Antipolyphosphate monoclonal antibodies derived from autoimmune mice.

Background: Inorganic polyphosphates (polyPs) are linear chains of phosphates that accelerate blood clotting. Targeting polyP in vivo has been shown to reduce thrombosis. Objectives: To identify and characterize anti-polyP monoclonal antibodies that could be used as analytical tools and as antithrombotic agents. Methods: Hybridomas were prepared from spleen cells from autoimmune NZBWF1/J female mice and screened for anti-polyP antibodies. Antibodies that bound polyP using enzyme-linked immunosorbent assay and pull-down assays were further characterized with plate binding, surface plasmon resonance, and plasma-based clotting assays. Antithrombotic potential was evaluated in a murine ferric chloride-induced carotid artery thrombosis model. Results: Of 4 antibodies that bound polyP in our pull-down assay, 2 (PP2069 and PP2099) were available for further characterization. While analyzing these anti-polyP antibodies, we found secretory leukocyte peptidase inhibitor (SLPI) to be a common contaminant of these antibodies and that SLPI binds polyP. We removed SLPI quantitatively from our purified immunoglobulin G. Both PP2069 and PP2099 immunoglobulin G displayed high affinity for polyP but also bound to other polyanions such as DNA, heparin, and certain other glycosaminoglycans, indicating limited specificity. Both antibodies inhibited polyP-initiated plasma clotting in vitro. When tested in vivo in a mouse thrombosis model, however, neither PP2069 nor PP2099 exhibited a significant antithrombotic effect. Conclusion: Autoimmune mice spontaneously produce antibodies against polyP. The 2 examples of anti-polyP monoclonal antibodies studied here not only bound to polyP with high affinity but also cross-reacted with DNA and heparin. Neither antibody protected against thrombosis in a mouse model, but they might have some utility for in vitro studies of polyP.

INHIBITORS OF INORGANIC POLYPHOSPHATE AND NUCLEIC ACIDS ATTENUATE IN VITRO THROMBIN GENERATION IN PLASMA FROM TRAUMA PATIENTS.

ABSTRACT: Background: Inorganic polyphosphate (polyP) is a procoagulant polyanion. We assessed the impact of polyP inhibition on thrombin generation after trauma using the novel polyP antagonists, macromolecular polyanion inhibitor 8 (MPI 8), and universal heparin reversal agent 8 (UHRA-8). Methods: Plasma thrombin generation (calibrated automated thrombogram, CAT), in 56 trauma patients and 39 controls +/- MPI 8 and UHRA-8 (50 µg/mL), was expressed as lag time (LT, minutes), peak height (PH, nM), and time to peak (ttPeak, minutes), with change in LT (ΔLT) and change in ttPeak (ΔttPeak) quantified. Results expressed in median and quartiles [Q1, Q3], Wilcoxon matched-pairs testing, P < 0.05 significant. Results: Trauma patients had greater baseline PH than controls (182.9 [121.0, 255.2]; 120.5 [62.1, 174.8], P < 0.001). MPI 8 treatment prolonged LT and ttPeak in trauma (7.20 [5.88, 8.75]; 6.46 [5.45, 8.93], P = 0.020; 11.28 [8.96, 13.14]; 11.00 [8.95, 12.94], P = 0.029) and controls (7.67 [6.67, 10.50]; 6.33 [5.33, 8.00], P < 0.001; 13.33 [11.67, 15.33]; 11.67 [10.33, 13.33], P < 0.001). UHRA-8 treatment prolonged LT and ttPeak and decreased PH in trauma (9.09 [7.45, 11.33]; 6.46 [5.45, 8.93]; 14.02 [11.78, 17.08]; 11.00 [8.95, 12.94]; 117.4 [74.5, 178.6]; 182.9 [121.0, 255.2]) and controls (9.83 [8.00, 12.33]; 6.33 [5.33, 8.00]; 16.67 [14.33, 20.00]; 11.67 [10.33, 13.33]; 55.3 [30.2, 95.9]; 120.5 [62.1, 174.8]), all P < 0.001. Inhibitor effects were greater for controls (greater ΔLT and ΔttPeak for both inhibitors, P < 0.001). Conclusion: PolyP inhibition attenuates thrombin generation, though to a lesser degree in trauma than in controls, suggesting that polyP contributes to accelerated thrombin generation after trauma.

