Coagulation potential and the integrated omics of extracellular vesicles from COVID-19 positive patient plasma.

Extracellular vesicles (EVs) participate in cell-to-cell communication and contribute toward homeostasis under physiological conditions. But EVs can also contribute toward a wide array of pathophysiology like cancer, sepsis, sickle cell disease, and thrombotic disorders. COVID-19 infected patients are at an increased risk of aberrant coagulation, consistent with elevated circulating levels of ultra-high molecular weight VWF multimers, D-dimer and procoagulant EVs. The role of EVs in COVID-19 related hemostasis may depend on cells of origin, vesicular cargo and size, however this is not well defined. We hypothesized that the procoagulant potential of EV isolates from COVID-19 (+) patient plasmas could be defined by thrombin generation assays. Here we isolated small EVs (SEVs) and large EVs (LEVs) from hospitalized COVID-19 (+) patient (n = 21) and healthy donor (n = 20) plasmas. EVs were characterized by flow cytometry, Transmission electron microscopy, nanoparticle tracking analysis, plasma thrombin generation and a multi-omics approach to define coagulation potential. These data were consistent with differences in EV metabolite, lipid, and protein content when compared to healthy donor plasma isolated SEVs and LEVs. Taken together, the effect of EVs on plasma procoagulant potential as defined by thrombin generation and supported by multi-omics is enhanced in COVID-19. Further, we observe that this effect is driven both by EV size and phosphatidyl serine.

Cross-talk between red blood cells and plasma influences blood flow and omics phenotypes in severe COVID-19.

Coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and can affect multiple organs, among which is the circulatory system. Inflammation and mortality risk markers were previously detected in COVID-19 plasma and red blood cells (RBCs) metabolic and proteomic profiles. Additionally, biophysical properties, such as deformability, were found to be changed during the infection. Based on such data, we aim to better characterize RBC functions in COVID-19. We evaluate the flow properties of RBCs in severe COVID-19 patients admitted to the intensive care unit by using microfluidic techniques and automated methods, including artificial neural networks, for an unbiased RBC analysis. We find strong flow and RBC shape impairment in COVID-19 samples and demonstrate that such changes are reversible upon suspension of COVID-19 RBCs in healthy plasma. Vice versa, healthy RBCs resemble COVID-19 RBCs when suspended in COVID-19 plasma. Proteomics and metabolomics analyses allow us to detect the effect of plasma exchanges on both plasma and RBCs and demonstrate a new role of RBCs in maintaining plasma equilibria at the expense of their flow properties. Our findings provide a framework for further investigations of clinical relevance for therapies against COVID-19 and possibly other infectious diseases.

Specialized interferon action in COVID-19.

The impacts of interferon (IFN) signaling on COVID-19 pathology are multiple, with both protective and harmful effects being documented. We report here a multiomics investigation of systemic IFN signaling in hospitalized COVID-19 patients, defining the multiomics biosignatures associated with varying levels of 12 different type I, II, and III IFNs. The antiviral transcriptional response in circulating immune cells is strongly associated with a specific subset of IFNs, most prominently IFNA2 and IFNG. In contrast, proteomics signatures indicative of endothelial damage and platelet activation associate with high levels of IFNB1 and IFNA6. Seroconversion and time since hospitalization associate with a significant decrease in a specific subset of IFNs. Additionally, differential IFN subtype production is linked to distinct constellations of circulating myeloid and lymphoid immune cell types. Each IFN has a unique metabolic signature, with IFNG being the most associated with activation of the kynurenine pathway. IFNs also show differential relationships with clinical markers of poor prognosis and disease severity. For example, whereas IFNG has the strongest association with C-reactive protein and other immune markers of poor prognosis, IFNB1 associates with increased neutrophil to lymphocyte ratio, a marker of late severe disease. Altogether, these results reveal specialized IFN action in COVID-19, with potential diagnostic and therapeutic implications.

