This study was performed according to the latest version of the declaration of Helsinki and guidelines for good clinical practice. The local independent ethical committee approved the study protocol (CMO 2020-6344, CMO 2020-6359, CMO 2016-2923). All patients admitted to the Radboud University Medical Center (Radboudumc) with a PCR-proven SARS-CoV-2 infection was asked for informed consent for participation in this study. After obtaining informed consent, ethylenediaminetetraacetic acid (EDTA) blood was collected and centrifuged for 10 min at 2,954 xg at room temperature (RT), plasma was collected and stored at −80°C for later analysis. For most patients in the non-ICU group, the blood samples collected between day 2 and 4 upon hospitalization were selected for this study except for the patients in the LMWH- group for whom the sample had to be collected immediately upon hospital admission prior to initiation of any standard medical intervention. Demographic data, medical history, and clinical laboratory measurements were collected from the medical file and processed in encoded form in electronic case report forms using Castor electronic data capture (Castor EDC, Amsterdam, Netherlands).

The activity of HPSE in EDTA plasma was determined by an in-house developed activity assay, which was optimized by the use of recombinant active human HPSE (Bio-techne, Abingdon, United Kingdom, Cat#7570-GH-005). In detail, Nunc maxisorp flat bottom 96 plates (Thermo scientific, Breda, Netherlands) were coated with 10 ug/ml heparan sulfate from bovine kidney (HSBK) (Sigma-Aldrich, Zwijndrecht, Netherlands) in optimized HS coating buffer, overnight in a humidified chamber at RT. Subsequently, plates were washed with 0.05% PBS-Tween 20 (Sigma-Aldrich) (PBST) and blocked for minimal 2 h with 1% bacto-gelatin (Difco laboratories, Detroit, Michigan, USA) in PBS at RT. Plates were washed with PBST, followed by a final washing step with PBS prior to 2 h incubation at 37°C with 4 times diluted plasma sample in HPSE buffer. Samples were randomly distributed over plates. The HPSE buffer consisted of 50 mM citric acid-sodium citrate (Merck, Zwijndrecht, Netherlands) buffer supplemented with 50 mM NaCl (Merck), 1 mM CaCl₂ (Sigma-Aldrich), and 1 mM DTT (Sigma-Aldrich) at final pH 5.0. Next, plates were washed with PBST and incubated with primary mouse anti-rat IgM HS antibody JM403 (Amsbio, Abingdon, United Kingdom, cat. no. #370730-S, RRID: AB_10890960, 1 μg/ml in PBST) for 1 h at RT. Subsequently, plates were washed with PBST and incubated with secondary goat anti-mouse IgM HRP antibody (Southern Biotech, Uden, Netherlands, cat. no. #1020-05, RRID: AB_2794201, dilution 1:10000 in PBST) for 1 h at RT. Finally, plates were washed with PBST and 3,3’,5,5’-tetramethylbenzidine (TMB) substrate (Invitrogen, Breda, Netherlands) was added and reaction was stopped by addition of 2 M sulfuric acid, and absorbance was measured at 450 nm. The HPSE activity in plasma was related to a standard curve of recombinant human HPSE in healthy control EDTA plasma.

For the in vitro HPSE inhibition experiment with dalteparin (Pfizer, Capelle a/d Ijssel, Netherlands, Fragmin 12,500 IU/0.5 ml), the HPSE activity was determined using the HPSE activity assay as outlined above. For inhibition studies 0–1 IU/ml dalteparin was used with a constant amount of 150 ng/ml recombinant human HPSE.

HS in EDTA plasma samples was quantified by a previously described HS competition assay (23, 24). Importantly, this assay is specific to HS, therefore the measurement is not affected by the presence of LMWH use. Plates were coated with HSBK and blocked with bacto-gelatin as outlined for the HPSE activity assay. Uncoated plates, blocked with bacto-gelatin, were washed with PBST. The plasma samples were four times diluted in PBST containing primary mouse anti-rat IgM HS antibody JM403 (1.3 μg/ml) and incubated for 1 h at RT. Samples were randomly distributed over plates. Subsequently, the samples were transferred from the uncoated plates to the HSBK-coated plates and incubated for 1 h at RT. Plates were washed with PBST and incubated with secondary goat anti-mouse IgM HRP antibody for 1 h at RT. Plates were developed and measured as outlined for the HPSE activity assay. The amount of HS detected in plasma is expressed in arbitrary units since HS from bovine kidney was coated and used to prepare the standard curve.