External Trigger Free Charge Switchable Cationic Ligands in the Design of Safe and Effective Universal Heparin Antidote.

Thrombosis, the formation of blood clots within a blood vessel, can lead to severe complications including pulmonary embolism, cardiac arrest, and stroke. The most widely administered class of anticoagulants is heparin-based anticoagulants such as unfractionated heparin, low-molecular weight heparins (LMWHs), and fondaparinux. Protamine is the only FDA-approved heparin antidote. Protamine has limited efficacy neutralizing LMWHs and no reversal activity against fondaparinux. The use of protamine can lead to complications, including excessive bleeding, hypotension, and hypersensitivity, and has narrow therapeutic window. In this work, a new concept in the design of a universal heparin antidote: switchable protonation of cationic ligands, is presented. A library of macromolecular polyanion inhibitors (MPIs) is synthesized and screened to identify molecules that can neutralize all heparins with high selectivity and reduced toxicity. MPIs are developed by assembling cationic binding groups possessing switchable protonation states onto a polymer scaffold. By strategically selecting the identity and modulating the density of cationic binding groups on the polymer scaffold, a superior universal heparin reversal agent is developed with improved heparin-binding activity and increased hemocompatibility profiles leading to minimal effect on hemostasis. The activity of this heparin antidote is demonstrated using in vitro and in vivo studies.

Survival, Growth, and Toxin Production of Bacillus cereus During Cooking and Storage of Fresh Rice Noodles.

Retail stores maintain fresh rice noodles (FRNs) at room temperature because refrigeration negatively impacts FRNs' texture. The room temperature and high water activity of FRNs help spore-forming Bacillus cereus to grow and produce toxins. In this study, the effect of steam cooking on survival and different storage temperatures on the growth and enterotoxins production of B. cereus in FRNs were investigated. White rice flour was used to make FRNs. Three treatments of FRNs were used in this study; uninoculated, inoculated (with 4.0 log CFU/ml of B. cereus spores), and autoclaved as a negative control. A slurry of rice flour, cornstarch, and water was steam cooked for 4 min at 90°C and incubated for 168 h at 4°C, and for 72 h at 22 and 32°C. Incubated FRNs were tested for pH, B. cereus growth, and enterotoxins production. Steam cooking reduced the total number of B. cereus spores by 0.7 ± 0.3 log CFU/g. Surviving B. cereus spores in inoculated and uninoculated FRNs germinated over 72 h of storage. No B. cereus was detected in negative controls. An interaction was observed across storage temperatures and time (p < 0.05). The B. cereus population in uninoculated FRNs increased by more than 7.0 log CFU/g at 22 and 32°C over 72 h, while inoculated FRNs showed a 5.0 log bacterial increase at these storage temperatures. No growth was observed at 4°C in both inoculated and uninoculated FRNs. The pH of inoculated FRNs was reduced from 6.9 ± 0.1 to 5.7 ± 0.0 at 32°C and to 6.2 ± 0.1 at 22°C, and the pH of uninoculated FRNs was reduced from 7.0 ± 0.1 to 5.8 ± 0.2 at 32°C and to 6.5 ± 0.0 at 22°C, indicative of FRNs spoilage. B. cereus in inoculated FRNs produced enterotoxins after 12 h of storage at 32°C, and over 24 h of storage at 22°C, while no toxin was detected at 4°C. Our findings show that storing FRNs at room temperature for 24 h leads to enterotoxin production, emphasizing the importance of proper FRN storage and their potential risk to consumers. Nevertheless, further research should investigate the effect of other foodborne pathogens on these products.

