Peptide 19-2.5 inhibits heparan sulfate-triggered inflammation in murine cardiomyocytes stimulated with human sepsis serum.

Myocardial dysfunction in sepsis has been linked to inflammation caused by pathogen-associated molecular patterns (PAMPs) as well as by host danger-associated molecular patterns (DAMPs). These include soluble heparan sulfate (HS), which triggers the devastating consequences of the pro-inflammatory cascades in severe sepsis and septic shock. Thus, there is increasing interest in the development of anti-infective agents, with effectiveness against both PAMPs and DAMPs. We hypothesized that a synthetic antimicrobial peptide (peptide 19-2.5) inhibits inflammatory response in murine cardiomyocytes (HL-1 cells) stimulated with PAMPs, DAMPs or serum from patients with septic shock by reduction and/or neutralization of soluble HS. In the current study, our data indicate that the treatment with peptide 19-2.5 decreases the inflammatory response in HL-1 cells stimulated with either PAMPs or DAMPs. Furthermore, our work shows that soluble HS in serum from patients with Gram-negative or Gram-positive septic shock induces a strong pro-inflammatory response in HL-1 cells, which can be effectively blocked by peptide 19-2.5. Based on these findings, peptide 19-2.5 is a novel anti-inflammatory agent interacting with both PAMPs and DAMPs, suggesting peptide 19-2.5 may have the potential for further development as a broad-spectrum anti-inflammatory agent in sepsis-induced myocardial inflammation and dysfunction.

Differences in the uptake and nuclear localization of anti-proliferative heparan sulfate between human lung fibroblasts and human lung carcinoma cells.

Heparan sulfate inhibits the proliferation of normal human lung fibroblasts (HFL-1) but not of a human lung carcinoma cell-line (A549). In this study we investigated possible mechanisms and structural requirements by which antiproliferative heparan sulfates exerts its effects on binding, uptake and subcellular localisation. Both HFL-1 and A549 cells were incubated with 125I- or rhodamine-labeled L-iduronate-rich antiproliferative heparan sulfate species as well as L-iduronate-poor inactive ones. The antiproliferative heparan sulfate was bound to the cell surface on both HFL-1 and A549 cells, but to a lesser extent and with less affinity to A549 cells. Both cell types bound the antiproliferative heparan sulfate with one high- and with one low affinity site. The L-iduronate-poor heparan sulfate bound to a lesser extent and with less affinity to both cell types compared to the antiproliferative heparan sulfate. The antiproliferative heparan sulfate accumulated in the cytoplasm of HFL-1 cells after 24 h incubation, but after 72 h it was found evenly distributed in the nucleus. The time-scale for antiproliferative activity correlated with nuclear localization. In contrast, in A549 cells it was only found near the nuclear membrane. The inactive heparan sulfate was taken up in considerably smaller amounts compared to the antiproliferative heparan sulfate and could not be detected in the nucleus of either HFL-1 or A549 cells. Our data suggest that the antiproliferative activity of L-iduronate-rich heparan sulfate on normal fibroblasts may be due to direct effects on nuclear processes, such as gene transcription.

Use of a low-molecular-weight heparinoid (danaparoid sodium) for continuous renal replacement therapy in intensive care patients.

The purpose of this study was to evaluate the efficacy and safety of danaparoid in the treatment of critically ill patients with acute renal failure and suspected heparin-induced thrombocytopenia (HIT) needing renal replacement therapy (RRT). We conducted a retrospective analysis of 13 consecutive intensive care patients with acute renal failure and suspected HIT who were treated with danaparoid for at least 3 days during RRT. In eight patients, continuous venovenous hemofiltration was performed. The mean infusion rate of danaparoid was 140 +/- 86 U/hour. Filter exchange was necessary every 37.5 hours. In five patients, continuous venovenous hemodialysis was used. A bolus injection of 750 U danaparoid was followed by a mean infusion rate of 138 +/- 122 U/hour. Filters were exchanged every 24 hours. In 7 of 13 patients, even a low mean infusion rate of 88 +/- 35 U/hour was efficient. Mean anti-Xa (aXa) levels were approximately 0.4 +/- 0.2 aXa U/mL. Persistent thrombocytopenia despite discontinuation of heparin treatment was observed in 9 of 13 patients, owing to disseminated intravascular coagulation (DIC). HIT was confirmed by an increase in platelet count and positive heparin-induced antibodies in 2 of 13 patients. No thromboembolic complications occurred, but major bleeding was observed in 6 of 13 patients, which could be explained by consumption of coagulation factors and platelets due to DIC in 5 of 6 patients. Nine of 13 patients died of multiorgan failure or sepsis, or both. In none of these patients was the fatal outcome related to danaparoid treatment. In critically ill patients with renal impairment and suspected HIT, a bolus injection of 750 U danaparoid followed by a mean infusion rate of 50 to 150 U/hour appears to be a safe and efficient treatment option when alternative anticoagulation is necessary.

