Increased complement activation is a distinctive feature of severe SARS-CoV-2 infection.

Complement activation has been implicated in the pathogenesis of severe SARS-CoV-2 infection. However, it remains to be determined whether increased complement activation is a broad indicator of critical illness (and thus, no different in COVID-19). It is also unclear which pathways are contributing to complement activation in COVID-19, and, if complement activation is associated with certain features of severe SARS-CoV-2 infection, such as endothelial injury and hypercoagulability. To address these questions, we investigated complement activation in the plasma from patients with COVID-19 prospectively enrolled at two tertiary care centers. We compared our patients to two non-COVID cohorts: (a) patients hospitalized with influenza, and (b) patients admitted to the intensive care unit (ICU) with acute respiratory failure requiring invasive mechanical ventilation (IMV). We demonstrate that circulating markers of complement activation (i.e., sC5b-9) are elevated in patients with COVID-19 compared to those with influenza and to patients with non-COVID-19 respiratory failure. Further, the results facilitate distinguishing those who are at higher risk of worse outcomes such as requiring ICU admission, or IMV. Moreover, the results indicate enhanced activation of the alternative complement pathway is most prevalent in patients with severe COVID-19 and is associated with markers of endothelial injury (i.e., Ang2) as well as hypercoagulability (i.e., thrombomodulin and von Willebrand factor). Our findings identify complement activation to be a distinctive feature of COVID-19, and provide specific targets that may be utilized for risk prognostication, drug discovery and personalized clinical trials.

A neutrophil activation signature predicts critical illness and mortality in COVID-19.

Pathologic immune hyperactivation is emerging as a key feature of critical illness in COVID-19, but the mechanisms involved remain poorly understood. We carried out proteomic profiling of plasma from cross-sectional and longitudinal cohorts of hospitalized patients with COVID-19 and analyzed clinical data from our health system database of more than 3300 patients. Using a machine learning algorithm, we identified a prominent signature of neutrophil activation, including resistin, lipocalin-2, hepatocyte growth factor, interleukin-8, and granulocyte colony-stimulating factor, which were the strongest predictors of critical illness. Evidence of neutrophil activation was present on the first day of hospitalization in patients who would only later require transfer to the intensive care unit, thus preceding the onset of critical illness and predicting increased mortality. In the health system database, early elevations in developing and mature neutrophil counts also predicted higher mortality rates. Altogether, these data suggest a central role for neutrophil activation in the pathogenesis of severe COVID-19 and identify molecular markers that distinguish patients at risk of future clinical decompensation.

Factor V activity in apheresis platelets: Implications for management of FV deficiency.

BACKGROUND: Allogeneic platelet (PLT) infusion is a strategy to raise Factor V (FV) levels in patients with congenital FV deficiency. However, since FV is labile in vitro, we hypothesized that FV activity could be low in PLT units. STUDY DESIGN AND METHODS: FV activity was tested using a prothrombin time-based platform in the supernatant and platelet lysate (PL) of apheresis PLT units (16 units stored in PLT additive solution with acetate and phosphate [PAS-C] and 10 units stored in plasma only), on post-collection days 3-6. Statistical analysis was performed using Student's t test (P < .05). RESULTS: FV activity was severely diminished in PAS-C PLTs (N = 16) supernatant (3.70% ± 1.02%) and PL (3.26% ± 1.02%). FV activity in plasma-only PLTs (N = 10) was lower in both supernatant (44.55% ± 6.46%) and lysate (39.67% ± 6.33%) relative to normal plasma levels, but both were significantly higher (P < .0001) compared to PAS-C PLTs. In a separate set of experiments, FV activity in PAS-C PLTs examined serially over storage time (N = 3 for these experiments) showed that FV levels were reduced by day 3 and not significantly different by day 5 of storage (Day 3 supernatant 5.03% ± 1.41%; Day 5 supernatant: 3.10% ± 0.57%; P = .2; Day 3 lysate: 3.89% ± 1.03%; Day 5 lysate: 2.61% ± 0.41%; P = .4). CONCLUSION: Plasma should be considered over PLTs as first-line therapy for non-complex FV deficiency-associated hemorrhage. If PLTs are considered for transfusion, plasma-only PLT units should be preferentially utilized, as PAS-C PLT have near-absent FV activity.