Profiling toxin genes and antibiotic resistance in Bacillus cereus isolated from pre-launch spacecraft.

Characterization of the microbiomes of pre-launch spacecraft in spacecraft assembly facilities is an important step in keeping crews healthy during journeys that can last several hundred days in small artificial environments in space. Bacillus cereus, a foodborne pathogenic bacterium, has the potential to be a significant source of food contamination in such environments. This bacterium is a spore-forming bacteria that resists different antimicrobial treatments in cleanrooms where spacecraft are assembled. This study evaluated 41 B. cereus isolates from four pre-launch spacecraft in spacecraft assembly facilities for their toxin gene profile and antibiotic resistance. Four enterotoxin genes (hlbC, cytK, nheA, and entFM) and two emetic toxin genes (ces and CER) were targeted for chromosomal DNA and plasmid DNA. Results showed 31.7, 7.3, 85, and 41.5% of isolates contained hblC, cytK, nheA, and entFM, respectively, in chromosomal or plasmid DNA. Overall, 37 isolates (90.2%) showed at least one enterotoxin gene. The emetic toxin gene, ces, was detected in the plasmid DNA of three isolates (7.3%). The antibiotic resistance of isolates was evaluated by the Kirby-Bauer disk diffusion procedure. All the isolates exhibited 100% susceptibility to gentamicin, 97% were susceptible to clindamycin, and 95% to chloramphenicol, imipenem, tetracycline, and vancomycin. The overall susceptibility average is 51%. However, 98% of the isolates were resistant to ß-lactam antibiotics, 97.5% were resistant to sulfamethoxazole/trimethoprim, and 80% were resistant to rifampin. This study provides important information on B. cereus isolates from spacecraft assembly facilities for use in microbial monitoring programs of spacecraft.

Inhibitors of Polyphosphate and Neutrophil Extracellular Traps.

The contact pathway of blood clotting has received intense interest in recent years as studies have linked it to thrombosis, inflammation, and innate immunity. Because the contact pathway plays little to no role in normal hemostasis, it has emerged as a potential target for safer thromboprotection, relative to currently approved antithrombotic drugs which all target the final common pathway of blood clotting. Research since the mid-2000s has identified polyphosphate, DNA, and RNA as important triggers of the contact pathway with roles in thrombosis, although these molecules also modulate blood clotting and inflammation via mechanisms other than the contact pathway of the clotting cascade. The most significant source of extracellular DNA in many disease settings is in the form of neutrophil extracellular traps (NETs), which have been shown to contribute to incidence and severity of thrombosis. This review summarizes known roles of extracellular polyphosphate and nucleic acids in thrombosis, with an emphasis on novel agents under current development that target the prothrombotic activities of polyphosphate and NETs.

Smart thrombosis inhibitors without bleeding side effects via charge tunable ligand design.

Current treatments to prevent thrombosis, namely anticoagulants and platelets antagonists, remain complicated by the persistent risk of bleeding. Improved therapeutic strategies that diminish this risk would have a huge clinical impact. Antithrombotic agents that neutralize and inhibit polyphosphate (polyP) can be a powerful approach towards such a goal. Here, we report a design concept towards polyP inhibition, termed macromolecular polyanion inhibitors (MPI), with high binding affinity and specificity. Lead antithrombotic candidates are identified through a library screening of molecules which possess low charge density at physiological pH but which increase their charge upon binding to polyP, providing a smart way to enhance their activity and selectivity. The lead MPI candidates demonstrates antithrombotic activity in mouse models of thrombosis, does not give rise to bleeding, and is well tolerated in mice even at very high doses. The developed inhibitor is anticipated to open avenues in thrombosis prevention without bleeding risk, a challenge not addressed by current therapies.

Utility of a "Lavender Lounge" to Reduce Stress Among Critical Care Registered Nurses: A Cross-Sectional Study.

