Thymidine Phosphorylase Mediates SARS-CoV-2 Spike Protein Enhanced Thrombosis in K18-hACE2TG Mice (preprint)

COVID-19, caused by SARS-CoV-2, is associated with arterial and venous thrombosis, thereby increasing mortality. SARS-CoV-2 spike protein (SP), a viral envelope structural protein, is implicated in COVID-19-associated thrombosis. However, the underlying mechanisms remain unknown. Thymidine phosphorylase (TYMP), a newly identified prothrombotic protein, is upregulated in the plasma, platelets, and lungs of patients with COVID-19 but its role in COVID-19-associated thrombosis is not defined. In this study, we found that wild-type SARS-CoV-2 SP significantly promoted arterial thrombosis in K18-hACE2TG mice. SP-accelerated thrombosis was attenuated by inhibition or genetic ablation of TYMP. SP increased the expression of TYMP, resulting in the activation of signal transducer and activator of transcription 3 (STAT3) in BEAS-2B cells, a human bronchial epithelial cell line. A siRNA-mediated knockdown of TYMP inhibited SP-enhanced activation of STAT3. Platelets derived from SP-treated K18-hACE2TG mice also showed increased STAT3 activation, which was reduced by TYMP deficiency. Activated STAT3 is known to potentiate glycoprotein VI signaling in platelets. While SP did not influence ADP- or collagen-induced platelet aggregation, it significantly shortened activated partial thromboplastin time and this change was reversed by TYMP knockout. Additionally, platelet factor 4 (PF4) interacts with SP, which also complexes with TYMP. TYMP enhanced the formation of the SP/PF4 complex, which may potentially augment the prothrombotic and procoagulant effects of PF4. We conclude that SP upregulates TYMP expression, and TYMP inhibition or knockout mitigates SP-enhanced thrombosis. These findings indicate that inhibition of TYMP may be a novel therapeutic strategy for COVID-19-associated thrombosis.

Aggregating Bipolar Opinions through Bipolar Assumption-based Argumentation (preprint)

We introduce a novel method to aggregate Bipolar Argumentation Frameworks expressing opinions of different parties in debates. We use Bipolar Assumption-based Argumentation (ABA) as an all-encompassing formalism for Bipolar Argumentation under different semantics. By leveraging on recent results on judgement aggregation in Social Choice Theory, we prove several preservation results for relevant properties of Bipolar ABA using quota and oligarchic rules. Specifically, we prove (positive and negative) results about the preservation of conflict-free, closed, admissible, preferred, complete, set-stable, well-founded and ideal extensions in Bipolar ABA, as well as the preservation of acceptability, acyclicity and coherence for individual assumptions. Finally, we illustrate our methodology and results in the context of a case study on opinion aggregation for the treatment of long COVID patients.

Unfolded Von Willebrand Factor Binds Protein S and Reduces Anticoagulant Activity (preprint)

Protein S (PS), the critical plasma cofactor for the anticoagulants tissue factor (TF) pathway inhibitor (TFPI) and activated protein C (APC), circulates in two functionally distinct pools: free (anticoagulant) or bound to complement component 4b-binding protein (C4BP) (anti-inflammatory). Acquired free PS deficiency is detected in several viral infections, but its cause is unclear. Here, we identified a shear-dependent interaction between PS and von Willebrand Factor (VWF) by mass spectrometry. Consistently, plasma PS and VWF comigrated in both native and agarose gel electrophoresis. The PS/VWF interaction was blocked by TFPI but not APC, suggesting an interaction with the C-terminal sex hormone binding globulin (SHBG) region of PS. Microfluidic systems, mimicking arterial laminar flow or disrupted turbulent flow, demonstrated that PS stably binds VWF as VWF unfolds under turbulent flow. PS/VWF complexes also localized to platelet thrombi under laminar arterial flow. In thrombin generation-based assays, shearing plasma decreased PS activity, an effect not seen in the absence of VWF. Finally, free PS deficiency in COVID-19 patients, measured using an antibody that binds near the C4BP binding site in SHBG, correlated with changes in VWF, but not C4BP, and with thrombin generation. Our data suggest that PS binds to a shear-exposed site on VWF, thus sequestering free PS and decreasing its anticoagulant activity, which would account for the increased thrombin generation potential. As many viral infections present with free PS deficiency, elevated circulating VWF, and increased vascular shear, we propose that the PS/VWF interaction reported here is a likely contributor to virus-associated thrombotic risk.

