Both UFH and NAH alleviate shedding of endothelial glycocalyx and coagulopathy in LPS-induced sepsis

Abstract

Sepsis commonly progresses to disseminated intravascular coagulation and induces the activation of heparanase (HPA) and the shedding of endothelial glycocalyx constituents, including syndecan-1 (SDC-1) and heparan sulphate (HS). However, the degradation of glycocalyx and its association with coagulation disorders remains undetermined. The present study aimed to evaluate the effect of unfractionated heparin (UFH) and N-acetylheparin (NAH), which is a non-anticoagulant heparin derivative, on endothelial glycocalyx and coagulation function in a lipopolysaccharide (LPS)-induced sepsis rat model, and to compare the differences observed in coagulation function between UFH and NAH. Experimental rats were randomly assigned to four groups: Control; LPS; UFH + LPS; and NAH + LPS. Rats were administered UFH or NAH and subsequently, ~1 min later, administered LPS (10 mg/kg; intravenous). The blood and lung tissues of rats were collected 0.5, 2 and 6 h after LPS injection, and were used for subsequent analysis. The results demonstrated that HPA activity and SDC-1 and HS levels increased, and this increase was associated with inflammatory cytokines and coagulation/fibrinolysis markers in the sepsis rat model. Histopathological examination was performed, and the lung injury score and lung wet/dry ratio indicated that UFH and NAH also significantly improved lung tissue injury. The results of the ELISA analysis demonstrated that UFH and NAH treatment: i) significantly decreased the levels of inflammatory cytokines including tumor necrosis factor-α and interleukin-6; ii) inhibited HPA activity and protected the integrity of the glycocalyx, which was identified by decreased HS and SDC-1 levels; and iii) decreased the levels of prothrombin fragment 1+2, thrombin-antithrombin complex, and plasminogen activator inhibitor-1 and increased the levels of fibrinogen and antithrombin-III. Preconditioning with UFH decreased the plasma activated partial thromboplastin time. These results indicated that UFH and NAH may alleviate sepsis-induced coagulopathy, and this effect may have been due to an inhibition of HPA activity and decrease in the shedding of the endothelial glycocalyx.

Keywords: N-acetylheparin; coagulation; glycocalyx; sepsis; unfractionated heparin.

Figure 1.

Effect of heparin on histopathologic changes and lung W/D ratio in LPS-induced lung injury. Sprague-Dawley male rats were injected i.v. with UFH (100 U/kg) or NAH (1 mg/kg) and subsequently treated with LPS (10 mg/kg; 1 ml/kg of body weight; i.v.). Rats in the control group were injected i.v. with an equal volume of NS. A total of 6 h following LPS injection, the lung histopathologic changes were observed using (A) hematoxylin and eosin staining (n=6 rats/group; magnification, ×200; scale bar, 50 µm). (B) Lung tissues from each experimental group were processed and the lung injury score was used for histological evaluation. (C) The lung W/D ratios in each treatment group were quantified. Data are presented as mean ± standard deviation from 3 independent experiments. *P<0.05 vs. the control group; ^#P<0.05 vs. the LPS 6 h group. Statistical comparisons were determined using a one-way analysis of variance followed by the Student-Newman-Keuls test for multiple group comparisons. W/D, wet/dry; LPS, lipopolysaccharide; UFH, unfractionated heparin; NAH, N-acetylheparin; NS, normal saline; i.v., intravenous.

Figure 2.

Effect of heparin on inflammatory cytokines in plasma of rats with LPS-induced sepsis. (A) TNF-α and (C) IL-6 were assessed using an ELISA at 0.5, 2 and 6 h after LPS injection, respectively. Effect of UFH or NAH on levels of (B) TNF-α and (D) IL-6 in plasma at 2 and 6 h after LPS injection. Data are presented as mean ± standard deviation. There were 6 animals in each group at 0.5, 2 and 6 h. *P<0.05 vs. the control group; ^#P<0.05 vs. the LPS group; ^§P<0.05 vs. the NAH + LPS group. The TNF-α data were analyzed using a Kruskal-Wallis test followed by a pairwise comparisons test. The IL-6 data were analyzed using a one-way analysis of variance followed by a Student-Newman-Keuls test for multiple group comparisons. LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; IL, interleukin; UFH, unfractionated heparin; NAH, N-acetylheparin.

