Persistent clotting protein pathology in Long COVID/Post-Acute Sequelae of COVID-19 (PASC) is accompanied by increased levels of antiplasmin

Background Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by acute clinical pathologies, including various coagulopathies that may be accompanied by hypercoagulation and platelet hyperactivation. Recently, a new COVID-19 phenotype has been noted in patients after they have ostensibly recovered from acute COVID-19 symptoms. This new syndrome is commonly termed Long COVID/Post-Acute Sequelae of COVID-19 (PASC). Here we refer to it as Long COVID/PASC. Lingering symptoms persist for as much as 6 months (or longer) after acute infection, where COVID-19 survivors complain of recurring fatigue or muscle weakness, being out of breath, sleep difficulties, and anxiety or depression. Given that blood clots can block microcapillaries and thereby inhibit oxygen exchange, we here investigate if the lingering symptoms that individuals with Long COVID/PASC manifest might be due to the presence of persistent circulating plasma microclots that are resistant to fibrinolysis. Methods We use techniques including proteomics and fluorescence microscopy to study plasma samples from healthy individuals, individuals with Type 2 Diabetes Mellitus (T2DM), with acute COVID-19, and those with Long COVID/PASC symptoms. Results We show that plasma samples from Long COVID/PASC still contain large anomalous (amyloid) deposits (microclots). We also show that these microclots in both acute COVID-19 and Long COVID/PASC plasma samples are resistant to fibrinolysis (compared to plasma from controls and T2DM), even after trypsinisation. After a second trypsinization, the persistent pellet deposits (microclots) were solubilized. We detected various inflammatory molecules that are substantially increased in both the supernatant and trapped in the solubilized pellet deposits of acute COVID-19 and Long COVID/PASC, versus the equivalent volume of fully digested fluid of the control samples and T2DM. Of particular interest was a substantial increase in α(2)-antiplasmin (α2AP), various fibrinogen chains, as well as Serum Amyloid A (SAA) that were trapped in the solubilized fibrinolytic-resistant pellet deposits. Conclusions Clotting pathologies in both acute COVID-19 infection and in Long COVID/PASC might benefit from following a regime of continued anticlotting therapy to support the fibrinolytic system function.

Persistent Clotting Protein Pathology in Long COVID/ Post-Acute Sequelae of COVID-19 (PASC) is Accompanied by Increased Levels of Antiplasmin

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by acute clinical pathologies, including various coagulopathies that may be accompanied by hypercoagulation and platelet hyperactivation. Recently, a new COVID-19 phenotype has been noted in patients after they have ostensibly recovered from acute COVID-19 symptoms. This new syndrome is commonly termed Long COVID/Post-Acute Sequelae of COVID-19 (PASC). Here we refer to it as Long COVID/PASC. Lingering symptoms persist for as much as 6 months (or longer) after acute infection, where COVID-19 survivors complain of recurring fatigue or muscle weakness, being out of breath, sleep difficulties, and anxiety or depression. MethodsGiven that blood clots can block microcapillaries and thereby inhibit oxygen exchange, we here investigate if the lingering symptoms that individuals with Long COVID/PASC manifest might be due to the presence of persistent circulating plasma clots that are resistant to fibrinolysis. We use techniques including proteomics and fluorescence microscopy to study plasma samples from healthy individuals, individuals with Type 2 Diabetes Mellitus (T2DM), with acute COVID-19, and those with Long COVID/PASC symptoms. ResultsWe show that plasma samples from Long COVID/PASC still contain large anomalous (amyloid) deposits (microclots). We also show that these microclots in both acute COVID-19 and Long COVID/PASC plasma samples are resistant to fibrinolysis (compared to plasma from controls and T2DM), even after trypsinisation. After a second trypsinization, the persistent pellet deposits were solubilized. We detected various inflammatory molecules that are substantially increased in both the supernatant and trapped in the solubilized pellet deposits of acute COVID-19 and Long COVID/PASC, versus the equivalent volume of fully digested fluid of the control samples and T2DM. Of particular interest was a substantial increase in α(2)-antiplasmin (α2AP), various fibrinogen chains, as well as Serum Amyloid A (SAA) that were trapped in the solubilized fibrinolytic-resistant pellet deposits. ConclusionsClotting pathologies in both acute COVID-19 infection and in Long COVID/PASC might therefore benefit from following a regime of continued anticlotting therapy to support the fibrinolytic system function.