IL-6 concentration was measured in plasma of COVID-19 patients using commercial ELISA kits for human IL-6 (Bio-techne, Abingdon, United Kingdom) according to manufacturer’s instruction.

Values are expressed as mean ± SEM. D’Agostino & Pearson normality test was performed to test for normality of data. Significance was determined by Fisher’s exact test to compare categorical variables, by Student’s t-test or Mann Whitney test to compare two groups and by Kruskal-Wallis test followed by Dunn’s test to compare more than two groups using GraphPad Prism V.8.4.2 (La Jolla, USA). P values less than 0.05 were considered as statistically significant.

Plasma was collected from 48 PCR-confirmed COVID-19 patients admitted to the ICU (n = 14) or to designated COVID-19 clinical wards (n = 34). More men than women were included (Table 1). All ICU patients received LMWH as part of standard ICU treatment. The non-ICU patients were further aggregated in those receiving prophylactic LMWH (LMWH+) (in general dalteparin 5,000 IU subcutaneously once daily) (n = 17) and those receiving either alternative anticoagulation (n = 8; vitamin K antagonist n = 6, direct oral anticoagulant n = 2) or patients for whom the sample collection was performed before initiation of any standard medical intervention (LMWH−) (n = 9). Hospital stay duration was significantly different between ICU and non-ICU groups as well as between LMWH− and LMWH+ groups, while number of deaths was not significantly different between the different subgroups. Notably, ICU patients had a significantly higher C-reactive protein concentration than non-ICU patients (Table 2). The LMWH+ group had significantly higher median D-dimer concentrations compared to LMWH− group, whereas the concentrations of the inflammatory markers C-reactive protein and serum ferritin were similar between LMWH+ and LMWH−.

Several (experimental) disease models have shown that increased HPSE activity can lead to endothelial barrier dysfunction, which may be involved in the development of ARDS and proteinuria/AKI (7, 25, 26). Measurement of plasma HPSE activity levels in COVID-19 patients and healthy controls revealed that HPSE activity was significantly elevated in COVID-19 patients compared to healthy controls (Figure 1A). In line with the increased HPSE activity, HS plasma levels were also significantly elevated in COVID-19 patients compared to healthy controls (Figure 1B). Similarly, we detected significantly elevated IL-6 levels in COVID-19 patients compared to healthy controls (Figure 1C). Overall, these results suggest that SARS-CoV-2 infection is associated with an increase in the activity of HPSE in plasma and an increase in plasma HS and IL-6 levels.

Next, we investigated whether HPSE activity levels were associated with COVID-19 disease severity. Plasma HPSE activity was significantly increased in both non-ICU and ICU patients compared to healthy controls, and HPSE levels in ICU patients, who all received mechanical ventilation, were higher than in non-ICU patients (Figure 2A). Moreover, HS (Figure 2B) and IL-6 levels (Figure 2C) in plasma were also higher in both non-ICU and ICU patients compared to healthy controls. Finally, plasma HPSE activity was significantly higher in patients with elevated LDH values (Figure 2D), and in patients with elevated serum creatinine values (Figure 2E). These findings reveal that patients with severe COVID-19 disease display higher plasma HPSE activity levels than patients with moderate COVID-19 disease.

Prophylactic treatment with LMWH is recommended for patients hospitalized with COVID-19 (27), whereas some experts recommend higher doses for critically ill patients (28). As LMWH inhibits HPSE activity and it is known that HPSE deficiency reduces expression of various cytokines (15), we analyzed the effect of prophylactic LMWH on HPSE activity, HS levels and IL-6 levels in plasma of COVID-19 patients. Markedly, non-ICU patients who received LMWH displayed significantly lower plasma HPSE activity compared to non-ICU patients without LMWH prophylaxis (Figure 3A), whereas no statistically significant differences could be observed on HS levels (Figure 3B) or IL-6 levels (Figure 3C) between non-ICU patients with or without LMWH prophylaxis. According to literature, a single injection of 5,000 units dalteparin would result in an estimated concentration of around 0.37 U/ml in vivo (29). We found a dose dependent inhibition of recombinant HPSE at concentrations between 0.0025 and 0.05 U/ml and full inhibition starting from 0.25 U/ml dalteparin in vitro (Figure 3D). These data suggest that the applied prophylactic LMWH dose is already effective in inhibition of HPSE activity within plasma of moderately diseased, while HPSE activity remained high in severely ill, COVID-19 patients.