key: cord-0890046-jz44o8tq authors: Buijsers, B.; Yanginlar, C.; Grondman, I.; de Nooijer, A.; Maciej-Hulme, M. L.; Jonkman, I.; Janssen, N.; Rother, N.; de Graaf, M.; Pickkers, P.; Kox, M.; Joosten, L.; Nijenhuis, T.; Netea, M. G.; Hillbrands, L.; van de Veerdonk, F.; Duivenvoorden, R.; de Mast, Q.; van der Vlag, J. title: Increased plasma heparanase activity in COVID-19 patients date: 2020-06-15 journal: nan DOI: 10.1101/2020.06.12.20129304 sha: 8b50747d7ff81687d722dc9d1aef5795c395ba2d doc_id: 890046 cord_uid: jz44o8tq Background: Several reports suggest endothelial dysfunction and loss of endothelial barrier function in COVID-19. It is well established that the endothelial glycocalyx-degrading enzyme heparanase (HPSE) contributes to vascular leakage and inflammation. Low molecular weight heparins (LMWH) serve as an inhibitor of heparanase. We hypothesize that heparanase contributes to the pathogenesis of COVID-19, and that HPSE may be inhibited by the use of LMWH. Methods: Heparanase activity and heparan sulfate levels were measured in plasma of healthy controls (n=10) and COVID-19 patients (n=48). Findings: Heparanase activity and heparan sulfate levels were significantly elevated in plasma of COVID-19 patients. There was an association between heparanase activity and disease severity including the need for intensive care and mechanical ventilation, lactate dehydrogenase levels and creatinine levels. Use of prophylactic low molecular weight heparin in non-ICU patients was associated with a reduced HPSE activity. Interpretation: Prophylactic doses of low molecular weight heparin reduces heparanase activity in COVID-19. In addition to HPSE inhibition, low molecular weight heparin contributes to anti-coagulation and may exert anti-inflammatory effects. Since there is no other clinically applied heparanase inhibitor currently available, treatment of COVID-19 patients with low molecular weight heparins should be explored. Funding: This study was financially supported by the Radboudumc PhD fellow program, consortium grant LSHM16058-SGF (GLYCOTREAT; a collaborative project financed by the PPP allowance made available by Top Sector Life Sciences & Health to the Dutch Kidney Foundation to stimulate public-private partnerships), ERC Advanced grant (#833247) and a Spinoza Grant of the Netherlands Organization for Scientific Research. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 15, 2020. . https://doi.org/10.1101/2020.06.12.20129304 doi: medRxiv preprint The coronavirus disease-2019 (COVID-19) pandemic caused by the severe acute 84 respiratory syndrome coronavirus 2 (SARS-CoV-2) has impacted widely on global health. 1 85 Severe COVID-19 usually manifests as pneumonitis or acute respiratory distress syndrome 86 (ARDS). 2,3 Moreover, severe COVID-19 can lead to multi-organ dysfunction. Case series 87 showed that upon hospital admission 59% of COVID-19 patients had proteinuria 4 , and 22% 88 of the non-ventilated patients and 90% of the ventilated patients developed acute kidney 89 injury (AKI). 5 Therefore, patients with more severe COVID-19 disease show a higher 90 incidence of AKI, highlighting AKI as a negative prognostic factor for the survival of COVID-91 19 patients. 5 conditions. 14 Degradation of HS by heparanase (HPSE), the only known mammalian HS-105 degrading enzyme, disrupts the endothelial glycocalyx. As such, shedding of the glycocalyx 106 and subsequent loss of endothelial barrier function, as observed in ARDS and proteinuric 107 kidney diseases, can be attributed to increased HPSE activity. 7,15-17 In addition to declining 108 barrier function, HPSE generates a pro-inflammatory glycocalyx that promotes the binding of 109 chemokines, cytokines and leukocytes to the endothelial cell surface. Inhibition of HPSE 110 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Taken together, we hypothesize that increased HPSE activity is one of the driving forces in 115 severe COVID-19 manifestation, including ARDS and proteinuria/AKI, and that HPSE may 116 be inhibited by the use of LMWH in COVID-19. 117 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Subsequently, plates were washed with PBST and incubated with secondary goat anti-145 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 15, 2020. . https://doi.org/10.1101/2020.06.12.20129304 doi: medRxiv preprint mouse IgM HRP antibody (Southern Biotech, cat. no. #1020-05, RRID: AB_2794201, 146 dilution 1:10000 in PBST) for 1 hour at RT. Finally, plates were washed with PBST and 147 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Invitrogen) was added and reaction was 148 stopped by addition of 2M sulfuric acid, and absorbance was measured at 450 nm. The 149 HPSE activity in plasma was related to a standard curve of recombinant human HPSE (R&D 150 systems, Cat#7570-GH-005) in healthy control EDTA plasma. 