. Clinical identification of a TMA requires documentation of microangiopathic hemolysis accompanied by thrombocytopenia. DIC must be recognized and treated before it is possible to discriminate among the other 3 major TMAs. STEC-HUS can be excluded through testing for Shiga toxin-producing E. coli. aHUS can be distinguished from TTP on the basis of ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity, with a severe decrease characteristic of TTP. This test, as both an activity assay and an inhibitor assay, should be ordered before the initiation of plasma therapy in any patient presenting with a TMA. Finally, it is important to recognize that aHUS remains a clinical diagnosis, but in complex scenarios, tissue biopsy may be a useful adjunct in diagnosis.

Role of the skin biopsy in the diagnosis of atypical hemolytic uremic syndrome.

INTRODUCTION: Atypical hemolytic uremic syndrome (aHUS) is a prototypic thrombotic microangiopathy attributable to complement dysregulation. In the absence of complement inhibition, progressive clinical deterioration occurs. The authors postulated that a biopsy of normal skin could corroborate the diagnosis of aHUS through the demonstration of vascular deposits of C5b-9. MATERIALS AND METHODS: Biopsies of normal skin from 22 patients with and without aHUS were processed for routine light microscopy and immunofluorescent studies. An assessment was made for vascular C5b-9 deposition immunohistochemically and by immunofluorescence. The biopsies were obtained primarily from the forearm and/or deltoid. RESULTS: Patients with classic features of aHUS showed insidious microvascular changes including loose luminal platelet thrombi, except in 2 patients in whom a striking thrombogenic vasculopathy was apparent in biopsied digital ulcers. Extensive microvascular deposits of the membrane attack complex/C5b-9 were identified, excluding 1 patient in whom eculizumab was initiated before biopsy. In 5 of the 7 patients where follow-up was available, the patients exhibited an excellent treatment response to eculizumab. Patients without diagnostic clinical features of aHUS failed to show significant vascular deposits of complement, except 2 patients with thrombotic thrombocytopenic purpura including 1 in whom a Factor H mutation was identified. CONCLUSIONS: In a clinical setting where aHUS is an important diagnostic consideration, extensive microvascular deposition of C5b-9 supports the diagnosis of either aHUS or a subset of thrombotic thrombocytopenic purpura patients with concomitant complement dysregulation; significant vascular C5b-9 deposition predicts clinical responsiveness to eculizumab.

Hematopoietic transplant-associated thrombotic microangiopathy: case report and review of diagnosis and treatments.

Transplant-associated thrombotic microangiopathy (TA-TMA) refers to inflammatory and thrombotic diseases of the microvasculature characterized by hemolytic anemia, thrombocytopenia, and evidence of organ damage, particularly acute renal failure. This syndrome occurs in 10% to 20% of patients with allogeneic hematopoietic stem cell transplants (HSCTs). It is much less frequent in the autologous setting. TA-TMAs present diagnostic challenges because they may not clearly fall into one of the categories of the 2 major TMAs: atypical hemolytic uremic syndrome (aHUS) and thrombotic thrombocytopenic purpura (TTP). In addition, complications of the transplant itself, including infection, graft-versus-host disease, and disseminated intravascular coagulation, as well as the side effects of immunosuppressive drugs, can mimic a TMA. Because the pathophysiology of TA-TMA is poorly understood, current treatment options are suboptimal, and the condition carries a very high mortality rate. In 3 recent case summaries, the median acute response rate to plasma exchange was as high as 55%, but this therapy failed to alter underlying disease pathology and had little impact on overall mortality, which was approximately 80%. Indeed, the vast majority of TA-TMA patients lack suppression of ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity to less than 5% to 10% of normal and do not have a complete response to plasma exchange, characteristics indicating that a TTP-like disorder is not involved. Recent advances in the treatment of aHUS may offer a therapeutic option in the aHUS-like TMAs associated with HSCTs. These issues are discussed in the context of a patient recently evaluated and treated at our institution; the case serves to illustrate the difficulties associated with the diagnosis and treatment of TA-TMA.

Defibrotide mitigates endothelial cell injury induced by plasmas from patients with COVID-19 and related vasculopathies.

BACKGROUND AND OBJECTIVES: COVID-19 progression is characterized by systemic small vessel arterial and venous thrombosis. Microvascular endothelial cell (MVEC) activation and injury, platelet activation, and histopathologic features characteristic of acute COVID-19 also describe certain thrombotic microangiopathies, including atypical hemolytic-uremic syndrome (aHUS), thrombotic thrombocytopenic purpura (TTP), and hematopoietic stem cell transplant (HSCT)-associated veno-occlusive disease (VOD). We explored the effect of clinically relevant doses of defibrotide, approved for HSCT-associated VOD, on MVEC activation/injury. METHODS: Human dermal MVEC were exposed to plasmas from patients with acute TMAs or acute COVID-19 in the presence and absence of defibrotide (5µg/ml) and caspase 8, a marker of EC activation and apoptosis, was assessed. RNAseq was used to explore potential mechanisms of defibrotide activity. RESULTS: Defibrotide suppressed TMA plasma-induced caspase 8 activation in MVEC (mean 60.2 % inhibition for COVID-19; p = 0.0008). RNAseq identified six major cellular pathways associated with defibrotide's alteration of COVID-19-associated MVEC changes: TNF-α signaling; IL-17 signaling; extracellular matrix (ECM)-EC receptor and platelet receptor interactions; ECM formation; endothelin activity; and fibrosis. Communications across these pathways were revealed by STRING analyses. Forty transcripts showing the greatest changes induced by defibrotide in COVID-19 plasma/MVEC cultures included: claudin 14 and F11R (JAM), important in maintaining EC tight junctions; SOCS3 and TNFRSF18, involved in suppression of inflammation; RAMP3 and transgelin, which promote angiogenesis; and RGS5, which regulates caspase activation and apoptosis. CONCLUSION: Our data, in the context of a recent clinical trial in severe COVID-19, suggest benefits to further exploration of defibrotide and these pathways in COVID-19 and related endotheliopathies.

