Increased Plasma Heparanase Activity in COVID-19 Patients

Abstract

Reports suggest a role of endothelial dysfunction and loss of endothelial barrier function in COVID-19. It is well established that the endothelial glycocalyx-degrading enzyme heparanase 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 heparanase may be inhibited by LMWH. To test this hypothesis, heparanase activity and heparan sulfate levels were measured in plasma of healthy controls (n = 10) and COVID-19 patients (n = 48). Plasma heparanase activity and heparan sulfate levels were significantly elevated in COVID-19 patients. Heparanase activity was associated with disease severity including the need for intensive care, lactate dehydrogenase levels, and creatinine levels. Use of prophylactic LMWH in non-ICU patients was associated with a reduced heparanase activity. Since there is no other clinically applied heparanase inhibitor currently available, therapeutic treatment of COVID-19 patients with low molecular weight heparins should be explored.

Keywords: COVID-19; LMWH (low molecular weight heparin); glycocalyx damage; heparanase; inflammation; vascular leakage.

Graphical Abstract

Heparin and low molecular weight heparins (LMWH) inhibit heparanase activity which is associated with disease severity in COVID-19.

Figure 1

COVID-19 patients display increased HPSE activity, and elevated levels of heparan sulfate and IL-6 in plasma. (A) HPSE activity was increased in plasma of COVID-19 patients compared to healthy controls. HPSE activity was measured using an in-house developed ELISA with a specific anti-HS antibody. (B) HS levels were increased in plasma of COVID-19 patients compared to healthy controls. HS levels were measured by an in-house developed competition ELISA using a specific anti-HS antibody. (C) IL-6 levels were increased in plasma of COVID-19 patients compared to healthy controls. IL-6 levels were measured using a commercial IL-6 ELISA. Data were presented as mean ± SEM and tested for normal distribution with D’Agostino & Pearson omnibus normality test and statistical differences were calculated using Mann Whitney test (n = 10 healthy; n = 48 COVID-19; **p < 0.01; ****p < 0.0001). HPSE, heparanase; HS, heparan sulfate; Healthy, healthy controls; COVID-19, coronavirus disease-19 patients; AU, arbitrary units.

Figure 2

Increased plasma HPSE activity associates with COVID-19 disease severity. (A) Plasma HPSE activity was significantly higher in ICU and non-ICU patients compared to healthy controls, and higher in ICU patients compared to non-ICU patients (n = 10 healthy; n = 34 non-ICU; n = 14 ICU). (B) HS plasma levels were significantly increased in plasma of ICU and non-ICU patients compared to healthy controls (n = 10 healthy; n = 34 non-ICU; n = 14 ICU). (C) IL-6 plasma levels were significantly increased in plasma of ICU and non-ICU patients compared to healthy controls (n = 10 healthy; n = 34 non-ICU; n = 14 ICU). (D) HPSE activity was significantly higher in plasma of patients with elevated LDH (>280 U/L) values compared to patients with normal LDH levels (n = 15 normal LDH; n = 26 elevated LDH). (E) HPSE activity was significantly higher in plasma of patients with elevated creatinine (>110 µmol/L for men and >90 µmol/L for women) values compared to patients with normal creatinine values (n = 30 normal creatinine; n = 11 elevated creatinine; patients with history of renal disease were excluded from this analysis). HPSE activity was measured using an in-house developed ELISA with a specific anti-HS antibody and HS plasma levels were measured using an in-house developed competition ELISA with a specific anti-HS antibody. Data were presented as mean ± SEM and tested for normal distribution with D’Agostino & Pearson omnibus normality test and statistical differences were calculated using Kruskal Wallis test followed by Dunn’s multiple comparison test, unpaired one-tailed Student’s t-test or unpaired one-tailed Mann Whitney test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). HPSE, heparanase; HS, heparan sulfate; LDH, lactate dehydrogenase; Healthy, healthy controls; non-ICU, COVID-19 patients in normal hospital ward; ICU, COVID-19 patients in ICU; AU, arbitrary units.

Figure 3

LMWH reduces plasma HPSE activity, but not plasma HS or IL-6 levels in moderately diseased COVID-19 patients. (A) LMWH reduces HPSE activity in plasma of non-ICU patients with COVID-19, which was measured using in-house developed HPSE activity assay (n = 17 for both groups, **p < 0.01). (B) LMWH does not reduce HS levels in plasma of non-ICU patients with COVID-19, which was measured with an in-house developed competition ELISA with a specific anti-HS antibody. (C) LMWH does not reduce IL-6 levels in plasma of non-ICU patients with COVID-19, which was measured using an in-house developed competition ELISA with a specific anti-HS antibody. Data were presented as mean ± SEM and tested for normal distribution with D’Agostino & Pearson omnibus normality test and statistical difference was calculated using unpaired one-tailed Mann Whitney test. (D) LMWH inhibits recombinant HPSE activity in vitro in a dose-dependent manner. HPSE activity was measured using an in-house developed ELISA with a specific anti-HS antibody (n = 5). HPSE, heparanase; HS, heparan sulfate; LMWH, low molecular weight heparin.