Comparison of the coagulopathies associated with COVID-19 and sepsis.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is associated with activation of coagulation that mainly presents as thrombosis. Sepsis is also associated with activation of coagulation that mainly presents as disseminated intravascular coagulation. Many studies have reported increased levels of plasma d-dimer in patients with COVID-19 that is associated with severity, thrombosis, and mortality. OBJECTIVES: The aim of this study was to compare levels of circulating extracellular vesicle tissue factor (EVTF) activity and active plasminogen activator inhibitor 1 (PAI-1) in plasma from patients with COVID-19 or sepsis. METHODS: We measured levels of d-dimer, EVTF activity, and active PAI-1 in plasma samples from patients with COVID-19 (intensive care unit [ICU], N = 15; and non-ICU, N = 20) and patients with sepsis (N = 35). RESULTS: Patients with COVID-19 had significantly higher levels of d-dimer, EVTF activity, and active PAI-1 compared with healthy controls. Patients with sepsis had significantly higher levels of d-dimer and EVTF activity compared with healthy controls. Levels of d-dimer were significantly lower in patients with COVID-19 compared with patients with sepsis. Levels of EVTF activity were significantly higher in ICU patients with COVID-19 compared with patients with sepsis. Levels of active PAI-1 were significantly higher in patients with COVID-19 compared with patients with sepsis. CONCLUSIONS: High levels of both EVTF activity and active PAI-1 may promote thrombosis in patients with COVID-19 due to simultaneous activation of coagulation and inhibition of fibrinolysis. The high levels of active PAI-1 in patients with COVID-19 may limit plasmin degradation of crosslinked fibrin and the release of d-dimer. This may explain the lower levels of D-dimer in patients with COVID-19 compared with patients with sepsis.

Tissue factor activity of small and large extracellular vesicles in different diseases

Background Circulating procoagulant extracellular vesicles (EVs) are increased in diseases, such as cancer, sepsis and coronavirus 2019 (COVID-19). EV tissue factor (TF) activity is associated with disseminated intravascular coagulation in sepsis and venous thrombosis in patients with pancreatic cancer and COVID-19. EVs are commonly isolated by centrifugation at ∼20,000 g. In this study, we analyzed TF activity of two EV populations enriched for large and small EVs in patients with either sepsis, pancreatic cancer or COVID-19. Methods EVs were isolated from plasma by sequential centrifugation at 20,000 g (large EVs, LEVs) and then 100,000 g (small EVs, SEVs). We analyzed EVs from plasma prepared from whole blood samples from healthy individuals with or without lipopolysaccharide (LPS) stimulation as well as EVs from plasma samples from patients with either sepsis, pancreatic cancer or COVID-19. TF-dependent (EV-TF activity) and TF-independent factor Xa (FXa) generation of the EVs was measured. Results LPS increased EV-TF activity in LEVs but not SEVs. Similarly, in two patients with sepsis that had EV-TF activity above the background of the assay we observed EV-TF activity in LEVs but not SEVs. Patients with pancreatic cancer or COVID-19 had circulating EV-TF activity in both LEVs and SEVs. Conclusion We recommend that EVs are isolated from plasma from patients by centrifugation at 100,000 g rather than 20,000 g to obtain a more accurate measure of levels of circulating EV-TF activity.

Tissue factor activity of small and large extracellular vesicles in different diseases.

Background: Circulating procoagulant extracellular vesicles (EVs) are increased in diseases, such as cancer, sepsis, and COVID-19. EV tissue factor (TF) activity is associated with disseminated intravascular coagulation in sepsis and venous thrombosis in patients with pancreatic cancer and COVID-19. EVs are commonly isolated by centrifugation at ∼20,000 g. Objectives: In this study, we analyzed the TF activity of 2 EV populations enriched for large and small EVs in patients with either sepsis, pancreatic cancer, or COVID-19. Methods: EVs were isolated from plasma by sequential centrifugation at 20,000 g (large EVs, LEVs) and then 100,000 g (small EVs, SEVs). We analyzed EVs from plasma prepared from whole blood samples from healthy individuals with or without lipopolysaccharide (LPS) stimulation as well as EVs from plasma samples from patients with either sepsis, pancreatic cancer, or COVID-19. TF-dependent (EV-TF activity) and TF-independent factor Xa (FXa) generation of the EVs was measured. Results: LPS increased EV-TF activity in LEVs but not SEVs. Similarly, in 2 patients with sepsis who had EV-TF activity above the background of the assay we observed EV-TF activity in LEVs but not SEVs. Patients with pancreatic cancer or COVID-19 had circulating EV-TF activity in both LEVs and SEVs. Conclusion: We recommend that EVs are isolated from plasma from patients by centrifugation at 100,000 g rather than 20,000 g to obtain a more accurate measure of levels of circulating EV-TF activity.