151 For the in vitro HPSE inhibition experiment with dalteparin (Pfizer, Fragmin 12,500 IU/0.5 152 ml), the HPSE activity was determined using the HPSE activity assay described above. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Kruskal-Wallis test followed by Dunn's test to compare more than two groups using 177 GraphPad Prism V.8.4.2 (La Jolla, USA). P values less than 0.05 were considered as 178 statistically significant. 179 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 15, 2020. . https://doi.org/10.1101/2020.06.12.20129304 doi: medRxiv preprint Plasma was collected from 48 PCR-confirmed COVID-19 patients admitted to the ICU (n= 182 14) or to designated COVID-19 clinical wards (n= 34). More men than women were included 183 (Table 1) Next, we investigated whether HPSE activity levels were associated with COVID-19 disease 206 severity. More specifically, potential associations were assessed between HPSE activity 207 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 15, 2020. . https://doi.org/10.1101/2020.06.12.20129304 doi: medRxiv preprint levels and the need for intensive care or for mechanical ventilation, lactate dehydrogenase 208 (LDH) values as a measure of tissue damage and creatinine values as a measure of kidney 209 function. Plasma HPSE activity was significantly higher in both non-ICU and ICU patients 210 compared to healthy controls, and HPSE levels in ICU patients were significantly higher than 211 in non-ICU patients (Figure 2A ). HS levels in plasma were also significantly higher in both 212 non-ICU and ICU patients compared to healthy controls ( Figure 2B ). HPSE activity was also 213 significantly elevated in the plasma of patients in need of mechanical ventilation ( Figure 2C ). 214 Importantly, plasma HPSE activity was increased in patients with elevated LDH values (>280 215 U/l) compared to patients with LDH values within the normal range ( Figure 2D ). Patients with 216 elevated serum creatinine values (>110 µmol/ for men and >90 µmol/ for women) also 217 displayed increased plasma HPSE activity ( Figure 2E ). These findings reveal that patients 218 with severe COVID-19 disease, such as those admitted to the ICU department, have higher 219 plasma HPSE activity levels than patients with moderate, such as non-ICU patients, COVID-220 Recent studies show a high rate of thromboembolic complications in patients with severe 224 COVID-19. Autopsy studies in COVID-19 patients have identified the presence of 225 coagulation in the microvasculature, which might also contribute to organ failure. 26,27 226 Prophylactic treatment with LMWH is therefore recommended for patients hospitalized with 227 COVID-19 28 , whereas some experts recommend higher doses for critically ill patients. As 228 LMWH possesses HPSE inhibiting properties, the effect of prophylactic LMWH on HPSE 229 activity in plasma of COVID-19 patients was analyzed. Markedly, non-ICU patients who 230 received LMWH displayed significantly lower HPSE activity compared to non-ICU patients 231 without LMWH prophylaxis ( Figure 3A) . According to literature a single injection of 5000 232 units dalteparin would result in an estimated concentration of around 0.37 U/ml in vivo. 29 We 233 found a dose dependent inhibition of recombinant HPSE at concentrations between 0.0025 234 and 0.05 U/ml and full inhibition starting from 0.25 U/ml dalteparin in vitro ( Figure 3B ). These 235 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. figure 1 ). 248 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. COVID-19 appears to be a disease that leads to endothelial dysfunction and disruption of 250 the endothelial barrier, which may underly development of ARDS and proteinuria/AKI. 11,33 251 Here, we report increased HPSE activity and HS levels in plasma of COVID-19 patients, 252 which were also associated with severity of the disease. IL-1, independent of HS-degrading activity. 