BACKGROUND: Work-related stressors are present in almost every profession, but many believe nurses caring for critically ill patients experience additional and unique stresses. Results of previous studies have demonstrated the potential benefits of various interventions to reduce stress among intensive care nurses. However, the practicality of nurses taking time out from a busy unit to meet their own needs remains in question. OBJECTIVES: To assess intensive care nurses' perceptions of the usability of a lounge designed to support them in refreshing and renewing themselves after stressful clinical situations. METHODS: This study used a descriptive, cross-sectional design and survey methodology with a convenience sample of registered nurses from a medical intensive care unit. RESULTS: Of 250 registered nurses eligible for participation, 54 (21.6%) completed surveys, and of those, 31 (57%) reported having used the lounge within the past month. Nurses reported having coverage provided by colleagues, visiting during their lunch break, and having low unit acuity were facilitators of lounge use. Barriers included high unit acuity, high unit census, and high patient care demands with no one available to cover patient assignments. CONCLUSIONS: The variables that lead to stress and burnout among medical intensive care unit nurses also prevent nurses from using a "relaxation room." A more effective approach may be organizational change that supports reduction of workload through increased staffing, prearranged breaks during shifts, and increased recovery time between shifts by limiting work to no more than 40 hours per week.

A site on factor XII required for productive interactions with polyphosphate.

BACKGROUND: During plasma contact activation, factor XII (FXII) binds to surfaces through its heavy chain and undergoes conversion to the protease FXIIa. FXIIa activates prekallikrein and factor XI (FXI). Recently, we showed that the FXII first epidermal growth factor-1 (EGF1) domain is required for normal activity when polyphosphate is used as a surface. OBJECTIVES: The aim of this study was to identify amino acids in the FXII EGF1 domain required for polyphosphate-dependent FXII functions. METHODS: FXII with alanine substitutions for basic residues in the EGF1 domain were expressed in HEK293 fibroblasts. Wild-type FXII (FXII-WT) and FXII containing the EGF1 domain from the related protein Pro-HGFA (FXII-EGF1) were positive and negative controls. Proteins were tested for their capacity to be activated, and to activate prekallikrein and FXI, with or without polyphosphate, and to replace FXII-WT in plasma clotting assays and a mouse thrombosis model. RESULTS: FXII and all FXII variants were activated similarly by kallikrein in the absence of polyphosphate. However, FXII with alanine replacing Lys73, Lys74, and Lys76 (FXII-Ala73,74,76) or Lys76, His78, and Lys81 (FXII-Ala76,78,81) were activated poorly in the presence of polyphosphate. Both have <5% of normal FXII activity in silica-triggered plasma clotting assays and have reduced binding affinity for polyphosphate. Activated FXIIa-Ala73,74,76 displayed profound defects in surface-dependent FXI activation in purified and plasma systems. FXIIa-Ala73,74,76 reconstituted FXII-deficient mice poorly in an arterial thrombosis model. CONCLUSION: FXII Lys73, Lys74, Lys76, and Lys81 form a binding site for polyanionic substances such as polyphosphate that is required for surface-dependent FXII function.

Walk before you run: Feasibility challenges and lessons learned from the PROCLAIM study, a multicenter randomized controlled trial of misoprostol for prevention of recurrent Clostridioides difficile during COVID-19.

We analyzed our challenging experience with a randomized controlled trial of misoprostol for prevention of recurrent C. difficile. Despite careful prescreening and thoughtful protocol modifications to facilitate enrollment, we closed the study early after enrolling just 7 participants over 3 years. We share lessons learned, noting the importance of feasibility studies, inclusion of biomarker outcomes, and dissemination of such findings to inform future research design and implementation successes.

Lopinavir/ritonavir for treatment of non-hospitalized patients with COVID-19: a randomized clinical trial.