Understanding COVID-19-associated coagulopathy.

COVID-19-associated coagulopathy (CAC) is a life-threatening complication of SARS-CoV-2 infection. However, the underlying cellular and molecular mechanisms driving this condition are unclear. Evidence supports the concept that CAC involves complex interactions between the innate immune response, the coagulation and fibrinolytic pathways, and the vascular endothelium, resulting in a procoagulant condition. Understanding of the pathogenesis of this condition at the genomic, molecular and cellular levels is needed in order to mitigate thrombosis formation in at-risk patients. In this Perspective, we categorize our current understanding of CAC into three main pathological mechanisms: first, vascular endothelial cell dysfunction; second, a hyper-inflammatory immune response; and last, hypercoagulability. Furthermore, we pose key questions and identify research gaps that need to be addressed to better understand CAC, facilitate improved diagnostics and aid in therapeutic development. Finally, we consider the suitability of different animal models to study CAC.

Homogeneous surrogate virus neutralization assay to rapidly assess neutralization activity of anti-SARS-CoV-2 antibodies.

The COVID-19 pandemic triggered the development of numerous diagnostic tools to monitor infection and to determine immune response. Although assays to measure binding antibodies against SARS-CoV-2 are widely available, more specific tests measuring neutralization activities of antibodies are immediately needed to quantify the extent and duration of protection that results from infection or vaccination. We previously developed a 'Serological Assay based on a Tri-part split-NanoLuc® (SATiN)' to detect antibodies that bind to the spike (S) protein of SARS-CoV-2. Here, we expand on our previous work and describe a reconfigured version of the SATiN assay, called Neutralization SATiN (Neu-SATiN), which measures neutralization activity of antibodies directly from convalescent or vaccinated sera. The results obtained with our assay and other neutralization assays are comparable but with significantly shorter preparation and run time for Neu-SATiN. As the assay is modular, we further demonstrate that Neu-SATiN enables rapid assessment of the effectiveness of vaccines and level of protection against existing SARS-CoV-2 variants of concern and can therefore be readily adapted for emerging variants.

COVID-19 generates hyaluronan fragments that directly induce endothelial barrier dysfunction.

Vascular injury has emerged as a complication contributing to morbidity in coronavirus disease 2019 (COVID-19). The glycosaminoglycan hyaluronan (HA) is a major component of the glycocalyx, a protective layer of glycoconjugates that lines the vascular lumen and regulates key endothelial cell functions. During critical illness, as in the case of sepsis, enzymes degrade the glycocalyx, releasing fragments with pathologic activities into circulation and thereby exacerbating disease. Here, we analyzed levels of circulating glycosaminoglycans in 46 patients with COVID-19 ranging from moderate to severe clinical severity and measured activities of corresponding degradative enzymes. This report provides evidence that the glycocalyx becomes significantly damaged in patients with COVID-19 and corresponds with severity of disease. Circulating HA fragments and hyaluronidase, 2 signatures of glycocalyx injury, strongly associate with sequential organ failure assessment scores and with increased inflammatory cytokine levels in patients with COVID-19. Pulmonary microvascular endothelial cells exposed to COVID-19 milieu show dysregulated HA biosynthesis and degradation, leading to production of pathological HA fragments that are released into circulation. Finally, we show that HA fragments present at high levels in COVID-19 patient plasma can directly induce endothelial barrier dysfunction in a ROCK- and CD44-dependent manner, indicating a role for HA in the vascular pathology of COVID-19.

Mechanisms of immunothrombosis in COVID-19.

PURPOSE OF REVIEW: Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus-2. Over the past year, COVID-19 has posed a significant threat to global health. Although the infection is associated with mild symptoms in many patients, a significant proportion of patients develop a prothrombotic state due to a combination of alterations in coagulation and immune cell function. The purpose of this review is to discuss the pathophysiological characteristics of COVID-19 that contribute to the immunothrombosis. RECENT FINDINGS: Endotheliopathy during COVID-19 results in increased multimeric von Willebrand factor release and the potential for increased platelet adhesion to the endothelium. In addition, decreased anticoagulant proteins on the surface of endothelial cells further alters the hemostatic balance. Soluble coagulation markers are also markedly dysregulated, including plasminogen activator inhibitor-1 and tissue factor, leading to COVID-19 induced coagulopathy. Platelet hyperreactivity results in increased platelet-neutrophil and -monocyte aggregates further exacerbating the coagulopathy observed during COVID-19. Finally, the COVID-19-induced cytokine storm primes neutrophils to release neutrophil extracellular traps, which trap platelets and prothrombotic proteins contributing to pulmonary thrombotic complications. SUMMARY: Immunothrombosis significantly contributes to the pathophysiology of COVID-19. Understanding the mechanisms behind COVID-19-induced coagulopathy will lead to future therapies for patients.