Figure 3.

Effect of heparin on heparanase activity in rats with LPS-induced sepsis. (A) Heparanase activity was determined using an ELISA at 0.5, 2 and 6 h after LPS injection. (B) Effect of UFH or NAH on heparanase activity in plasma at 2 and 6 h after LPS injection. Data are presented as mean ± standard deviation. There were 6 animals in each group at 0.5, 2 and 6 h. *P<0.05 vs. the control group; ^#P<0.05 vs. the LPS group. The HPA data were analyzed using a one-way analysis of variance, followed by a Student-Newman-Keuls test for multiple group comparisons. LPS, lipopolysaccharide; UFH, unfractionated heparin; NAH, N-acetylheparin.

Figure 4.

Effect of heparin on endothelial glycocalyx degradation products in an LPS-induced sepsis rat model. (A) HS and (C) SDC-1 in plasma was determined using an ELISA at 0.5, 2 and 6 h after LPS injection. Effect of UFH or NAH on (B) HS and (D) SDC-1 in plasma at 2 and 6 h after LPS injection. The distribution of (E) HS (red) and (F) SDC-1 (red) in rat lungs was assessed using staining with specific antibodies. Endothelial cell marker of pulmonary vascular was assessed using BDCA-3 (green) staining (magnification, ×200; scale bar, 50 µm). (G) Fluorescence intensity analyses of the images presented in (E). (H) Fluorescence intensity analyses of the images presented in (F). Data are presented as mean ± standard deviation for 3 independent experiments. *P<0.05 vs. the control group; ^#P<0.05 vs. the LPS group. The HS and SDC-1 data were determined using a one-way analysis of variance followed by a Student-Newman-Keuls test for multiple group comparisons. LPS, lipopolysaccharide; HS, heparan sulphate; SDC-1, syndecan-1; UFH, unfractionated heparin; NAH, N-acetylheparin; BDCA-3, thrombomodulin.

Figure 5.

Effect of heparin on activated coagulation parameters in an LPS-induced sepsis rat model. (A) F1+2 and (C) TAT were determined using an ELISA at 0.5, 2 and 6 h after LPS injection, respectively. Effect of UFH or NAH on (B) F1+2 and (D) TAT in the plasma at 2 and 6 h after LPS injection. Data are presented as mean ± standard deviation. There were 6 animals in each group at 0.5, 2 and 6 h. *P<0.05 vs. the control group; ^#P<0.05 vs. the LPS group. The F1+2 data were analyzed using a Kruskal-Wallis test along with Bonferroni correction. The TAT data were analyzed using a one-way analysis of variance followed by a Student-Newman-Keuls test for multiple group comparisons. LPS, lipopolysaccharide; F1+2, prothrombin fragment 1+2; TAT, thrombin-antithrombin complex; UFH, unfractionated heparin; NAH, N-acetylheparin.

Figure 6.

Effect of heparin on anti-coagulation and fibrinolysis parameters in an LPS-induced sepsis rat model. (A) AT and (C) PAI-1 were determined using an ELISA at 0.5, 2 and 6 h after LPS injection, respectively. Effect of UFH or NAH on (B) AT and (D) PAI-1 in plasma at 2 and 6 h after LPS injection. Data are presented as mean ± standard deviation. There were 6 animals in each group at 0.5, 2 and 6 h, respectively. *P<0.05 vs. the control group; ^#P<0.05 vs. the LPS group. The AT data were analyzed using a Kruskal-Wallis test along with Bonferroni correction. The PAI-1 data were analyzed using a one-way analysis of variance followed by a Student-Newman-Keuls test for multiple group comparisons. LPS, lipopolysaccharide; AT, antithrombin; PAI-1, plasminogen activator inhibitor-1; UFH, unfractionated heparin; NAH, N-acetylheparin.