Noncanonical Wnt signaling promotes osteoclast differentiation and is facilitated by the human immunodeficiency virus protease inhibitor ritonavir.

Wnt proteins that signal via the canonical Wnt/ß-catenin pathway directly regulate osteoblast differentiation. In contrast, most studies of Wnt-related effects on osteoclasts involve indirect changes. While investigating bone mineral density loss in the setting of human immunodeficiency virus (HIV) infection and its treatment with the protease inhibitor ritonavir (RTV), we observed that RTV decreased nuclear localization of ß-catenin, critical to canonical Wnt signaling, in primary human and murine osteoclast precursors. This occurred in parallel with upregulation of Wnt5a and Wnt5b transcripts. These Wnts typically stimulate noncanonical Wnt signaling, and this can antagonize the canonical Wnt pathway in many cell types, dependent upon Wnt receptor usage. We now document RTV-mediated upregulation of Wnt5a/b protein in osteoclast precursors. Recombinant Wnt5b and retrovirus-mediated expression of Wnt5a enhanced osteoclast differentiation from human and murine monocytic precursors, processes facilitated by RTV. In contrast, canonical Wnt signaling mediated by Wnt3a suppressed osteoclastogenesis. Both RTV and Wnt5b inhibited canonical, ß-catenin/T cell factor-based Wnt reporter activation in osteoclast precursors. RTV- and Wnt5-induced osteoclast differentiation were dependent upon the receptor-like tyrosine kinase Ryk, suggesting that Ryk may act as a Wnt5a/b receptor in this context. This is the first demonstration of a direct role for Wnt signaling pathways and Ryk in regulation of osteoclast differentiation, and its modulation by a clinically important drug, ritonavir. These studies also reveal a potential role for noncanonical Wnt5a/b signaling in acceleration of bone mineral density loss in HIV-infected individuals, and illuminate a potential means of influencing such processes in disease states that involve enhanced osteoclast activity.

Atypical hemolytic uremic syndrome (aHUS): making the diagnosis.

Atypical hemolytic uremic syndrome (aHUS) is a major thrombotic microangiopathy (TMA). A TMA is recognized by the laboratory signs of microangiopathic hemolysis, as indicated by schistocytes, elevated lactate dehydrogenase, low haptoglobin, and low hemoglobin, plus thrombocytopenia and accompanying signs and symptoms of organ system involvement. aHUS results from chronic, uncontrolled activity of the alternative complement pathway. In most patients, this defect is related to a genetic deficiency in one or more soluble and/or membrane-bound complement regulatory proteins. Complement factor H is most frequently implicated. Clinically, aHUS is often indistinguishable from the other TMAs: Shiga toxin­producing Escherichia coli (STEC) hemolytic uremic syndrome and thrombotic thrombocytopenic purpura (TTP). TTP and aHUS are associated with high morbidity and mortality. aHUS has a distinct pathology from TTP. In nearly all patients, aHUS can be distinguished from TTP on the basis of an ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) enzyme activity measurement. It is essential that aHUS and TTP be differentiated quickly, as they require markedly divergent treatments. The standard treatment for TTP is plasma exchange, a therapy that has no role for patients with a diagnosis of aHUS established by ADAMTS13 activity levels.

Long COVID endotheliopathy: hypothesized mechanisms and potential therapeutic approaches.

SARS-CoV-2-infected individuals may suffer a multi-organ system disorder known as "long COVID" or post-acute sequelae of SARS-CoV-2 infection (PASC). There are no standard treatments, the pathophysiology is unknown, and incidence varies by clinical phenotype. Acute COVID-19 correlates with biomarkers of systemic inflammation, hypercoagulability, and comorbidities that are less prominent in PASC. Macrovessel thrombosis, a hallmark of acute COVID-19, is less frequent in PASC. Female sex at birth is associated with reduced risk for acute COVID-19 progression, but with increased risk of PASC. Persistent microvascular endotheliopathy associated with cryptic SARS-CoV-2 tissue reservoirs has been implicated in PASC pathology. Autoantibodies, localized inflammation, and reactivation of latent pathogens may also be involved, potentially leading to microvascular thrombosis, as documented in multiple PASC tissues. Diagnostic assays illuminating possible therapeutic targets are discussed.