Early Changes in Crayfish Hemocyte Proteins after Injection with a ß-1,3-Glucan, Compared to Saline Injected and Naive Animals

Early changes in hemocyte proteins in freshwater crayfish Pacifastacus leniusculus, in response to an injection with the fungal pattern recognition protein β-1,3-glucan (laminarin) were investigated, as well as changes after saline (vehicle) injection and in naïve animals. Injection of saline resulted in rapid recruitment of granular hemocytes from surrounding tissues, whereas laminarin injection on the other hand induced an initial dramatic drop of hemocytes. At six hours after injection, the hemocyte populations therefore were of different composition. The results show that mature granular hemocytes increase in number after saline injection as indicated by the high abundance of proteins present in granular cell vesicles, such as a vitelline membrane outer layer protein 1 homolog, mannose-binding lectin, masquerade, crustin 1 and serine protease homolog 1. After injection with the β-1,3-glucan, only three proteins were enhanced in expression, in comparison with saline-injected animals and uninjected controls. All of them may be associated with immune responses, such as a new and previously undescribed Kazal proteinase inhibitor. One interesting observation was that the clotting protein was increased dramatically in most of the animals injected with laminarin. The number of significantly affected proteins was very few after a laminarin injection when compared to uninjected and saline-injected crayfish. This finding may demonstrate some problematic issues with gene and protein expression studies from other crustaceans receiving injections with pathogens or pattern recognition proteins. If no uninjected controls are included and no information about hemocyte count (total or differential) is given, expressions data for proteins or mRNAs are very difficult to properly interpret.

Persistent clotting protein pathology in Long COVID/ Post-Acute Sequelae of COVID-19 (PASC) is accompanied by increased levels of antiplasmin

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by acute clinical pathologies, including various coagulopathies that may be accompanied by hypercoagulation and platelet hyperactivation. Recently, a new COVID-19 phenotype has been noted in patients after they have ostensibly recovered from acute COVID-19 symptoms. This new syndrome is commonly termed Long COVID/Post-Acute Sequelae of COVID-19 (PASC). Here we refer to it as Long COVID/PASC. Lingering symptoms persist for as much as 6 months (or longer) after acute infection, where COVID-19 survivors complain of recurring fatigue or muscle weakness, being out of breath, sleep difficulties, and anxiety or depression. Given that blood clots can block microcapillaries and thereby inhibit oxygen exchange, we here investigate if the lingering symptoms that individuals with Long COVID/PASC manifest might be due to the presence of persistent circulating plasma clots that are resistant to fibrinolysis. We use techniques including proteomics and fluorescence microscopy to study plasma samples from healthy individuals, individuals with Type 2 Diabetes Mellitus (T2DM), with acute COVID-19, and those with Long COVID/PASC symptoms. We show that plasma samples from Long COVID/PASC still contain large anomalous (amyloid) deposits. We also show that these anomalous deposits in both acute COVID-19 and Long COVID/PASC plasma samples are resistant to fibrinolysis (compared to plasma from controls and T2DM), even after trypsinisation. After a second trypsinization, the persistent pellet deposits were solubilized. We detected various inflammatory molecules that are substantially increased in both the supernatant and trapped in the solubilized pellet deposits of acute COVID-19 and Long COVID/PASC, versus the equivalent volume of fully digested fluid of the control samples. Of particular interest was a substantial increase in α(2)-antiplasmin (α2AP), various fibrinogen chains, as well as Serum Amyloid A (SAA) that were trapped in the solubilized fibrinolytic-resistant pellet deposits. Clotting pathologies in both acute COVID-19 infection and in Long COVID/PASC might therefore benefit from following a regime of continued anticlotting therapy to support the fibrinolytic system function.

Fibrinogen function achieved through multiple covalent states

Disulfide bonds link pairs of cysteine amino acids and their formation is assumed to be complete in the mature, functional protein. Here, we test this assumption by quantifying the redox state of disulfide bonds in the blood clotting protein fibrinogen. The disulfide status of fibrinogen from healthy human donor plasma and cultured human hepatocytes are measured using differential cysteine alkylation and mass spectrometry. This analysis identifies 13 disulfide bonds that are 10–50% reduced, indicating that fibrinogen is produced in multiple disulfide-bonded or covalent states. We further show that disulfides form upon fibrin polymerization and are required for a robust fibrin matrix that withstands the mechanical forces of flowing blood and resists premature fibrinolysis. The covalent states of fibrinogen are changed by fluid shear forces ex vivo and in vivo, indicating that the different states are dynamic. These findings demonstrate that fibrinogen exists and functions as multiple covalent forms.

Molecular packing structure of fibrin fibers resolved by X-ray scattering and molecular modeling

The blood clotting protein fibrin has extraordinary elastomeric properties due to its hierarchical structure. SAXS is combined with computational molecular modeling, providing insight in fibrin elasticity and guidelines for designing new polymers.