Neutrophil adhesion to vessel walls impairs pulmonary circulation in COVID-19 pathology (preprint)

Microthrombus formation is associated with COVID-19 severity; however, the detailed mechanism remains unclear. In this study, we investigated mouse models with severe pneumonia caused by SARS-CoV-2 infection by using our in vivo two-photon imaging system. In the lungs of SARS-CoV-2-infected mice, increased expression of adhesion molecules in intravascular neutrophils prolonged adhesion time to the vessel wall, resulting in platelet aggregation and impaired lung perfusion. Re-analysis of scRNA-seq data from peripheral blood mononuclear cells from COVID-19 cases revealed increased expression levels of CD44 and SELL in neutrophils in severe COVID-19 cases compared to a healthy group, consistent with our observations in the mouse model. These findings suggest that pulmonary perfusion defects caused by neutrophil adhesion to pulmonary vessels contribute to COVID-19 severity.

Discovering collective narratives shifts in online discussions (preprint)

Narrative is a foundation of human cognition and decision making. Because narratives play a crucial role in societal discourses and spread of misinformation and because of the pervasive use of social media, the narrative dynamics on social media can have profound societal impact. Yet, systematic and computational understanding of online narratives faces critical challenge of the scale and dynamics; how can we reliably and automatically extract narratives from massive amount of texts? How do narratives emerge, spread, and die? Here, we propose a systematic narrative discovery framework that fill this gap by combining change point detection, semantic role labeling (SRL), and automatic aggregation of narrative fragments into narrative networks. We evaluate our model with synthetic and empirical data two-Twitter corpora about COVID-19 and 2017 French Election. Results demonstrate that our approach can recover major narrative shifts that correspond to the major events.

Proteome profiling of nasopharynx reveals pathophysiological signature of COVID-19 disease severity (preprint)

An aberrant innate immune system caused by the beta coronavirus SARS-CoV-2 is a characteristic manifestation of severe coronavirus disease 2019 (COVID-19). Here, we performed proteome profiling of nasopharyngeal (NP) swabs from 273 hospitalized patients with mild and severe COVID-19 symptoms, including non-survivors. We identified depletion in STAT1-mediated type I interferon response, retinol metabolism and NRF2 antioxidant system that are associated with disease severity in our patient demography. We found that the dysregulation of glucocorticoid signaling and renin-angiotensin-aldosterone system (RAAS) contribute to the pathophysiology of COVID-19 fatality. Hyperactivation of host innate immune system was observed in severe patients, marked by elevated proteins involved in neutrophil degranulation and platelet aggregation. Our study using high-throughput proteomics on the nasopharynx of COVID-19 patients provides additional evidence on the SARS-CoV-2-induced pathophysiological signatures of disease severity and fatality.

Dysregulated Platelet Function and Thrombosis in Patients with Post-Acute Sequelae of COVID-19 (preprint)

Objective: Post-acute sequelae of COVID-19 (PASC, also referred as Long-COVID) sometimes follows COVID-19, a disease caused by SARS-CoV-2. While SARS-CoV-2 is well-known to promote a prothrombotic state, and especially to activate platelets acutely, less is known about the thrombosis risk in PASC. Approach and Results: PASC patients and age-matched healthy controls were enrolled in the study on average 15 months after documented SARS-CoV-2 infection. Platelet activation was evaluated by Light Transmission Aggregometry (LTA) and flow cytometry in response to platelet surface receptor agonists. Thrombosis in platelet-deplete plasma was evaluated by Factor Xa activity. A microfluidics system assessed thrombosis in whole blood under venous shear stress conditions. While only a mild increase in platelet aggregation in PASC patients through the thromboxane receptor was observed platelet activation through the glycoprotein VI (GPVI) receptor was markedly decreased in PASC patients compared to age- and sex-matched healthy controls. Thrombosis under venous shear conditions as well as Factor Xa activity were reduced in PASC patients. Plasma from PASC patients was an extremely potent activator of washed, healthy platelets - a phenomenon not observed using age- and sex-matched platelets from healthy individuals. Conclusions: PASC patients demonstrate dysregulated responses in platelets and coagulation in plasma, likely caused by a circulating plasma-derived molecule that promotes thrombosis. A hitherto undescribed protective response appears to exists in PASC patients to counterbalance ongoing thrombosis that is common to SARS-CoV-2 infection.