OBJECTIVES: Effective and widely available therapies are still needed for outpatients with COVID-19. We aimed to evaluate the efficacy and safety of lopinavir/ritonavir (LPV/r) for early treatment of non-hospitalized individuals diagnosed with COVID-19. METHODS: This randomized, placebo (Plb)-controlled, double-blind, multi-site decentralized clinical trial enrolled non-hospitalized adults with confirmed SARS-CoV-2 infection and six or fewer days of acute respiratory infection symptoms who were randomized to either twice-daily oral LPV/r (400 mg/100 mg) or Plb for 14 days. Daily surveys on study days 1 through 16 and again on study day 28 evaluated symptoms, daily activities, and hospitalization status. The primary outcome was longitudinal change in an ordinal scale based on a combination of symptoms, activity, and hospitalization status through day 15 and was analyzed by use of a Bayesian longitudinal proportional odds logistic regression model for estimating the probability of a superior recovery for LPV/r over Plb (odds ratio >1). RESULTS: Between June 2020 and December 2021, 448 participants were randomized to receive either LPV/r (n = 216) or Plb (n = 221). The mean symptom duration before randomization was 4.3 days (SD 1.3). There were no differences between treatment groups through the first 15 days for the ordinal primary outcome (odds ratio 0.96; 95% credible interval: 0.66 to 1.41). There were 3.2% (n = 7) of LPV/r and 2.7% (n = 6) of Plb participants hospitalized by day 28. Serious adverse events did not differ between groups. CONCLUSION: LPV/r did not significantly improve symptom resolution or reduce hospitalization in non-hospitalized participants with COVID-19. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04372628.

Resonant Acoustic Rheometry to Measure Coagulation Kinetics in Hemophilia A and Healthy Plasma: A Novel Viscoelastic Method.

Compared with conventional coagulation tests and factor-specific assays, viscoelastic hemostatic assays (VHAs) can provide a more thorough evaluation of clot formation and lysis but have several limitations including clot deformation. In this proof-of-concept study, we test a noncontact technique, termed resonant acoustic rheometry (RAR), for measuring the kinetics of human plasma coagulation. Specifically, RAR utilizes a dual-mode ultrasound technique to induce and detect surface oscillation of blood samples without direct physical contact and measures the resonant frequency of the surface oscillation over time, which is reflective of the viscoelasticity of the sample. Analysis of RAR results of normal plasma allowed defining a set of parameters for quantifying coagulation. RAR detected a flat-line tracing of resonant frequency in hemophilia A plasma that was corrected with the addition of tissue factor. Our RAR results captured the kinetics of plasma coagulation and the newly defined RAR parameters correlated with increasing tissue factor concentration in both healthy and hemophilia A plasma. These findings demonstrate the feasibility of RAR as a novel approach for VHA, providing the foundation for future studies to compare RAR parameters to conventional coagulation tests, factor-specific assays, and VHA parameters.

Influence of Steric Shield on Biocompatibility and Antithrombotic Activity of Dendritic Polyphosphate Inhibitor.

The polyanion, inorganic polyphosphate (polyP), is a procoagulant molecule which has become a promising therapeutic target in the development of antithrombotics. Neutralizing polyP's prothrombotic activity using polycationic inhibitors is one of the viable strategies to design new polyP inhibitors. However, in this approach, a fine balance between the electrostatic interaction of polyP and the inhibitor is needed. Any unprotected polycations are known to interact with negatively charged blood components, potentially resulting in platelet activation, cellular toxicity, and bleeding. Thus, designing potent polycationic polyP inhibitors with good biocompatibility is a major challenge. Building on our previous research on universal heparin reversal agent (UHRA), we report polyP inhibitors with a modified steric shield design. The molecular weight, number of cationic binding groups, and the length of the polyethylene glycol (PEG) chains were varied to arrive at the desired inhibitor. We studied two different PEG lengths (mPEG-750 versus mPEG-350) on the polyglycerol scaffold and investigated their influence on biocompatibility and polyP neutralization activity. The polyP inhibitor with mPEG-750 brush layer, mPEG750 UHRA-10, showed superior biocompatibility compared to its mPEG-350 analogs by a number of measured parameters without losing its neutralization activity. An increase in cationic binding groups (25 groups in mPEG750 UHRA-8 and 32 in mPEG750 UHRA-10 [HC]) did not alter the neutralization activity, which suggested that the mPEG-750 shield layer provides significant protection of cationic binding groups and thus helps to minimize unwanted nonspecific interactions. Furthermore, these modified polyP inhibitors are highly biocompatible compared to conventional polycations that have been previously used as polyP inhibitors (e.g., PAMAM dendrimers and polyethylenimine). Through this study, we demonstrated the importance of the design of steric shield toward highly biocompatible polyP inhibitors. This approach can be exploited in the design of highly biocompatible macromolecular inhibitors.