RESPON IMUNITAS BENIH LOBSTER, Panulirus homarus DENGAN PENGGUNAAN PROBIOTIK PADA PAKAN MOIST

Pemeliharaan benih lobster P. homarus masih menghadapi beberapa permasalahan, di antaranya infeksi penyakit bakteri (red body disease) dan mortalitas yang tinggi. Tujuan penelitian ini adalah untuk mengkaji respons imunitas benih lobster P. homarus yang diberi pakan pelet basah (moist diets) dengan penambahan probiotik. Pemeliharaan benih lobster dilakukan secara individu (1 ekor/keranjang). Lama pemeliharaan selama tiga bulan. Bobot awal puerulus P. homarus adalah 0,37 ± 0,05 g. Perlakuan meliputi pemberian pakan moist yang ditambahkan (A) ragi Saccharomyces cerevisiae, (B) kombinasi probiotik, Alteromonas sp. BY-9 dan Bacillus cereus BC, dan (C) tanpa probiotik. Respons imunitas dianalisis dengan RT-qPCR melalui tujuh gen target terkait ekspresi imunitas, setelah diuji tantang dengan Vibrio harveyi (penyebab red body disease). Hasil penelitian menunjukkan bahwa sintasan benih lobster sebesar (A) 32,22%; (B) 29,63%; dan (C) 33,33%. Pertumbuhan panjang dan bobot benih lobster tidak berbeda nyata (P>0,05). Respons imunitas benih lobster P. homarus pada perlakuan A dan B menunjukkan nilai ekspresi imun yang lebih tinggi dibandingkan dengan perlakuan C (tanpa probiotik). Ekspresi gen penyandi anti lipopolisakarida (ALFHa-1) meningkat pada (A) rata-rata sebesar 3,44 kali dan (B) 3,25 kali dibandingkan dengan perlakuan C (2,43 kali). Kelipatan ekspresi profenoloksidase (proPO) benih lobster meningkat pada perlakuan A (penambahan ragi) rata-rata sebesar 5,27 kali, sedangkan pada perlakuan B (kombinasi probiotik) sebesar 12,92 kali. Ekspresi Clotting sistem (transglutaminase, clotting protein) dan antioxidant defense mechanism (glutathione peroxidase/GPO) dan SAA juga mengalami peningkatan pada perlakuan A dan B.A number of contrains including disease infections and significant mortality have been occurring in lobster aquaculture. The aim of this research was to observe the immune response of juvenile lobster P. homarus culture fed by moist pellet supplemented with probiotic. Experimental juveniles were reared in individual system (one juvenile/basket). The experiment was conducted for three months. The treatments comprised (A) whole cell of yeast Saccharomyces cerevisiae, (B) combination of probiotics Alteromonas sp. BY-9 and Bacillus sp. BC, and (C) without probiotic as control. Initial weight of juveniles were 0.37 ± 0.05 g. Immunity responses were analyzed using seven immunity related genes expression by RT-qPCR. The results showed that the survival rate of juvenile for treatments A, B, and C were 32.22%, 29.63%, and 33.33% respectively. The weight and length gain of the juvenile were not significantly different (P>0.05) among treatments. Based on immunity related gene expression analysis, it revealed that A and B treatments have shown differences in the increament of immunity responses. Expressions of ALFHa-1 genes were increased on (A) treatment with average of 3.44 fold and (B) treatment (3.25 fold) and higher than C treatment (2.03 fold). Prophenoloxidase (ProPO) expression was increase average up to 5.27 fold on A (yeast supplementated) treatment and B (combination of probiotic) were 12.92 fold. Gene expression on Clotting system (transglutaminase, clotting protein) and antioxidant defense mechanism (glutathione peroxidase/GPO) was increased on A and B treatments.

Physiological and immune response of Litopenaeus vannamei undergoing the acute phase of the necrotizing hepatopancreatitis disease and after being treated with oxytetracycline and FF

The physiological and immune responses of adult shrimp (Litopenaeus vannamei) undergoing the acute phase of the necrotizing hepatopancreatitis (NHP) disease and the efficiency of oxytetracycline (OXI) and florfenicol (FF) to eliminate the pathogen were evaluated. Four shrimp groups were considered: three groups infected with necrotizing hepatopancreatitis bacteria (NHP-B) (two treated with antibiotics and a positive control) and one group non-infected (negative control). Hemolymph concentration of glucose, lactate, acylglycerides, cholesterol, total protein, aminotransferases, superoxide dismutase, and the transcriptional expression of several immune related genes were monitored at the acute phase of the disease, and at 15 and 20 days after administration of antibiotics (daa). Shrimp from the positive control registered a mortality of 100%. NHP-B infection affected the immu-nophysiological response of shrimp; herein, most of the parameters were significantly up regulated in infected shrimp before the use of antibiotics, compared to the negative control. Increased transcriptional levels of clotting protein, lipopolysaccharide and β-1-3-glucan binding protein (LGBP), serine protease, peroxinectin, lysozyme, heat shock proteins (HSP) 60 and 70 were detected in shrimp treated with OXI. At 20 daa NHP-B was still detected in FF-treated shrimp, but not in OXI-treated shrimp. It is hypothesized that despite none of the antibiotics per se eliminated the bacterium, both had a negative effect on its virulence. OXI seems to have a greater effect, allowing shrimp to integrate a better immune response at 15 daa.