Recombinant ADAMTS13 reduces abnormally up-regulated von Willebrand factor in plasma from patients with severe COVID-19

Abstract

Thrombosis affecting the pulmonary and systemic vasculature is common during severe COVID-19 and causes adverse outcomes. Although thrombosis likely results from inflammatory activation of vascular cells, the mediators of thrombosis remain unconfirmed. In a cross-sectional cohort of 36 severe COVID-19 patients, we show that markedly increased plasma von Willebrand factor (VWF) levels were accompanied by a partial reduction in the VWF regulatory protease ADAMTS13. In all patients we find this VWF/ADAMTS13 imbalance to be associated with persistence of ultra-high-molecular-weight (UHMW) VWF multimers that are highly thrombogenic in some disease settings. Incubation of plasma samples from patients with severe COVID-19 with recombinant ADAMTS13 (rADAMTS13) substantially reduced the abnormally high VWF activity, reduced overall multimer size and depleted UHMW VWF multimers in a time and concentration dependent manner. Our data implicate disruption of normal VWF/ADAMTS13 homeostasis in the pathogenesis of severe COVID-19 and indicate that this can be reversed ex vivo by correction of low plasma ADAMTS13 levels. These findings suggest a potential therapeutic role for rADAMTS13 in helping restore haemostatic balance in COVID-19 patients.

Keywords: ADAMTS13; COVID-19; Endothelium; Inflammation; SARS-COV-2; Thrombosis; rADAMTS13; von Willebrand factor.

Fig. 1

VWF and ADAMTS13 levels in 36 patients with severe COVID-19. A: Summary statistics presented as median and range alongside reference intervals obtained from analysis of healthy volunteer samples. The UHMW VWF multimer quantitation parameter is the proportion of the migration distance of the VWF dimers that was occupied by all VWF multimers in each sample lane expressed as a percentage value of that in the healthy control plasma lane from the same gel. VWF - von Willebrand factor; UHMW - ultra high molecular weight; ADAMTS13 - a disintegrin and metalloprotease with a thrombospondin type 1 motif member 13; n.a.- not applicable. * normal human plasma is devoid of UHMW VWF multimers. B: Assay results from all patient samples and controls. COVID-19 patient samples (circles); Acute phase TTP control (triangle); Normal Plasma control (squares); the grey boxes indicate the reference intervals.

Fig. 2

Relationship between von Willebrand Factor laboratory parameters and ADAMTS13 activity in the COVID-19 samples. A: Graphical relationship between the laboratory parameters showing lines of best fit form the correlation analysis using the Pearson test. B: Correlation estimates as r-values and associated p-values. Correlations were classified as strong (r: 0.7 to 1.0), moderate (r: 0.5 to 0.7), weak (r: 0.3 to 0.5) or no correlation (r: 0 to 0.3).

Fig. 3

Electrophoretic analysis of von Willebrand factor multimers from patients with severe COVID-19. A: COVID-19 plasma samples (B17-B24) were separated by electrophoresis on a 1% agarose gel. VWF multimers were visualised after immunostaining with an anti-VWF primary antibody followed by a secondary goat-anti-rabbit ALP conjugate. Normal control 1- pooled normal plasma after virus inactivation by heat treatment; Normal control 2- pooled normal plasma without heat inactivation; Normal control 3- volunteer healthy donor travelling control plasma; Acute TTP control- sample from a patient with acute autoimmune TTP. The solid lines indicate the fastest migrating band in each lane which corresponds to VWF dimers. The broken lines indicate the upper limit of the stainable part of each lane indicating the largest VWF multimers. B: Representative densitometric scan of the sample lane for a COVID-19 patient (B24), for a patient with acute autoimmune TTP and for a pooled normal plasma sample. The x axis represents the distance in arbitrary units from the upper end of the separation gel (designated 0.0) and the lowest molecular weight band corresponding to the VWF dimers designated as 1.0. The y axis is the optical density.

Fig. 4

Example electrophoretic determination of multimer size in plasma from patient B26. The plasma sample from the patient was adjusted to 1 IU/ mL VWF:Ag and separated on a 1% agarose gel followed by immunostaining and densitometry. The distance between the top of the separation well and the lowest multimer band (VWF dimers) was assigned a migration value of 1.0. In this example the relative migration distance (Rf) of the largest VWF multimer was 0.192 that of the lowest multimer band. The proportion of the total migration distance of the VWF dimer band that is occupied by larger VWF multimers is therefore 1-Rf, in this example calculated as 1.000–0.192 = 0.808. The 1-Rf value for normal plasma separated on the same gel was 0.729 (not shown). Therefore, the proportion of the patient sample lane containing VWF multimers (UHMW multimer quantitation parameter) is 111% (0.808/0.729*100 = 111%) that of control reflecting the UHMW multimers near the top of the lane.

Fig. 5

VWF multimers from patients with severe COVID-19 visualised using the semi-automated electrophoresis system. A: Plasma samples of 9 patients with COVID-19 were separated by semi-automated electrophoresis using the HYDRAGEL von WILLEBRAND FACTOR MULTIMERS kit and a HYDRASYS 2 instrument. Each sample was adjusted to 1 IU VWF:Ag per mL, separated and stained for multimers in parallel. Control samples from a patient with acute TTP and a healthy volunteer was applied to the same gel. The broken line indicates the largest stainable part of the normal human plasma control. The migration distances of differently sized ultra-high molecular weight multimers were less pronounced as with the home-cast low resolution gels. Although, this prevented reproducible quantitation, the UHMW VWF multimers were clearly evident as abnormal immunostaining material above the dotted line. B: UHMW VWF quantified from the same samples from home cast gels using the analysis method presented in Fig. 4.

Fig. 6

Incubation of plasma samples with 0.5 or 1.0 IU/mL rADAMTS13. A: Plasma from illustrative severe COVID-19 patient S12 (VWF/ADAMTS13 ratio of 7.4) was incubated with 0.5 IU/mL (triangles) or 1 IU/mL (squares) rADAMTS13 or without ADAMTS13 (diamonds). Sub-samples were taken immediately after addition of rADAMTS13, and at 2 and 5 h and analysed for VWF activity by the collagen binding assay. B: Pooled absolute VWF:CB results from plasma samples from 10 severe COVID-19 patients labelled as in graph A. C: Pooled VWF:CBA values labelled as in graph A but with values expressed as a percentage of the value at 0 h. For both of the pooled data, the points are means and the error bars are standard deviations. There was a significant decrease in VWF:CBA (P < 0.001; t-value −7.04) after incubation for 5 h compared with baseline values.

Fig. 7

Incubation of plasma sample B1 with 1.0 or 10.0 IU/mL rADAMTS13. Plasma from severe COVID-19 patient B1 (VWF/ADAMTS13 ratio of 13.4) was incubated with 1.0 IU/mL (triangles) or 10.0 IU/mL (squares) rADAMTS13 or without ADAMTS13 (diamonds). Samples were taken immediately after addition of rADAMTS13, and at 2 and 5 h. A: VWF activity by the collagen binding assay; B: multimer composition visualised using the semi-automated electrophoresis gel system (Sebia) C: The corresponding gel densitograms with the VWF dimer peaks arrowed and high molecular weight VWF multimers to the right of each trace. The vertical axis represents the intensity of gel staining in arbitrary units. The control samples were normal human plasma, B1 plasma without the addition of any reagents and no incubation, and B1 plasma incubated under identical conditions but without the addition of rADAMTS13.