Cytokine release syndrome in COVID-19: Innate immune, vascular, and platelet pathogenic factors differ in severity of disease and sex.

COVID-19 rapidly emerged as a crippling public health crisis in the last few months, which has presented a series health risk. Understanding of the immune response and biomarker analysis is needed to progress toward understanding disease pathology and developing improved treatment options. The goal of this study is to identify pathogenic factors that are linked to disease severity and patient characteristics. Patients with COVID-19 who were hospitalized from March 17 to June 5, 2020 were analyzed for clinical features of disease and soluble plasma cytokines in association with disease severity and sex. Data from COVID-19 patients with acute illness were examined along with an age- and gender-matched control cohort. We identified a group of 16 soluble factors that were found to be increased in COVID-19 patients compared to controls, whereas 2 factors were decreased. In addition to inflammatory cytokines, we found significant increases in factors known to mediate vasculitis and vascular remodeling (PDGF-AA, PDGF-AB-BB, soluble CD40L (sCD40L), FGF, and IP10). Four factors such as platelet-derived growth factors, fibroblast growth factor-2, and IFN-γ-inducible protein 10 were strongly associated with severe disease and ICU admission. Th2-related factors (IL-4 and IL-13) were increased with IL-4 and sCD40L present at increased levels in males compared with females. Our analysis revealed networking clusters of cytokines and growth factors, including previously unknown roles of vascular and stromal remodeling, activation of the innate immunity, as well activation of type 2 immune responses in the immunopathogenesis of COVID-19. These data highlight biomarker associations with disease severity and sex in COVID-19 patients.

COVID-19 and Sepsis Are Associated With Different Abnormalities in Plasma Procoagulant and Fibrinolytic Activity.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) is associated with derangement in biomarkers of coagulation and endothelial function and has been likened to the coagulopathy of sepsis. However, clinical laboratory metrics suggest key differences in these pathologies. We sought to determine whether plasma coagulation and fibrinolytic potential in patients with COVID-19 differ compared with healthy donors and critically ill patients with sepsis. Approach and Results: We performed comparative studies on plasmas from a single-center, cross-sectional observational study of 99 hospitalized patients (46 with COVID-19 and 53 with sepsis) and 18 healthy donors. We measured biomarkers of endogenous coagulation and fibrinolytic activity by immunoassays, thrombin, and plasmin generation potential by fluorescence and fibrin formation and lysis by turbidity. Compared with healthy donors, patients with COVID-19 or sepsis both had elevated fibrinogen, d-dimer, soluble TM (thrombomodulin), and plasmin-antiplasmin complexes. Patients with COVID-19 had increased thrombin generation potential despite prophylactic anticoagulation, whereas patients with sepsis did not. Plasma from patients with COVID-19 also had increased endogenous plasmin potential, whereas patients with sepsis showed delayed plasmin generation. The collective perturbations in plasma thrombin and plasmin generation permitted enhanced fibrin formation in both COVID-19 and sepsis. Unexpectedly, the lag times to thrombin, plasmin, and fibrin formation were prolonged with increased disease severity in COVID-19, suggesting a loss of coagulation-initiating mechanisms accompanies severe COVID-19. CONCLUSIONS: Both COVID-19 and sepsis are associated with endogenous activation of coagulation and fibrinolysis, but these diseases differently impact plasma procoagulant and fibrinolytic potential. Dysregulation of procoagulant and fibrinolytic pathways may uniquely contribute to the pathophysiology of COVID-19 and sepsis.

Is there a role for the ACE2 receptor in SARS-CoV-2 interactions with platelets?

There is an urgent need to understand the underlying mechanisms contributing to thrombotic and inflammatory complications during COVID-19. Data from independent groups have identified that platelets are hyperreactive during COVID-19. Platelet hyperreactivity is accompanied by changes in platelet gene expression, and enhanced interactions between platelets and leukocytes. In some patients, SARS-CoV-2 mRNA has been detected in platelets. Together, this suggests that SARS-CoV-2 may interact with platelets. However, controversy remains on which receptors mediate SARS-CoV-2 platelet interactions. Most, but not all, transcriptomic and proteomic analyses fail to observe the putative SARS-CoV-2 receptor, angiotensin converting enzyme-2, or the cellular serine protease necessary for viral entry, TMPRSS2, on platelets and megakaryocytes. Interestingly, platelets express other known SARS-CoV-2 receptors, which induce similar patterns of activation to those observed when platelets are incubated with SARS-CoV-2. This article explores these findings and discusses ongoing areas of controversy and uncertainty with regard to SARS-CoV-2 platelet interactions.

COVID-19 patients exhibit reduced procoagulant platelet responses.