Endothelium-protective, histone-neutralizing properties of the polyanionic agent defibrotide.

Neutrophil-mediated activation and injury of the endothelium play roles in the pathogenesis of diverse disease states ranging from autoimmunity to cancer to COVID-19. Neutralization of cationic proteins (such as neutrophil extracellular trap-derived [NET-derived] histones) with polyanionic compounds has been suggested as a potential strategy for protecting the endothelium from such insults. Here, we report that the US Food and Drug Administration-approved polyanionic agent defibrotide (a pleiotropic mixture of oligonucleotides) directly engages histones and thereby blocks their pathological effects on endothelium. In vitro, defibrotide counteracted endothelial cell activation and pyroptosis-mediated cell death, whether triggered by purified NETs or recombinant histone H4. In vivo, defibrotide stabilized the endothelium and protected against histone-accelerated inferior vena cava thrombosis in mice. Mechanistically, defibrotide demonstrated direct and tight binding to histone H4 as detected by both electrophoretic mobility shift assay and surface plasmon resonance. Taken together, these data provide insights into the potential role of polyanionic compounds in protecting the endothelium from thromboinflammation with potential implications for myriad NET- and histone-accelerated disease states.

Thrombin generation abnormalities in commonly encountered platelet function disorders.

INTRODUCTION: Studies of thrombin generation (TG) with platelet-rich plasma (PRP) and platelet-poor plasma (PPP) have provided insights on bleeding disorders. We studied TG for a cohort with commonly encountered platelet function disorders (PFD). METHODS: Participants included 40 controls and 31 with PFD due to: nonsyndromic dense granule (DG) deficiency (PFD-DGD, n = 9), RUNX1 haploinsufficiency (n = 6) and aggregation defects from other, uncharacterized causes (n = 16). TG was tested with PRP and PPP samples. As DG store ADP and polyphosphate that enhance platelet-dependent TG, PFD-DGD PRP TG was tested for correction with ADP, polyphosphate and combined additives. Tissue factor pathway inhibitor (TFPI), platelet factor V (FV), and platelet TFPI and ANO6 transcript levels were also evaluated. Findings were tested for associations with TG endpoints and bleeding. RESULTS: PFD samples had impaired PRP TG, but also impaired PPP TG, with strong associations between their PRP and PPP TG endpoints (P ≤ .005). PFD-DGD PRP TG endpoints showed associations to PPP TG endpoints but not to DG counts, and were improved, but not fully corrected, by adding polyphosphate and agonists. PFD participants had increased plasma TFPI and reduced platelet TFPI (P ≤ .02) but normal levels of platelet FV, and platelet TFPI and ANO6 transcripts levels. PFD plasma TFPI levels showed significant association to several PPP TG endpoints (P ≤ .04). Several PFD PRP TG endpoints showed significant associations to bleeding symptoms, including wound healing problems and prolonged bleeding from minor cuts (P ≤ .04). CONCLUSION: TG is impaired in commonly encountered PFD, with their PRP TG findings showing interesting associations to symptoms.

Effect of a pragmatic home-based mobile health exercise intervention after transcatheter aortic valve replacement: a randomized pilot trial.