Incidence of anti-platelet factor4/polyanionic antibodies, thrombocytopenia, and thrombosis after COVID-19 vaccination with ChAdOx1 nCoV-19 in Thais (preprint)

Background The prevalence of anti-platelet factor 4 (PF4)/polyanionic antibodies occurring after vaccination with ChAdOx1 nCoV-19 is low. Most of these antibodies are not associated with vaccine-induced thrombotic thrombocytopenia. It remains unknown whether these antibodies are preexisting or occur as a result of vaccination. In this study, we demonstrated the incidence of anti-PF4/polyanionic antibodies, thrombocytopenia, and thrombosis after vaccination with ChAdOx1 nCoV-19 in a large cohort of Thais.Methods We conducted a prospective study in a cohort of health care workers and members of the general population who received COVID-19 vaccination with ChAdOx1 nCoV-19. Blood collection for complete blood count, D-dimer, and anti-PF4/polyanionic antibodies was performed before vaccination (day 0), day 10, and day 28 after vaccination. Anti-PF4/polyanionic antibodies were detected using enzyme-link immunosorbent assay (ELISA). Functional assay was performed for all positive ELISA tests.Results A total of 720 participants were included in the study. 214 participants received both the first and second doses, 91 participants received only the first, 51 received only the second, and 364 received the third booster dose of ChAdOx1 nCoV-19. Median age was 42 years (IQR, 34–53). 67% of participants were female. Three participants developed seroconversion, yielding an incidence of vaccination-induced anti-PF4/polyanionic antibodies of 0.42% (95% confidence interval 0.08, 1.23). Fourteen (1.9%) participants had preexisting anti-PF4/polyanionic antibodies before the vaccination but their optical density of anti-PF4/polyanionic antibodies did not significantly increase over time. None of the anti-PF4/polyanionic positive sera induced platelet aggregation. Abnormal D-dimer levels following vaccination were not different among the positive and negative anti-PF4/polyanionic groups (11.8% vs 13.2%, p = 0.86). Thrombocytopenia occurred in one person with negative anti-PF4/polyanionic antibodies. No clinical thrombosis or bleeding occurred.Conclusion We found a low incidence of seroconversion of anti-PF4/polyanionic antibodies after vaccination with ChAdOx1 nCoV-19 in Thais. Most of the anti-PF4/polyanionic antibodies were preexisting and did not significantly increase after vaccination with ChAdOx1 nCoV-19. Following vaccination, some participants with anti-PF4/polyanionic antibodies had elevated D-dimer levels, while only one developed thrombocytopenia and no thrombotic events were observed.

Treatment of Long COVID symptoms with triple anticoagulant therapy (preprint)