Evidence for structural protein damage and membrane lipid remodeling in red blood cells from COVID-19 patients

The SARS-CoV-2 beta coronavirus is the etiological driver of COVID-19 disease, which is primarily characterized by shortness of breath, persistent dry cough, and fever. Because they transport oxygen, red blood cells (RBCs) may play a role in the severity of hypoxemia in COVID-19 patients. The present study combines state-of-the-art metabolomics, proteomics, and lipidomics approaches to investigate the impact of COVID-19 on RBCs from 23 healthy subjects and 29 molecularly-diagnosed COVID-19 patients. RBCs from COVID-19 patients had increased levels of glycolytic intermediates, accompanied by oxidation and fragmentation of ankyrin, spectrin beta, and the N-terminal cytosolic domain of band 3 (AE1). Significantly altered lipid metabolism was also observed, especially short and medium chain saturated fatty acids, acyl-carnitines, and sphingolipids. Nonetheless, there were no alterations of clinical hematological parameters, such as RBC count, hematocrit, and mean corpuscular hemoglobin concentration, with only minor increases in mean corpuscular volume. Taken together, these results suggest a significant impact of SARS-CoV-2 infection on RBC structural membrane homeostasis at the protein and lipid levels. Increases in RBC glycolytic metabolites are consistent with a theoretically improved capacity of hemoglobin to off-load oxygen as a function of allosteric modulation by high-energy phosphate compounds, perhaps to counteract COVID-19-induced hypoxia. Conversely, because the N-terminus of AE1 stabilizes deoxyhemoglobin and finely tunes oxygen off-loading, RBCs from COVID-19 patients may be incapable of responding to environmental variations in hemoglobin oxygen saturation when traveling from the lungs to peripheral capillaries and, as such, may have a compromised capacity to transport and deliver oxygen.

The COVIDome Explorer researcher portal.

COVID-19 pathology involves dysregulation of diverse molecular, cellular, and physiological processes. To expedite integrated and collaborative COVID-19 research, we completed multi-omics analysis of hospitalized COVID-19 patients, including matched analysis of the whole-blood transcriptome, plasma proteomics with two complementary platforms, cytokine profiling, plasma and red blood cell metabolomics, deep immune cell phenotyping by mass cytometry, and clinical data annotation. We refer to this multidimensional dataset as the COVIDome. We then created the COVIDome Explorer, an online researcher portal where the data can be analyzed and visualized in real time. We illustrate herein the use of the COVIDome dataset through a multi-omics analysis of biosignatures associated with C-reactive protein (CRP), an established marker of poor prognosis in COVID-19, revealing associations between CRP levels and damage-associated molecular patterns, depletion of protective serpins, and mitochondrial metabolism dysregulation. We expect that the COVIDome Explorer will rapidly accelerate data sharing, hypothesis testing, and discoveries worldwide.

Seroconversion stages COVID19 into distinct pathophysiological states.

COVID19 is a heterogeneous medical condition involving diverse underlying pathophysiological processes including hyperinflammation, endothelial damage, thrombotic microangiopathy, and end-organ damage. Limited knowledge about the molecular mechanisms driving these processes and lack of staging biomarkers hamper the ability to stratify patients for targeted therapeutics. We report here the results of a cross-sectional multi-omics analysis of hospitalized COVID19 patients revealing that seroconversion status associates with distinct underlying pathophysiological states. Low antibody titers associate with hyperactive T cells and NK cells, high levels of IFN alpha, gamma and lambda ligands, markers of systemic complement activation, and depletion of lymphocytes, neutrophils, and platelets. Upon seroconversion, all of these processes are attenuated, observing instead increases in B cell subsets, emergency hematopoiesis, increased D-dimer, and hypoalbuminemia. We propose that seroconversion status could potentially be used as a biosignature to stratify patients for therapeutic intervention and to inform analysis of clinical trial results in heterogenous patient populations.

Serum Proteomics in COVID-19 Patients: Altered Coagulation and Complement Status as a Function of IL-6 Level.

Over 5 million people around the world have tested positive for the beta coronavirus SARS-CoV-2 as of May 29, 2020, a third of which are in the United States alone. These infections are associated with the development of a disease known as COVID-19, which is characterized by several symptoms, including persistent dry cough, shortness of breath, chills, muscle pain, headache, loss of taste or smell, and gastrointestinal distress. COVID-19 has been characterized by elevated mortality (over 100 thousand people have already died in the US alone), mostly due to thromboinflammatory complications that impair lung perfusion and systemic oxygenation in the most severe cases. While the levels of pro-inflammatory cytokines such as interleukin-6 (IL-6) have been associated with the severity of the disease, little is known about the impact of IL-6 levels on the proteome of COVID-19 patients. The present study provides the first proteomics analysis of sera from COVID-19 patients, stratified by circulating levels of IL-6, and correlated to markers of inflammation and renal function. As a function of IL-6 levels, we identified significant dysregulation in serum levels of various coagulation factors, accompanied by increased levels of antifibrinolytic components, including several serine protease inhibitors (SERPINs). These were accompanied by up-regulation of the complement cascade and antimicrobial enzymes, especially in subjects with the highest levels of IL-6, which is consistent with an exacerbation of the acute phase response in these subjects. Although our results are observational, they highlight a clear increase in the levels of inhibitory components of the fibrinolytic cascade in severe COVID-19 disease, providing potential clues related to the etiology of coagulopathic complications in COVID-19 and paving the way for potential therapeutic interventions, such as the use of pro-fibrinolytic agents. Raw data for this study are available through ProteomeXchange with identifier PXD020601.