Prospective randomised open-label comparison of danaparoid with dextran 70 in the treatment of heparin-induced thrombocytopaenia with thrombosis: a clinical outcome study.

AIM: To compare clinical outcomes in a randomised comparison of treatment with danaparoid sodium (a heparinoid), or dextran 70, for heparin-induced thrombocytopaenia (HIT) plus thrombosis. METHODS: Forty-two patients with recent thrombosis and a clinical diagnosis of probable HIT who presented at ten Australian hospitals during a study period of six and one half years were randomly assigned to open-label treatment with intravenous danaparoid or dextran 70, each combined with oral warfarin. Thirty-four patients (83%) had a positive platelet aggregation or 14C-serotonin release test for HIT antibody. Twenty-five received danaparoid as a bolus injection of 2400 anti-Xa units followed by 400 units per hour for 2 h, 300 units per hour for 2 h, and then 200 units per hour for five days. Seventeen received 1000 mL dextran 70 on day one and then 500 mL on days 2-5. Patients were reviewed daily for clinical evidence of thrombus progression or resolution, fresh thrombosis or embolism, bleeding or other complications. The primary trial endpoint was the proportion of thromboembolic events with complete clinical resolution by the time of discharge from hospital. RESULTS: With danaparoid, there was complete clinical recovery from 56% of thromboembolic events compared to 14% after dextran 70 (Odds Ratio 10.53, 95% Confidence Interval 1.6-71.4; p = 0.02). Clinical recovery with danaparoid was complete or partial in 86% of thromboembolic events compared with 53% after dextran 70 (Odds Ratio 4.55, 95% Confidence Interval 1.2-16.7; p = 0.03). Overall clinical effectiveness of danaparoid was rated as high or moderate in 88% of patients compared with 47% for dextran 70 (p = 0.01). One patient given danaparoid died of thrombosis compared with three patients given dextran 70. The platelet count returned to normal after a mean of 6.7 days with danaparoid and 7.3 days with dextran 70. There was no major bleeding with either treatment. CONCLUSION: danaparoid plus warfarin treatment for HIT with thrombosis is effective, safe, and superior to dextran 70 plus warfarin.

Neurotoxicity of the 22 kDa thrombin-cleavage fragment of apolipoprotein E and related synthetic peptides is receptor-mediated.

Potent neurotoxicity is associated with both apolipoprotein E (apoE)-related synthetic peptides and the 22 kDa N-terminal thrombin-cleavage fragment of apoE. Furthermore, the E4 isoform of the 22 kDa fragment is significantly more toxic than the same fragment derived from the E3 isoform, suggesting the possibility of a direct role of apoE-associated neurotoxicity in the pathophysiology of Alzheimer's disease. In the present study, the potential role of cell surface receptors in mediating neurotoxicity was assessed by using a variety of agents that should block the heparin-binding and receptor-binding activity of apoE. Effective inhibitors of neurotoxicity of both the apoE peptides and the apoE fragment include heparin, heparan sulfate, sodium chlorate and heparinase, the low-density lipoprotein (LDL) receptor-related protein receptor-associated protein, and a polyclonal anti-LDL receptor-related protein antibody. These results suggest that the neurotoxicity of the 22 kDa thrombin cleavage fragment of apoE and related peptides is receptor-mediated, and that the most likely candidate receptor is a heparan sulfate proteoglycan-LDL receptor-related protein complex.

Comparative antithrombotic and hemorrhagic effects of dermatan sulfate, heparan sulfate and heparin.

Dermatan sulfate and heparan sulfate are currently under development as potential antithrombotic drugs. In our studies we have evaluated the relative antithrombotic and bleeding effects of these two agents in comparison to heparin, the commonly used anticoagulant. In a rabbit model of stasis thrombosis, a 500 micrograms/kg IV dose of dermatan or heparan produced 50-60% inhibition of induced in vivo thrombosis. At 750 micrograms/kg, both agents produced greater than 75% inhibition of thrombosis. Ex vivo measurement of plasma samples obtained from these animals demonstrated variable clotting effects at the lower dose and a proportional increase in the clotting activity at the higher dose. No anti-Xa or anti-IIa activity was observed in any sample. In contrast, animals treated with only 100 micrograms/kg heparin showed complete inhibition of induced thrombosis with significant anti-Xa and anti-IIa activities as well as prolongation of the clotting assays (APTT, TT and HeptestR). In the hemorrhagic studies utilizing a rabbit ear blood loss model, a 5.0 mg/kg IV dose of dermatan or heparan produced much less blood loss than heparin. On a gravimetric basis, dermatan and heparan were 10 fold less hemorrhagic than heparin. These results indicate that the relative contribution of plasmatic and cellular sites to the mediation of the antithrombotic action of heparin, dermatan and heparan differ. Although the antithrombotic dosages of dermatan and heparan are higher than heparin, due to the different mechanisms of action of each agent, a better safety index may be provided by dermatan and heparan than heparin.

