Cohen, M. J. | Search bioRxiv

bioRxiv2021

Increased Histone-DNA Complexes and Endothelial-Dependent Thrombin Generation in Severe COVID-19

ObjectiveCoagulopathy in severe COVID-19 is common but poorly understood. The purpose of this study was to determine how SARS-CoV-2 infection impacts histone levels, fibrin structure, and endogenous thrombin potential in the presence and absence of endothelial cells. ApproachWe studied individuals with SARS-CoV-2 infection and acute respiratory distress syndrome at the time of initiation of mechanical ventilation compared to healthy controls. Blood samples were assayed for levels of histone-DNA complexes. Confocal microscopy was used to evaluate fibrin structure in clots formed from recalcified plasma samples using fluorescently-labeled fibrinogen. Endogenous thrombin potential was measured by calibrated automated thrombin assays in the presence of tissue factor and phospholipid (PCPS) or cultured human endothelial cells. ResultsCirculating nucleosomes were elevated in the plasma of COVID-19 patients relative to healthy controls (n=6, each group). COVID-19 patient plasma thrombin generation was also altered. Despite having an increased endogenous thrombin potential, patient plasma samples exhibited prolonged lag times and times to peak thrombin in the presence of added tissue factor and PCPS. Strikingly different results were observed when endothelial cells were used in place of tissue factor and PCPS. Control plasma samples did not generate measurable thrombin (lag time >60 min); in contrast, plasma samples from COVID-19+ patients generated thrombin (mean lag time [~]20 min). Consistent with the observed alterations in thrombin generation, clots from COVID-19 subjects exhibited a denser fibrin network, thinner fibers and lower fibrin resolvability. ConclusionsElevated histones, aberrant fibrin formation, and increased endothelial-dependent thrombin generation in COVID-19 may contribute to coagulopathy. HIGHLIGHTSO_LIHistone-DNA complexes are significantly elevated in the plasma of patients with severe SARS-CoV-2 infection. C_LIO_LIMeasures of thrombin generation by calibrated automated thrombography and fibrin clots formed in situ are altered in severe COVID-19. C_LIO_LIPlasma from COVID-19 patients promotes thrombin generation on cultured endothelial cells in the absence of added tissue factor or phospholipids. C_LIO_LIThe additive effects of histones on thrombin generation and endothelial cell function may play a major role in the thrombotic complications observed in severe SARS-CoV-2 infection. C_LI

biochemistry↗

bioRxiv2020

Enzymatic analysis of yeast cell wall-resident GAPDH and its secretion

In yeast, many proteins are found both in the cytoplasmic and extracellular compartments, and consequently it can be difficult to distinguish non-conventional secretion from cellular leakage. We therefore monitored extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity of intact cells as a specific marker for non-conventional secretion. Extracellular GAPDH activity was proportional to the number of cells assayed, increased with incubation time, and was dependent on added substrates. Preincubation of intact cells with 100M dithiothreitol increased the reaction rate, consistent with increased access of the enzyme after reduction of cell wall disulfide crosslinks. Such treatment did not increase cell permeability to propidium iodide, in contrast to effects of higher concentrations of reducing agents. An amine-specific membrane-impermeant biotinylation reagent specifically inactivated extracellular GAPDH. The enzyme was secreted again after a 30-60-minute lag following the inactivation, and there was no concomitant increase in propidium iodide staining. There were about 4 x 104 active GAPDH molecules per cell at steady state, and secretion studies showed replenishment to that level one hour after inactivation. These results establish conditions for specific quantitative assays of cell wall proteins in the absence of cytoplasmic leakage and for subsequent quantification of secretion rates in intact cells. ImportanceEukaryotic cells secrete many proteins, including many proteins that do not follow the classical secretion pathway. Among these, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is unexpectedly found in the walls of yeasts and other fungi, and in extracellular space in mammalian cell cultures. It is difficult to quantify extracellular GAPDH, because leakage of just a little of the very large amount of cytoplasmic enzyme can invalidate the determinations. We used enzymatic assays of intact cells, while also maintaining membrane integrity. The results lead to estimates of the amount of extracellular enzyme, and its rate of secretion to the wall in intact cells. Therefore, enzyme assays under controlled conditions can be used to investigate non-conventional secretion more generally.

cell biology↗