AIMS: Impaired physical function is common in patients undergoing transcatheter aortic valve replacement (TAVR) and associated with worse outcomes. Participation in centre-based cardiac rehabilitation (CR) after cardiovascular procedures is sub-optimal. We aimed to test a home-based mobile health exercise intervention as an alternative or complementary approach. METHODS AND RESULTS: At five centres, after a run-in period, eligible individuals treated with TAVR were randomized 1:1 at their 1-month post-TAVR visit to an intervention group [activity monitor (AM) with personalized daily step goal and resistance exercises] or a control group for 6 weeks. Among 50 participants, average age was 76 years, 34% were female, average STS score was 2.91.8, and 40% had Short Physical Performance Battery (SPPB) 9. Daily compliance with wearing the AM and performing exercises averaged 8590%. In the intention to treat population, there was no evidence that the intervention improved the co-primary endpoints: daily steps +769 (95% CI 244 to +1783); SPPB +0.68 (0.27 to 1.53); and Kansas City Cardiomyopathy Questionnaire 1.7 (9.1 to 7.1). The intervention did improve secondary physical activity parameters, including moderate-to-intense daily active minutes (P<0.05). In a pre-specified analysis including participants who did not participate in CR (n=30), the intervention improved several measures of physical activity: +1730 (1003360) daily steps; +66 (28105) daily active minutes; +53 (2780) moderate-to-intense active minutes; and 157 (265 to 50) sedentary minutes. CONCLUSION: Among selected participants treated with TAVR, this study did not provide evidence that a pragmatic home-based mobile health exercise intervention improved daily steps, physical performance or QoL for the overall cohort. However, the intervention did improve several measures of daily activity, particularly among individuals not participating in CR. TRIAL REGISTRY: Clinicaltrials.gov NCT03270124.

Endothelium-protective, histone-neutralizing properties of the polyanionic agent defibrotide.

Neutrophil-mediated activation and injury of the endothelium play a role in the pathogenesis of diverse disease states ranging from autoimmunity to cancer to COVID-19. Neutralization of cationic proteins (such as neutrophil extracellular trap/NET-derived histones) with polyanionic compounds has been suggested as a potential strategy for protecting the endothelium from such insults. Here, we report that the FDA-approved polyanionic agent defibrotide (a pleiotropic mixture of oligonucleotides) directly engages histones and thereby blocks their pathological effects on endothelium. In vitro , defibrotide counteracted endothelial cell activation and pyroptosis-mediated cell death, whether triggered by purified NETs or recombinant histone H4. In vivo , defibrotide stabilized the endothelium and protected against histone-accelerated inferior vena cava thrombosis in mice. Mechanistically, defibrotide demonstrated direct and tight binding to histone H4 as detected by both electrophoretic mobility shift assay and surface plasmon resonance. Taken together, these data provide insights into the potential role of polyanionic compounds in protecting the endothelium from thromboinflammation with potential implications for myriad NET- and histone-accelerated disease states.

Prothrombotic autoantibodies in serum from patients hospitalized with COVID-19.