BACKGROUND: Emerging evidence implicates dysfunctional platelet responses in thrombotic complications in COVID-19 patients. Platelets are important players in inflammation-induced thrombosis. In particular, procoagulant platelets support thrombin generation and mediate thromboinflammation. OBJECTIVES: To examine if procoagulant platelet formation is altered in COVID-19 patients and if procoagulant platelets contribute to pulmonary thrombosis. PATIENTS/METHODS: Healthy donors and COVID-19 patients were recruited from the University of Utah Hospital System. Platelets were isolated and procoagulant platelet formation measured by annexin V binding as well as mitochondrial function were examined. We utilized mice lacking the ability to form procoagulant platelets (CypDplt-/- ) to examine the role of procoagulant platelets in pulmonary thrombosis. RESULTS AND CONCLUSIONS: We observed that platelets isolated from COVID-19 patients had a reduced ability to become procoagulant compared to those from matched healthy donors, as evidenced by reduced mitochondrial depolarization and phosphatidylserine exposure following dual stimulation with thrombin and convulxin. To understand what impact reduced procoagulant platelet responses might have in vivo, we subjected mice with a platelet-specific deletion of cyclophilin D, which are deficient in procoagulant platelet formation, to a model of pulmonary microvascular thrombosis. Mice with platelets lacking cyclophilin D died significantly faster from pulmonary microvascular thrombosis compared to littermate wild-type controls. These results suggest dysregulated procoagulant platelet responses may contribute to thrombotic complications during SARS-CoV-2 infection.

Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome.

COVID-19 affects millions of patients worldwide, with clinical presentation ranging from isolated thrombosis to acute respiratory distress syndrome (ARDS) requiring ventilator support. Neutrophil extracellular traps (NETs) originate from decondensed chromatin released to immobilize pathogens, and they can trigger immunothrombosis. We studied the connection between NETs and COVID-19 severity and progression. We conducted a prospective cohort study of COVID-19 patients (n = 33) and age- and sex-matched controls (n = 17). We measured plasma myeloperoxidase (MPO)-DNA complexes (NETs), platelet factor 4, RANTES, and selected cytokines. Three COVID-19 lung autopsies were examined for NETs and platelet involvement. We assessed NET formation ex vivo in COVID-19 neutrophils and in healthy neutrophils incubated with COVID-19 plasma. We also tested the ability of neonatal NET-inhibitory factor (nNIF) to block NET formation induced by COVID-19 plasma. Plasma MPO-DNA complexes increased in COVID-19, with intubation (P < .0001) and death (P < .0005) as outcome. Illness severity correlated directly with plasma MPO-DNA complexes (P = .0360), whereas Pao2/fraction of inspired oxygen correlated inversely (P = .0340). Soluble and cellular factors triggering NETs were significantly increased in COVID-19, and pulmonary autopsies confirmed NET-containing microthrombi with neutrophil-platelet infiltration. Finally, COVID-19 neutrophils ex vivo displayed excessive NETs at baseline, and COVID-19 plasma triggered NET formation, which was blocked by nNIF. Thus, NETs triggering immunothrombosis may, in part, explain the prothrombotic clinical presentations in COVID-19, and NETs may represent targets for therapeutic intervention.

Platelet gene expression and function in patients with COVID-19.

There is an urgent need to understand the pathogenesis of coronavirus disease 2019 (COVID-19). In particular, thrombotic complications in patients with COVID-19 are common and contribute to organ failure and mortality. Patients with severe COVID-19 present with hemostatic abnormalities that mimic disseminated intravascular coagulopathy associated with sepsis, with the major difference being increased risk of thrombosis rather than bleeding. However, whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection alters platelet function to contribute to the pathophysiology of COVID-19 remains unknown. In this study, we report altered platelet gene expression and functional responses in patients infected with SARS-CoV-2. RNA sequencing demonstrated distinct changes in the gene-expression profile of circulating platelets of COVID-19 patients. Pathway analysis revealed differential gene-expression changes in pathways associated with protein ubiquitination, antigen presentation, and mitochondrial dysfunction. The receptor for SARS-CoV-2 binding, angiotensin-converting enzyme 2 (ACE2), was not detected by messenger RNA (mRNA) or protein in platelets. Surprisingly, mRNA from the SARS-CoV-2 N1 gene was detected in platelets from 2 of 25 COVID-19 patients, suggesting that platelets may take-up SARS-COV-2 mRNA independent of ACE2. Resting platelets from COVID-19 patients had increased P-selectin expression basally and upon activation. Circulating platelet-neutrophil, -monocyte, and -T-cell aggregates were all significantly elevated in COVID-19 patients compared with healthy donors. Furthermore, platelets from COVID-19 patients aggregated faster and showed increased spreading on both fibrinogen and collagen. The increase in platelet activation and aggregation could partially be attributed to increased MAPK pathway activation and thromboxane generation. These findings demonstrate that SARS-CoV-2 infection is associated with platelet hyperreactivity, which may contribute to COVID-19 pathophysiology.