Removal of COVID-19 Spike Protein, Whole Virus, Exosomes, and Exosomal MicroRNAs by the Hemopurifier® Lectin-Affinity Cartridge in Critically Ill Patients With COVID-19 Infection

Coronavirus−19 (COVID-19) has rapidly spread throughout the world resulting in a significant amount of morbidity and mortality. Despite advances in therapy, social distancing, masks, and vaccination many places in the world continue to see an increase in the number of cases and deaths. Viremia is commonly present in severely ill patients with COVID-19 infections and is associated with organ dysfunction and poor outcomes. Exosomes released by activated cells have been implicated in the pathogenesis of COVID-19 infection. We report the experience of two cases of critically ill COVID-19 patients treated with the Hemopurifier; a lectin affinity cartridge designed to remove mannosylated viruses and exosomes. Both patients tolerated the Hemopurifier sessions without adverse effects. In the first patient removal of exosomes and exosomal microRNAs was associated with improved coagulopathy, oxygenation, and clinical recovery, while in a second patient removal of COVID-19 by the Hemopurifier cartridge was observed. The Hemopurifier is currently under further investigation in up to 40-patients in a safety and feasibility study in ICU patients with COVID-19 infection.

Removal of COVID-19 Spike Protein, Whole Virus, Exosomes and Exosomal microRNAs by the Hemopurifier® Lectin-Affinity Cartridge in Critically Ill Patients with COVID-19 Infection

Coronavirus − 19 (COVID-19) has rapidly spread throughout the world resulting in a significant amount of morbidity and mortality. Despite advances in therapy, social distancing, masks, and vaccination many places in the world continue to see an increase in the number of cases and deaths. Viremia is commonly present in severely ill patients with COVID-19 infections and is associated with organ dysfunction and poor outcomes. Exosomes released by activated cells have been implicated in the pathogenesis of COVID-19 infection. We report the experience of two cases of critically ill COVID-19 patients treated with the Hemopurifier; a lectin affinity cartridge designed to remove mannosylated viruses and exosomes. Both patients tolerated the Hemopurifier sessions without adverse effects. In the first patient removal of exosomes and exosomal microRNAs was associated with improved coagulopathy, oxygenation, and clinical recovery, while in a second patient removal of COVID-19 by the Hemopurifier cartridge was observed. The Hemopurifier is currently under further investigation in up to 40-patients in a safety and feasibility study in ICU patients with COVID-19 infection.

A Retrospective Analysis of the Cartilage Kunitz Protease Inhibitory Proteins Identifies These as Members of the Inter-α-Trypsin Inhibitor Superfamily with Potential Roles in the Protection of the Articulatory Surface

Aim: The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors (SPIs) were related to the Kunitz inter-α-trypsin inhibitor (ITI) family. Methods: Ovine articular cartilage was finely diced and extracted in 6 M urea and SPIs isolated by sequential anion exchange, HA affinity and Sephadex G100 gel permeation chromatography. Selected samples were also subjected to chymotrypsin and concanavalin-A affinity chromatography. Eluant fractions from these isolation steps were monitored for protein and trypsin inhibitory activity. Inhibitory fractions were assessed by affinity blotting using biotinylated trypsin to detect SPIs and by Western blotting using antibodies to α1-microglobulin, bikunin, TSG-6 and 2-B-6 (+) CS epitope generated by chondroitinase-ABC digestion. Results: 2-B-6 (+) positive 250, 220,120, 58 and 36 kDa SPIs were detected. The 58 kDa SPI contained α1-microglobulin, bikunin and chondroitin-4-sulfate stub epitope consistent with an identity of α1-microglobulin-bikunin (AMBP) precursor and was also isolated by concanavalin-A lectin affinity chromatography indicating it had N-glycosylation. Kunitz protease inhibitor (KPI) species of 36, 26, 12 and 6 kDa were autolytically generated by prolonged storage of the 120 and 58 kDa SPIs; chymotrypsin affinity chromatography generated the 6 kDa SPI. KPI domain 1 and 2 SPIs were separated by concanavalin lectin affinity chromatography, domain 1 displayed affinity for this lectin indicating it had N-glycosylation. KPI 1 and 2 displayed potent inhibitory activity against trypsin, chymotrypsin, kallikrein, leucocyte elastase and cathepsin G. Localisation of versican, lubricin and hyaluronan (HA) in the surface regions of articular cartilage represented probable binding sites for the ITI serine proteinase inhibitors (SPIs) which may preserve articulatory properties and joint function. Discussion/Conclusions: The Kunitz SPI proteins synthesised by articular chondrocytes are members of the ITI superfamily. By analogy with other tissues in which these proteins occur we deduce that the cartilage Kunitz SPIs may be multifunctional proteins. Binding of the cartilage Kunitz SPIs to HA may protect this polymer from depolymerisation by free radical damage and may also protect other components in the cartilage surface from proteolytic degradation preserving joint function.

ProbingN-glycoprotein microheterogeneity by lectin affinity purification-mass spectrometry analysis

A lectin affinity purification-mass spectrometry approach to characterize lectin-reactive glycoproteoforms and elucidate lectin specificities at the intact protein level.