Patients with COVID-19 are at high risk for thrombotic arterial and venous occlusions. Lung histopathology often reveals fibrin-based blockages in the small blood vessels of patients who succumb to the disease. Antiphospholipid syndrome is an acquired and potentially life-threatening thrombophilia in which patients develop pathogenic autoantibodies targeting phospholipids and phospholipid-binding proteins (aPL antibodies). Case series have recently detected aPL antibodies in patients with COVID-19. Here, we measured eight types of aPL antibodies in serum samples from 172 patients hospitalized with COVID-19. These aPL antibodies included anticardiolipin IgG, IgM, and IgA; anti-ß2 glycoprotein I IgG, IgM, and IgA; and anti-phosphatidylserine/prothrombin (aPS/PT) IgG and IgM. We detected aPS/PT IgG in 24% of serum samples, anticardiolipin IgM in 23% of samples, and aPS/PT IgM in 18% of samples. Antiphospholipid autoantibodies were present in 52% of serum samples using the manufacturer's threshold and in 30% using a more stringent cutoff (≥40 ELISA-specific units). Higher titers of aPL antibodies were associated with neutrophil hyperactivity, including the release of neutrophil extracellular traps (NETs), higher platelet counts, more severe respiratory disease, and lower clinical estimated glomerular filtration rate. Similar to IgG from patients with antiphospholipid syndrome, IgG fractions isolated from patients with COVID-19 promoted NET release from neutrophils isolated from healthy individuals. Furthermore, injection of IgG purified from COVID-19 patient serum into mice accelerated venous thrombosis in two mouse models. These findings suggest that half of patients hospitalized with COVID-19 become at least transiently positive for aPL antibodies and that these autoantibodies are potentially pathogenic.

Neutrophil extracellular traps in stored canine red blood cell units.

BACKGROUND: Neutrophil extracellular traps (NETs), webs of DNA and citrullinated histones extruded from activated neutrophils cause transfusion-related acute lung injury. Supernatants of stored red blood cell (RBC) units might promote NETosis in neutrophils from the units or from transfusion recipients. HYPOTHESES: (1) NETs form during storage of canine RBC, (2) leukoreduction (LR) before storage of RBC reduces NETosis, and (3) supernatant from stored, nonleukoreduced (NLR) RBC units induces NETosis in healthy canine neutrophils modeling transfusion recipients. ANIMALS: Six healthy purpose-bred research dogs were utilized for blood donation. METHODS: Prospective controlled study. RBC units were collected from each dog, aseptically divided into 2 equal subunits, 1 of which was leukoreduced, and stored for 42 days. Stored units were sampled biweekly for quantification of NET markers citrullinated histone H3 (Western blot) and cell-free DNA (cfDNA) (DNA dye binding). Unit supernatants were applied ex vivo to canine neutrophils and extracellular DNA release representing NETosis was assessed. RESULTS: Markers of NETs increased during RBC storage (cfDNA P < .0001 and citrullinated H3 P = .0002) and were higher in NLR than LR units (day 42 LR cfDNA 0.34 ± 0.82 ng/mL vs day 42 NLR 1361.07 ± 741.00 ng/mL, P < .0001; day 42 LR citrullinated H3 0.19 ± 0.13 AU vs NLR 0.57 ± 0.34 AU, P = .007). Isolated neutrophils did not form NETs when exposed to stored canine RBC supernatant. CONCLUSIONS AND CLINICAL IMPORTANCE: NETosis occurs in stored canine NLR RBC units, and is attenuated by LR before storage. NETs might be mediators of transfusion reactions.

Bacterial polyphosphates interfere with the innate host defense to infection.

Polyphosphates are linear polymers and ubiquitous metabolites. Bacterial polyphosphates are long chains of hundreds of phosphate units. Here, we report that mouse survival of peritoneal Escherichia coli sepsis is compromised by long-chain polyphosphates, and improves with bacterial polyphosphatekinase deficiency or neutralization using recombinant exopolyphosphatase. Polyphosphate activities are chain-length dependent, impair pathogen clearance, antagonize phagocyte recruitment, diminish phagocytosis and decrease production of iNOS and cytokines. Macrophages bind and internalize polyphosphates, in which their effects are independent of P2Y1 and RAGE receptors. The M1 polarization driven by E. coli derived LPS is misdirected by polyphosphates in favor of an M2 resembling phenotype. Long-chain polyphosphates modulate the expression of more than 1800 LPS/TLR4-regulated genes in macrophages. This interference includes suppression of hundreds of type I interferon-regulated genes due to lower interferon production and responsiveness, blunted STAT1 phosphorylation and reduced MHCII expression. In conclusion, prokaryotic polyphosphates disturb multiple macrophage functions for evading host immunity.