Background: Fibrin(ogen) amyloid microclots and platelet hyperactivation are key pathological findings in patients with acute COVID-19 infection and also in those with Long COVID/Post-Acute Sequelae of COVID-19 (PASC). These pathologies may represent a suitable target for pharmacological treatment of Long COVID.  Methods: Here we report on the symptoms displayed by a cohort of 91 South African Long COVID patients at baseline and after a clinician-initiated anticoagulant regime was completed. For laboratory analysis, patients provided a blood sample before and after treatment. Fibrinaloid microclot presence was studied by adding thioflavin T to platelet poor plasma (PPP), whilst platelet hyperactivation was studied using two platelet markers- PAC1 and CD62P (P-selectin). The anticoagulant regime included dual antiplatelet therapy (DAPT- Clopidogrel 75mg + Aspirin 75mg) once a day, and a direct oral anticoagulant (DOAC- Apixaban) 5mg twice a day. A proton pump inhibitor (PPI) pantoprazole 40 mg/day was also prescribed for gastric protection. Each of the treated cases reported their main Long COVID symptoms, and whether their symptoms resolved following treatment or not.  Results: In our cohort a most participants did not report any comorbidities before acute COVID-19 infection. Hypertension and dyslipidaemia were the commonest underlying illnesses, whilst the most commonly reported Long COVID symptoms included fatigue, cognitive dysfunction, shortness of breath, and joint and muscle pains. Following completion of treatment, each of the different symptoms resolved in the majority of patients. This was also reflected in the laboratory analysis, where a decrease in the severity of fibrin amyloid microclotting and the degree of platelet pathology was noted. No serious adverse bleeding events were reported.  Conclusions: Fibrin amyloid microclots, platelet hyperactivation/ aggregation, and  widespread endothelialitis inhibit the transport of oxygen at a capillary/cellular level. This provides a ready explanation for the symptoms of Long COVID. By normalizing the failed clotting physiology and reversal of the endothelialitis, triple anticoagulant therapy represents a promising treatment option that appears to be highly efficacious, and warrants controlled clinical studies. We caution that such a regime must only be followed under expert medical supervision in view of the risk of  bleeding.

Possible Role of P-selectin Adhesion in Long-COVID: A Case of Recovery After Long-COVID (preprint)

Background: Long-term outcomes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are now recognized as an emerging public health challenge - a condition termed post-acute coronavirus 2019 syndrome (PACS) or Long-COVID. The pathophysiology of Long-COVID remains to be established, several mechanisms in study focus on the role of P-selectin, an inflammation-induced protein expressed by platelets and endothelial cells. Functional P-selectin activity, potentially implicated in COVID-19 and Long-COVID sequelae, was measured for a Long-COVID subject at 68 weeks from the SARS-CoV-2 infection after fully recover from the syndrome. It was compared with the results from the same subject at 20 weeks post-infection, when subject experienced severe Long-COVID symptoms. Methods: Flow adhesion of whole blood or isolated white blood cells to P-selectin (FA-WB-Psel and FA-WBC-Psel) was measured using a standardized microfluidics clinical assay; impedance aggregometry with a collagen agonist was measured using model 590 Chrono-Log impedance aggregometer; standard laboratory assays were performed to evaluate changes in blood chemistries. Results: After recovery from Long-COVID, RBC count and D-dimer remained elevated and other blood chemistry results remained within the normal range as compared to 20 weeks post infection when severe Long-COVID symptoms were present. Total iron and transferrin-iron saturation percentage values that were elevated when symptoms were present, declined to normal range. Whole blood aggregometry results indicate an absence of previously present platelet hyperactivity. FA-WB-PSel that was significantly elevated during Long-COVID (590 +/- 260 cells/mm2) was significantly reduced after the resolution of Long-COVID symptoms (98 +/- 38 cells/mm2). However, supplementation of whole blood with crizanlizumab did not result in any measurable inhibition of cell adhesion to P-selectin, similarly with previously reported. Similar to what was observed for the subject when Long-COVID symptoms were present, crizanlizumab, even at a dose 10-fold lower than clinical, induced pronounced inhibition of FA-WBC-Psel when tested in buffer, but not in patient own plasma. Conclusions: This report documents the changes in leukocyte adhesion properties for a patient at more than a year from the initial infection after the gradual resolution of Long-COVID symptoms, as compared to when Long-COVID symptoms were present. Recovery from Long-COVID may be associated with normalization of platelet activity, but not necessarily with complete alleviation of endothelial activation. It remains to be determined to what extent changes in leukocyte adhesion to P-selectin may represent a new risk factor for a mechanism driving Long-COVID syndrome.