Circulating markers of angiogenesis and endotheliopathy in COVID-19.

Increase in thrombotic and microvascular complications is emerging to be a key feature of patients with critical illness associated with COVID-19 infection. While endotheliopathy is thought to be a key factor of COVID-19-associated coagulopathy, markers indicative of this process that are prognostic of disease severity have not been well-established in this patient population. Using plasma profiling of patients with COVID-19, we identified circulating markers that segregated with disease severity: markers of angiogenesis (VEGF-A, PDGF-AA and PDGF-AB/BB) were elevated in hospitalized patients with non-critical COVID-19 infection, while markers of endothelial injury (angiopoietin-2, FLT-3L, PAI-1) were elevated in patients with critical COVID-19 infection. In survival analysis, elevated markers of endothelial injury (angiopoietin-2, follistatin, PAI-1) were strongly predictive of in-hospital mortality. Our findings demonstrate that non-critical and critical phases of COVID-19 disease may be driven by distinct mechanisms involving key aspects of endothelial cell function, and identify drivers of COVID-19 pathogenesis and potential targets for future therapies.

A neutrophil activation signature predicts critical illness and mortality in COVID-19.

Pathologic immune hyperactivation is emerging as a key feature of critical illness in COVID-19, but the mechanisms involved remain poorly understood. We carried out proteomic profiling of plasma from cross-sectional and longitudinal cohorts of hospitalized patients with COVID-19 and analyzed clinical data from our health system database of over 3,300 patients. Using a machine learning algorithm, we identified a prominent signature of neutrophil activation, including resistin, lipocalin-2, HGF, IL-8, and G-CSF, as the strongest predictors of critical illness. Neutrophil activation was present on the first day of hospitalization in patients who would only later require transfer to the intensive care unit, thus preceding the onset of critical illness and predicting increased mortality. In the health system database, early elevations in developing and mature neutrophil counts also predicted higher mortality rates. Altogether, we define an essential role for neutrophil activation in the pathogenesis of severe COVID-19 and identify molecular neutrophil markers that distinguish patients at risk of future clinical decompensation.

Circulating Markers of Angiogenesis and Endotheliopathy in COVID-19.

Despite over 9.3 million infected and 479,000 deaths, the pathophysiological factors that determine the wide spectrum of clinical outcomes in COVID-19 remain inadequately defined. Importantly, patients with underlying cardiovascular disease have been found to have worse clinical outcomes,1 and autopsy findings of endotheliopathy as well as angiogenesis in COVID-19 have accumulated.2,3 Nonetheless, circulating vascular markers associated with disease severity and mortality have not been reliably established. To address this limitation and better understand COVID-19 pathogenesis, we report plasma profiling of factors related to the vascular system from a series of patients admitted to Yale-New Haven Hospital with confirmed diagnosis of COVID-19 via PCR, which demonstrate significant increase in markers of angiogenesis and endotheliopathy in patients hospitalized with COVID-19.

Endotheliopathy in COVID-19-associated coagulopathy: evidence from a single-centre, cross-sectional study.