Evidence of Structural Protein Damage and Membrane Lipid Remodeling in Red Blood Cells from COVID-19 Patients.

The SARS-CoV-2 beta coronavirus is the etiological driver of COVID-19 disease, which is primarily characterized by shortness of breath, persistent dry cough, and fever. Because they transport oxygen, red blood cells (RBCs) may play a role in the severity of hypoxemia in COVID-19 patients. The present study combines state-of-the-art metabolomics, proteomics, and lipidomics approaches to investigate the impact of COVID-19 on RBCs from 23 healthy subjects and 29 molecularly diagnosed COVID-19 patients. RBCs from COVID-19 patients had increased levels of glycolytic intermediates, accompanied by oxidation and fragmentation of ankyrin, spectrin beta, and the N-terminal cytosolic domain of band 3 (AE1). Significantly altered lipid metabolism was also observed, in particular, short- and medium-chain saturated fatty acids, acyl-carnitines, and sphingolipids. Nonetheless, there were no alterations of clinical hematological parameters, such as RBC count, hematocrit, or mean corpuscular hemoglobin concentration, with only minor increases in mean corpuscular volume. Taken together, these results suggest a significant impact of SARS-CoV-2 infection on RBC structural membrane homeostasis at the protein and lipid levels. Increases in RBC glycolytic metabolites are consistent with a theoretically improved capacity of hemoglobin to off-load oxygen as a function of allosteric modulation by high-energy phosphate compounds, perhaps to counteract COVID-19-induced hypoxia. Conversely, because the N-terminus of AE1 stabilizes deoxyhemoglobin and finely tunes oxygen off-loading and metabolic rewiring toward the hexose monophosphate shunt, RBCs from COVID-19 patients may be less capable of responding to environmental variations in hemoglobin oxygen saturation/oxidant stress when traveling from the lungs to peripheral capillaries and vice versa.

Evidence for structural protein damage and membrane lipid remodeling in red blood cells from COVID-19 patients.

The SARS-CoV-2 beta coronavirus is the etiological driver of COVID-19 disease, which is primarily characterized by shortness of breath, persistent dry cough, and fever. Because they transport oxygen, red blood cells (RBCs) may play a role in the severity of hypoxemia in COVID-19 patients. The present study combines state-of-the-art metabolomics, proteomics, and lipidomics approaches to investigate the impact of COVID-19 on RBCs from 23 healthy subjects and 29 molecularly-diagnosed COVID-19 patients. RBCs from COVID-19 patients had increased levels of glycolytic intermediates, accompanied by oxidation and fragmentation of ankyrin, spectrin beta, and the N-terminal cytosolic domain of band 3 (AE1). Significantly altered lipid metabolism was also observed, especially short and medium chain saturated fatty acids, acyl-carnitines, and sphingolipids. Nonetheless, there were no alterations of clinical hematological parameters, such as RBC count, hematocrit, and mean corpuscular hemoglobin concentration, with only minor increases in mean corpuscular volume. Taken together, these results suggest a significant impact of SARS-CoV-2 infection on RBC structural membrane homeostasis at the protein and lipid levels. Increases in RBC glycolytic metabolites are consistent with a theoretically improved capacity of hemoglobin to off-load oxygen as a function of allosteric modulation by high-energy phosphate compounds, perhaps to counteract COVID-19-induced hypoxia. Conversely, because the N-terminus of AE1 stabilizes deoxyhemoglobin and finely tunes oxygen off-loading, RBCs from COVID-19 patients may be incapable of responding to environmental variations in hemoglobin oxygen saturation when traveling from the lungs to peripheral capillaries and, as such, may have a compromised capacity to transport and deliver oxygen.