Direct Cryo-ET observation of platelet deformation induced by SARS-CoV-2 Spike protein (preprint)

SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic. Its high pathogenicity is due to SARS-CoV-2 spike protein (S protein) contacting host-cell receptors. A critical hallmark of COVID-19 is the occurrence of coagulopathies. Here, we report the direct observation of the interactions between S protein and platelets. Live imaging showed that the S protein triggers platelets to deform dynamically, in some cases, leading to their irreversible activation. Strikingly, cellular cryo-electron tomography revealed dense decorations of S protein on the platelet surface, inducing filopodia formation. Hypothesizing that S protein binds to filopodia-inducing integrin receptors, we tested the binding to RGD motif-recognizing platelet integrins and found that S protein recognizes integrin v{beta}3. Our results infer that the stochastic activation of platelets is due to weak interactions of S protein with integrin, which can attribute to the pathogenesis of COVID-19 and the occurrence of rare but severe coagulopathies.

The impact of the COVID-19 pandemic on the reporting of violence against children (preprint)

Objective To verify the impact (effect) of the COVID-19 pandemic on the rates of reporting of interpersonal violence against children aged 0-11 years old in Salvador, Bahia, from 2020 to 2021. Methods The study used two epidemiological approaches: a) temporal aggregation and b) an individual cross section, based on cases of interpersonal violence against children reported in SINAN from 2009 to 2021. Annual rates of reporting of interpersonal violence against children (per 10,000) and percentages were calculated according to different strata of each variable of interest. The temporal trend was analyzed using the simple linear regression method (R2=0.6955) applied to the rates from 2014 to 2019, the period in which they showed the most consistency. Results The rates of reporting of violence against children showed a large variation, with a mean of 4.7/10,000. In 2021, the rate was 7/10,000 (a 45.8% increase on the previous year). Regression analysis indicated a mean reduction of 0.337/10,000 a year, and expected rates of 4.62 and 4.28/10,000, respectively, for 2020 and 2021. Conclusion The occurrence of COVID-19 and, particularly, the increase in the number of reported cases of interpersonal violence against children in the second year of the pandemic in Bahia suggest that these events may be directly or indirectly related. More robust studies are needed to confirm this relationship.

Phenotypic alteration of low-density granulocytes in people with pulmonary post-acute sequalae of SARS-CoV-2 infection (preprint)

Low-density granulocytes (LDGs) are a distinct subset of neutrophils whose increased abundance is associated with the severity of COVID-19. However, the long-term effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on LDG levels and phenotypic alteration remain unexplored. Using participants naïve to SARS-CoV-2 (NP), infected with SARS-CoV-2 with no residual symptoms (NRS), and infected with SARS-CoV-2 with chronic pulmonary symptoms (PPASC), we compared LDG levels and their phenotype by measuring the expression of markers for activation, maturation, and neutrophil extracellular trap (NET) formation using flow cytometry. The number of LDGs was significantly elevated in PPASC compared to NP. Individuals infected with SARS-CoV-2 (NRS and PPASC) demonstrated increased CD10+ and CD16HI subset counts of LDGs compared to NP group. Further characterization of LDGs demonstrated that LDGs from PPASC displayed higher NET forming ability and aggregation with platelets compared to LDGs from NP and NRS. Our data demonstrates that mature neutrophils with a heightened activation phenotype remain in circulation long after initial SARS-CoV-2 infection. Persistent elevation of markers for neutrophil activation and NET formation on LDGs, as well as an enhanced proclivity for platelet-neutrophil aggregation (PNA) formation in individuals with PPASC may be associated with the development of long-term pulmonary sequelae.

Increased levels of inflammatory molecules in blood of Long COVID patients point to thrombotic endotheliitis (preprint)