Atypical hemolytic uremic syndrome after myomectomy: A case report.

Atypical hemolytic uremic syndrome (aHUS) is a rare form of thrombotic microangiopathy due to inability to regulate the complement cascade, resulting in thrombocytopenia, intravascular hemolysis, and end-organ damage. Over 70% of cases are associated with mutations in complement or complement regulatory proteins, and some two-thirds have recognized complement-activating conditions triggering an aHUS event. We describe a case of aHUS after abdominal myomectomy in a 42-year-old woman that was managed with plasma exchange and eculizumab (an anti-C5 monoclonal antibody). The diagnosis was confirmed by biopsy of normal-appearing deltoid skin that demonstrated microvascular C5b-9 deposition, diagnostic of systemic complement pathway activation. Although extremely uncommon following gynecologic surgery, aHUS should be considered in the setting of postoperative oliguric acute kidney injury, as prompt diagnosis is necessary to prevent significant morbidity and mortality.

The skin as a critical window in unveiling the pathophysiologic principles of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of coronavirus disease 2019 (COVID-19), is a single-stranded RNA virus whose sequence is known. COVID-19 is associated with a heterogeneous clinical phenotype ranging from asymptomatic to fatal disease. It appears that access to nasopharyngeal respiratory epithelia expressing angiotensin-converting enzyme (ACE) 2, the receptor for SARS-CoV-2, is followed by viral replication in the pulmonary alveolar septal capillary bed. We have demonstrated in earlier studies that incomplete viral particles, termed pseudovirions, dock to deep subcutaneous and other vascular beds, potentially contributing to the prothrombotic state and systemic complement activation that characterizes severe and critical COVID-19. A variety of skin eruptions have been described in the setting of SARS-CoV-2 infection and more recently, after COVID-19 vaccination. The vaccines deliver a laboratory-synthesized mRNA that encodes a protein that is identical to the spike glycoprotein of SARS-CoV-2, allowing the production of immunogenic spike glycoprotein that will then elicit T cell and B cell adaptive immune responses. In this contribution, we review an array of cutaneous manifestations of COVID-19 that provide an opportunity to study critical pathophysiologic mechanisms that underlie all clinical facets of COVID-19, ranging from asymptomatic/mild to severe and critical COVID-19. We classify cutaneous COVID-19 according to underlying pathophysiologic principles. In this regard we propose three main pathways: (1) complement mediated thrombotic vascular injury syndromes deploying the alternative and mannan binding lectin pathways and resulting in the elaboration of cytokines like interleukin 6 from endothelium in the setting of severe and critical COVID-19 and (2) the robust T cell and type I interferon-driven inflammatory and (3) humoral-driven immune complex mediated vasculitic cutaneous reactions observed with mild and moderate COVID-19. Presented are novel data on cutaneous vaccine reactions that manifest a clinical and morphologic parallel with similar eruptions observed in patients with mild and moderate COVID-19 and in some cases represent systemic eczematoid hypersensitivity reactions to a putative vaccine-based antigen versus unmasking subclinical hypersensitivity due to immune enhancing effects of the vaccine. Finally, we demonstrate for the first time the localization of human synthesized spike glycoprotein after the COVID-19 vaccine to the cutaneous and subcutaneous vasculature confirming the ability of SARS-CoV-2 spike glycoprotein to bind endothelium in the absence of intact virus.

Tissue factor upregulation is associated with SARS-CoV-2 in the lungs of COVID-19 patients.

BACKGROUND: A substantial proportion of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop severe/critical coronavirus disease 2019 (COVID-19) characterized by acute respiratory distress syndrome (ARDS) with thrombosis. OBJECTIVES: We tested the hypothesis that SARS-CoV-2--induced upregulation of tissue factor (TF) expression may be responsible for thrombus formation in COVID-19. METHODS: We compared autopsy lung tissues from 11 patients with COVID-19--associated ARDS with samples from 6 patients with ARDS from other causes (non-COVID-19 ARDS) and 11 normal control lungs. RESULTS: Dual RNA in situ hybridization for SARS-CoV-2 and TF identified sporadic clustered SARS-CoV-2 with prominent co-localization of SARS-CoV-2 and TF RNA. TF expression was 2-fold higher in COVID-19 than in non-COVID-19 ARDS lungs (P = .017) and correlated with the intensity of SARS-CoV-2 staining (R2  = .36, P = .04). By immunofluorescence, TF protein expression was 2.1-fold higher in COVID-19 versus non-COVID-19 ARDS lungs (P = .0048) and 11-fold (P < .001) higher than control lungs. Fibrin thrombi and thrombi positive for platelet factor 4 (PF4) were found in close proximity to regions expressing TF in COVID-19 ARDS lung, and correlated with TF expression (fibrin, R2  = .52, P < .001; PF4, R2  = .59, P < .001). CONCLUSIONS: These data suggest that upregulation of TF expression is associated with thrombus formation in COVID-19 lungs and could be a key therapeutic target. Correlation of TF expression with SARS-CoV-2 in lungs of COVID-19 patients also raises the possibility of direct TF induction by the virus.