45 HS fragments released by HPSE activity also 276 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 15, 2020. In summary, this cross-sectional study shows that HPSE activity and HS levels are 300 significantly elevated in plasma of COVID-19 patients, which is associated with the severity 301 of COVID-19. Targeting of HPSE activity could be beneficial for the clinical outcome of 302 COVID-19 patients, since it is well established that increased HPSE activity compromises 303 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 15, 2020. . https://doi.org/10.1101/2020.06.12.20129304 doi: medRxiv preprint the endothelial glycocalyx and endothelial barrier function and contributes to the 304 establishment of a pro-inflammatory cytokine milieu. Considering the fact that no specific 305 clinically approved heparanase inhibitors are currently available, prospective studies 306 evaluating the clinical outcome of COVID-19 patients treated with therapeutic doses of 307 LMWH are urgently needed. 308 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 15, 2020. BioRender.com. 316 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 15, 2020. . https://doi.org/10.1101/2020.06.12.20129304 doi: medRxiv preprint The authors have declared that no conflict of interest exists. 318 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 15, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 15, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 15, 2020. . https://doi.org/10.1101/2020.06.12.20129304 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 15, 2020. . https://doi.org/10.1101/2020.06.12.20129304 doi: medRxiv preprint 28 history of renal disease were excluded from this analysis). HPSE activity was measured 517 using an in-house developed ELISA with a specific anti-HS antibody. Data were presented 518 as mean±SEM and tested for normal distribution with D'Agostino & Pearson omnibus 519 normality test and statistical differences were calculated using Kruskal Wallis test followed 520 by Dunn's multiple comparison test, unpaired one-tailed Student's t-test or unpaired one-521 tailed Mann Whitney test (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001). HPSE, 522 heparanase; HS, heparan sulfate; LDH, lactate dehydrogenase; Healthy, healthy controls; (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 15, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 15, 2020. . https://doi.org/10.1101/2020.06.12.20129304 doi: medRxiv preprint CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-332 Acute Respiratory Distress Syndrome and Death in Patients With Coronavirus Disease What we know so far: COVID-19 current clinical knowledge and research Caution on Kidney Dysfunctions of 339 COVID-19 Patients Acute kidney 341 injury in patients hospitalized with COVID-19. Kidney International Kidney disease is 343 associated with in-hospital death of patients with COVID-19 Is COVID-19 an 357 endothelial disease? Clinical Endothelial glycocalyx dysfunction in disease: albuminuria 359 and increased microvascular permeability Versatile role of heparanase in inflammation Heparanase Is Essential for the Development of Acute Experimental Glomerulonephritis Heparanase is essential for the development of diabetic nephropathy in mice Heparanase-2 protects from LPS-mediated endothelial injury by inhibiting TLR4 signalling The Glomerular Endothelium in Diabetic Nephropathy: Role of 372 Heparanase: Pathophysiology and Clinical Aspects The potential of low 374 molecular weight heparin to mitigate cytokine storm in severe covid-19 patients: a 375 retrospective clinical study Inhibition of 380 heparanase-mediated degradation of extracellular matrix heparan sulfate by non-381 anticoagulant heparin species µg/l 973 All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted June 15, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted June 15, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted June 15, 2020. test and statistical differences were calculated using Mann Whitney test (n=10 healthy; n=48 501 COVID-19, *** p<0.001, **** p <0.0001). HPSE, heparanase; HS, heparan sulfate; Healthy, 502 healthy controls; COVID-19, coronavirus disease-19 patients; AU, arbitrary units. 503All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. WBC, x10 9 /l* 6.8 (5.2-9.1) + 6.7 (5.9-7.9) All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Measurements with missing values are indicated with * and the number of + signs indicates 550 the number of missing patients per characteristic and group. & 75% quartile is unknown due 551 to prolonged hospitalization of some patients. # 4 patients with history of renal disease were 552 excluded. 553 All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted June 15, 2020. . https://doi.org/10.1101/2020.06.12.20129304 doi: medRxiv preprint