The prevailing hypotheses for the persistent symptoms of Long COVID have been narrowed down to immune dysregulation and autoantibodies, widespread organ damage, viral persistence, and fibrinaloid microclots (entrapping numerous inflammatory molecules) together with platelet hyperactivation. Here we demonstrate significantly increased concentrations of Von Willebrand Factor, platelet factor 4, serum amyloid A, alpha-2-antiplasmin E-selectin, and platelet endothelial cell adhesion molecule-1, in the soluble part of the blood. It was noteworthy that the mean level of alpha-2-antiplasmin exceeded the upper limit of the laboratory reference range in Long COVID patients, and the other 5 were significantly elevated in Long COVID patients as compared to the controls. This is alarming if we take into consideration that a significant amount of the total burden of these inflammatory molecules has previously been shown to be entrapped inside fibrinolysis-resistant microclots (thus decreasing the apparent level of the soluble molecules). We also determined that by individually adding E-selectin and PECAM-1 to healthy blood, these molecules may indeed be involved in protein-protein interactions with plasma proteins (contributing to microclot formation) and platelet hyperactivation. This investigation was performed as a laboratory model investigation and the final exposure concentration of these molecules was chosen to mimic concentrations found in Long COVID. We conclude that presence of microclotting, together with relatively high levels of six inflammatory molecules known to be key drivers of endothelial and clotting pathology, points to thrombotic endotheliitis as a key pathological process in Long COVID. This has implications for the choice of appropriate therapeutic options in Long COVID.

SARS-CoV-2 and HSV-1 Induce Amyloid Aggregation in Human CSF (preprint)

The corona virus (SARS-CoV-2) pandemic and the resulting long-term neurological complications in patients, known as long COVID, have renewed the interest in the correlation between viral infections and neurodegenerative brain disorders. While many viruses can reach the central nervous system (CNS) causing acute or chronic infections (such as herpes simplex virus 1, HSV-1), the lack of a clear mechanistic link between viruses and protein aggregation into amyloids, a characteristic of several neurodegenerative diseases, has rendered such a connection elusive. Recently, we showed that viruses can induce aggregation of purified amyloidogenic proteins via the direct physicochemical mechanism of heterogenous nucleation (HEN). In the current study, we show that the incubation of HSV-1 and SARS-CoV-2 with human cerebrospinal fluid (CSF) leads to the amyloid aggregation of several proteins known to be involved in neurodegenerative diseases, such as: APLP1 (amyloid beta precursor like protein 1), ApoE, clusterin, 2-macroglobulin, PGK-1 (phosphoglycerate kinase 1), ceruloplasmin, nucleolin, 14-3-3, transthyretin and vitronectin. Importantly, UV-inactivation of SARS-CoV-2 does not affect its ability to induce amyloid aggregation, as amyloid formation is dependent on viral surface catalysis via HEN and not its ability to replicate. Our results show that viruses can physically induce amyloid aggregation of proteins in human CSF, and thus providing a potential mechanism that may account for the association between persistent and latent/reactivating brain infections and neurodegenerative diseases.

STIM1 and ORAI1 mutations leading to tubular aggregate myopathies are sensitive to the Store-operated Ca2+-entry modulators CIC-37 and CIC-39.

Gain-of-function mutations on STIM1 and ORAI1 genes are responsible for an increased store-operated calcium entry, and underlie the characteristic symptoms of three overlapping ultra-rare genetic disorders (i.e tubular aggregate myopathy, Stormorken syndrome, York platelet syndrome) that can be grouped as tubular aggregate myopathies. These mutations lead to a wide spectrum of defects, which usually include muscle weakness and cramps. Negative modulators of store-operated Ca2+-entry targeting wild-type STIM1 and ORAI1 have entered clinical trials for a different array of disorders, including pancreatitis, COVID-19, cancer, and autoimmune disorders and, while efficacy data is awaited, safety data indicates tolerability of this STIM1/ORAI1 mutations are amenable to pharmacological intervention. If this were so, given that there are no approved treatments or clinical trials ongoing for these rare disorders, it could be envisaged that these agents could also rehabilitate tubular aggregate myopathy patients. In the present contribution we characterized the Ca2+-entry patterns induced by eleven STIM1 and three ORAI1 mutations in heterologous systems or in patient-derived cells, i.e. fibroblasts and myotubes, and evaluated the effect of CIC-37 and CIC-39, two novel store-operated calcium entry modulators. Our data show that all STIM1 and ORAI1 gain-of-function mutations tested, with the possible exception of the R304Q STIM1 mutation, are amenable to inhibition, albeit with slightly different sensitivities, paving the way to the development of SOCE modulators in tubular aggregate myopathies.