Nephritogenicity of proteoglycans. II. A model of immune complex nephritis.

Antibodies to glomerular basement membrane, heparan sulfate-proteoglycans are nephrotoxic but possess a weak nephritogenic potential. In order to enhance the nephritogenic potential, the antibodies were intravenously administered into rats presensitized with heterologous rabbit IgG. This resulted in the integration of heterologous and autologous phases, the two phases characteristic of the traditional model of nephrotoxic serum nephritis. The presensitization caused a dramatic shift in the binding characteristics of the heterologous antibodies between the kidney and lymphoid tissues. A proliferative form of immune complex glomerulonephritis associated with a remarkable proteinuric response was observed. In addition, a moderate degree of hematuria was noted as well. The proteinuria was largely complement-dependent and may possibly be cell-mediated as well. The proteinuria became severe with increasing production of host IgG antibodies and with their subsequent sequestration in the glomeruli. The predominant glomerular lesions were in the form of epimembranous/subepithelial immune deposits, which became more frequent with timely increasing titer of host autologous IgG antibodies. These findings indicate that antibodies to heparan sulfate-proteoglycan, an authentic component of the basement membrane, are capable of mediating a glomerular injury with acquisition of nephritogenic potential in an appropriate environment of the host. At present, it seems that this is the sole constituent of the basement membrane whose antibodies are capable of inducing an immune complex nephritis.

Nephritogenicity of proteoglycans. III. Mechanism of immune deposit formation.

Administration of antibody, directed against glomerular basement membrane (GBM) heparan sulfate-proteoglycan, into a presensitized rat results in the induction of membranous nephropathy with subepithelial immune-complex deposits. In this investigation, we examined the mechanisms responsible for the formation of subepithelial immune-complex deposits in the anti-HS-PG model. In initial experiments, the intravenously administered radioiodinated antibody was seen exclusively localized in the regions of the glomerular capillary wall where the subepithelial deposits were observed. To determine their exclusive localization in the subepithelial space, kinetics of movement of the intravenously administered antibody was investigated. The antibody localized in the inner layers of the GBM within a few minutes after its administration. It equilibrated in the inner and outer layers of the GBM in a matter of a few hours. Then, after 24 hours, it gradually disappeared from the inner layers of the GBM and persisted in the outer layers only. The ready clearance of the antibody from the inner layers may be related to the differential in the kinetics of lateral intrinsic plasma fluid currents within the GBM. The persistence of heterologous antibody exclusively in the outer layers and the availability of host autologous antibodies probably resulted in the development of immune complex deposits in the subepithelial space. The glomeruli devoid of plasma water currents showed no change in the concentration of the antibody in the inner and outer layers of the GBM or mesangial matrix. Also, no antibody binding was observed with the plasmalemma of either the foot processes or visceral epithelia. The data suggest that the biochemical-biophysical properties of the glomerular capillary wall, in concert with its intraglomerular hemodynamics, most likely played a significant role in the development of subepithelial immune-complex deposits in this model.

Characterization of renal injury initiated by immunization of rats with heparan sulfate.

To investigate the role of antibody to heparan sulfate (HS) in the development of glomerular injury, male Lewis rats were immunized with HS and compared with unimmunized controls. In HS-immunized rats circulating antibodies that bound to renal basement membranes, an increase in serum creatinine (0.8 mg/dl versus 0.6 in controls P less than 0.01), and a 40% decline in creatinine clearance developed. In no animal did abnormal proteinuria develop. By histologic examination there was glomerular and interstitial capillary engorgement with erythrocytes, modest infiltration by polymorphonuclear leukocytes, and no proliferation of intrinsic glomerular cells. Immunofluorescence microscopy demonstrated deposits of rat IgG along the glomerular basement membrane. Bowman's capsule, and peritubular capillaries. Electron-microscopic examination revealed capillary engorgement with erythrocytes that appeared adherent to each other and contained entrapped areas of rarefied material. These observations demonstrate that binding of antibody to HS in the glomerulus induces a mild inflammatory reaction and a reduction in glomerular filtration rate, but no abnormal proteinuria.