BACKGROUND: An important feature of severe acute respiratory syndrome coronavirus 2 pathogenesis is COVID-19-associated coagulopathy, characterised by increased thrombotic and microvascular complications. Previous studies have suggested a role for endothelial cell injury in COVID-19-associated coagulopathy. To determine whether endotheliopathy is involved in COVID-19-associated coagulopathy pathogenesis, we assessed markers of endothelial cell and platelet activation in critically and non-critically ill patients admitted to the hospital with COVID-19. METHODS: In this single-centre cross-sectional study, hospitalised adult (≥18 years) patients with laboratory-confirmed COVID-19 were identified in the medical intensive care unit (ICU) or a specialised non-ICU COVID-19 floor in our hospital. Asymptomatic, non-hospitalised controls were recruited as a comparator group for biomarkers that did not have a reference range. We assessed markers of endothelial cell and platelet activation, including von Willebrand Factor (VWF) antigen, soluble thrombomodulin, soluble P-selectin, and soluble CD40 ligand, as well as coagulation factors, endogenous anticoagulants, and fibrinolytic enzymes. We compared the level of each marker in ICU patients, non-ICU patients, and controls, where applicable. We assessed correlations between these laboratory results with clinical outcomes, including hospital discharge and mortality. Kaplan-Meier analysis was used to further explore the association between biochemical markers and survival. FINDINGS: 68 patients with COVID-19 were included in the study from April 13 to April 24, 2020, including 48 ICU and 20 non-ICU patients, as well as 13 non-hospitalised, asymptomatic controls. Markers of endothelial cell and platelet activation were significantly elevated in ICU patients compared with non-ICU patients, including VWF antigen (mean 565% [SD 199] in ICU patients vs 278% [133] in non-ICU patients; p<0·0001) and soluble P-selectin (15·9 ng/mL [4·8] vs 11·2 ng/mL [3·1]; p=0·0014). VWF antigen concentrations were also elevated above the normal range in 16 (80%) of 20 non-ICU patients. We found mortality to be significantly correlated with VWF antigen (r = 0·38; p=0·0022) and soluble thrombomodulin (r = 0·38; p=0·0078) among all patients. In all patients, soluble thrombomodulin concentrations greater than 3·26 ng/mL were associated with lower rates of hospital discharge (22 [88%] of 25 patients with low concentrations vs 13 [52%] of 25 patients with high concentrations; p=0·0050) and lower likelihood of survival on Kaplan-Meier analysis (hazard ratio 5·9, 95% CI 1·9-18·4; p=0·0087). INTERPRETATION: Our findings show that endotheliopathy is present in COVID-19 and is likely to be associated with critical illness and death. Early identification of endotheliopathy and strategies to mitigate its progression might improve outcomes in COVID-19. FUNDING: This work was supported by a gift donation from Jack Levin to the Benign Hematology programme at Yale, and the National Institutes of Health.

Changes in inflammatory and immune drivers in response to immunomodulatory therapies in COVID-19.

As the global community strives to discover effective therapies for COVID-19, immunomodulatory strategies have emerged as a leading contender to combat the cytokine storm and improve clinical outcomes in patients with severe disease. Systemic corticosteroids and selective cytokine inhibitory agents have been utilized both as empiric therapies and in clinical trials. While multiple randomized, placebo controlled trials have now demonstrated that corticosteroids improve survival in patients with COVID-19,1, 2 IL-6 inhibition, which gained significant early interest based on observational studies, has not demonstrated reliable efficacy in randomized, placebo controlled trials.3, 4 To better understand the mechanistic basis of immunomodulatory therapies being implemented for treatment of COVID-19, we assessed longitudinal biochemical changes in response to such approaches in hospitalized patients with COVID-19. We demonstrate broad suppression of multiple immunomodulatory factors associated with adverse clinical outcomes in COVID-19 in patients who received corticosteroids, but no such response was seen in patients who either received tocilizumab or no immunomodulatory therapy. Our findings provide early insights into molecular signatures that correlate with immunomodulatory therapies in COVID-19 which may be useful in understanding clinical outcomes in future studies of larger patient cohorts.

Modified approach to fibrinogen replacement in the setting of dysfibrinogenaemia.

Most fibrinogen replacement strategies focus on quantitative deficiencies. A thrombin time (TT) mixing study helped to assess qualitative defects caused by dysfibrinogens. Plasma samples were collected from non-anticoagulated subjects (n=6) meeting laboratory criteria for suspected dysfibrinogenaemia (TT > 22 s; fibrinogen activity <180) and from a control group. TT mixing studies were performed on subject plasma with increasing volumes of pooled normal plasma at 1:2, 1:4 and 1:5 dilutions. No subjects with dysfibrinogenaemia demonstrated a complete TT correction at 1:2, but 50% corrected at 1:4 and 100% at 1:5 dilution. Based on these data, a correction factor (CF), defined as the reciprocal dilution yielding complete correction, was incorporated into our clinical practice formula for fibrinogen dosing in patients with dysfibrinogenaemias. Our study incorporates TT mixing studies for assessment of dysfibrinogens. The addition of a mix-derived CF to classical formulae may better approximate dosing in patients with dysfibrinogenaemia.