HMGB1 correlates with severity and death of COVID-19 patients (preprint)

SARS-CoV-2, the causative agent of the ongoing COVID-19, has spread worldwide since it was first identified in November 2019 in Wuhan. Since then, it was already demonstrated an exuberant inflammation, cytokine storm, endothelium dysfunction, platelets hyperactivation and aggregation, following T cell exhaustion leading to severe multi-organ damage and death of COVID-19 patients. Here, we sought to identify molecular biomarkers of disease severity in a Brazilian cohort of COVID-19 patients by measuring the serum levels of endogenous danger signals. Our data revealed that ICU patients that are critically ill, at the early hyperinflammatory phase of COVID-19 (around 12-25 days after hospital admission) display higher serum levels of the classical alarmin HMGB1. Serum levels of HMGB1 were positively correlated with cys-leukotrienes, D-dimer, AST, and ALT. Notably, we verified that HMGB1 levels above 125.4 ng/mL is the cut off that distinguishes the patients that are at higher risk of death. Serum levels of extracellular ATP, PGE<2, LTB4, cys-LTs, and tissue factor were also elevated in the serum of ICU patients. In conclusion, we propose that serum levels of HMGB1 serve as prognostic biomarker of risk of death in patients suffering from severe COVID-19.

The Delta variant SARS-CoV-2 spike protein uniquely promotes aggregation of pseudotyped viral particles (preprint)

Individuals infected with the SARS-CoV-2 Delta variant, lineage B.1.617.2, exhibit faster initial infection with a higher viral load than prior variants, and pseudotyped particles bearing the SARS-CoV-2 Delta variant spike protein induce a faster initial infection rate of target cells compared to those bearing other SARS-CoV-2 variant spikes. Here, we show that pseudotyped particles bearing the Delta variant spike form unique aggregates, as evidenced by negative stain and cryogenic electron microscopy (EM), flow cytometry, and nanoparticle tracking analysis. Viral particles pseudotyped with other SARS-CoV-2 spike variants do not show aggregation by any of these criteria. The contribution to infection kinetics of the Delta spike's unique property to aggregate is discussed with respect to recent evidence for collective infection by other viruses. Irrespective of this intriguing possibility, spike-dependent aggregation is a new functional parameter of spike-expressing viral particles to evaluate in future spike protein variants.

Possible Role of P-selectin Adhesion in Long-COVID: A Comparative Analysis of a Long-COVID Case Versus an Asymptomatic Post-COVID Case (preprint)

Background: Long-term outcomes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are now recognized as an emerging public health challenge - a condition termed Long-COVID. The pathophysiology of Long-COVID remains to be established. Functional P-selectin activity, implicated in COVID-19 sequalae, was measured between two convalescent COVID-19 subjects, one with (Long-COVID subject) and another without Long-COVID symptoms. Methods: Flow adhesion of whole blood or isolated white blood cells to P-selectin (FA-WB-Psel and FA-WBC-Psel) was measured using a standardized microfluidics clinical assay; impedance aggregometry with a collagen agonist was measured using model 590 Chrono-Log impedance aggregometer; standard laboratory assays were performed to evaluate changes in blood chemistries. Results: For both subjects, hemoglobin, WBC, platelet counts, electrolytes and ferritin were within normal reference ranges, with FA-WB-Psel significantly elevated compared to healthy controls (p<0.01). In vitro treatment of whole blood samples with crizanlizumab (anti-p-selectin monoclonal antibody) within the clinical dose range (10 g/ml) mL) inhibited FA-WB-Psel only in samples from asymptomatic post-COVID subject, with the Long-COVID subject sample requiring close to 5-fold elevated dose to achieve a response. Pronounced inhibition of P-selectin adhesion of isolated leukocytes was observed for both subjects in autologous platelet-poor plasma and buffer. Impedance aggregometry showed greater baseline platelet aggregation to collagen in the Long-COVID sample, although both samples responded similarly to aspirin-induced platelet inhibition. Conclusions: Presented results suggest that elevated platelet activation in Long-COVID subject may be associated with increased P-Selectin activity. The results are discussed in terms of possible use on P-selectin inhibition therapies in treating Long-COVID.