Abstract
In sickle cell disease (SCD), a mutation of the β-globin gene leads to abnormal polymerization of hemoglobin, resulting in formation of sickled red blood cells, hemolytic anemia, and vaso-occlusive crisis (VOC). These primary events produce clinical complications and trigger additional multiple pathologies driven by chronic oxidative stress, sterile inflammation, and activation of coagulation.
Heparins, highly sulfated forms of heparan sulfate (HS), are a group of polysaccharide compounds with great variance in structure. In addition to their anticoagulant effect, heparins have anti-adhesive and anti-inflammatory properties. These are determined by both sulfation pattern and length of the polysaccharide chain, which influence the ability to bind HMGB-1, histones, and P-selectin (Psel). This heterogeneity together with the short half-life and dosing regimen based on anticoagulant activity, limit the use of heparins as anti-adhesive and anti-inflammatory agents. To overcome these limitations, we previously developed a chemoenzymatic approach to synthesize a structurally defined HS oligosaccharides and demonstrated their ability to reduce sterile inflammation in animal models by binding HMGB-1 and histones. In the present study, we investigated the compound's anti-Psel properties in vitro and in a mouse model of SCD.
First, using a Psel inhibition ELISA assay, we determined that a heptadecasaccharide (17-mer) is the minimum polysaccharide chain length required for inhibition of Psel binding to Sialyl Lewis X polyacrylamide. Based on these data, we synthesized three 18-mer compounds with a different sulfation position on each monosaccharide ring (NS2S, NS6S and NS2S6S). To obtain 18-mers with no anticoagulant activity, we omitted 3-O-sulfation of glucosamine, which is important for binding antithrombin III (confirmed by anti-FXa activity assay). In the Psel inhibition ELISA assay, all compounds demonstrated dose dependent (0.1 - 1000 µg/mL) anti-Psel activity comparable to that observed for low molecular weight heparin (LMWH).
Psel is a key molecule mediating VOC by promoting formation of multicellular aggregates. Therefore, we evaluated the effect of 18-mers on Psel-mediated platelet/leukocyte aggregates (PLA) formation ex vivo. Leukocytes and platelets isolated from healthy donors were stimulated with PMA (100 nM) for 1 hour or thrombin (5 µg/mL) for 30 minutes, respectively, then incubated with vehicle, 18-mer compounds, or LMWH (0.5, 5, 50 and 500 µg/mL) for 15 minutes. After incubation, cells were combined to allow PLA formation for 15 minutes and analyzed by flow cytometry. At the highest tested 18-mer concentration, all compounds attenuated PLA formation. However only NS2S6S, the most highly sulfated compound, showed significant inhibition at all concentrations. NS2S6S decreased PLA formation to 82.8% 90.8%, 76.3% and 68.3%, lowest to highest concentrations respectively (p<0.01 for all concentrations versus vehicle). LMWH demonstrated significant decreases only at the two highest concentrations (82.1% and 63.8%, p<0.001). The number of circulating PLA was increased in sickle Townes HbSS mice by 6.1-fold (p<0.01) compared to non-sickle Townes HbAA controls. In ex vivo experiments, addition of NS2S6S (1 mg/ml) to the HbSS blood decreased PLA formation to 66.4% (p=0.03) compared to untreated HbSS blood.
Finally, we determined the effect of NS2S6S on heme-induced microvascular stasis in Townes HbSS mice. Sickle mice were implanted with a dorsal skinfold chamber to visualize dermal microvessels. PBS or NS2S6S (3 mg/kg, s.c.) were injected 15 min before infusion of heme (1.2 µmol/kg, iv). 0ne, 2, 3 and 4 hours after heme infusion, microvascular stasis was observed in 31.5, 20.5, 18.9 and 14.2% of preselected vessels in PBS treated sickle mice, and NS2S6S treatment reduced that numbers to 10.9, 6.2, 3.1 and 3.2%, respectively (p<0.01 for all time points).
In summary, we showed that NS2S6S prevents Psel dependent formation of PLA ex vivo and reduces heme-induced stasis in sickle mice. Together with previously described anti-HMGB1 and anti-histone effects, this compound is a good candidate for multi-modal therapy to mitigate the pathophysiology of SCD. However, like LMWH, NS2S6S has a short half-life which makes prophylactic treatment of SCD patients impractical. Studies to extend the half-life of HS are currently ongoing in our group.
Disclosures
Xu: Glycan Therapeutics: Current Employment. Belcher: Mitobridge/Astellas: Consultancy, Research Funding; CSL Behring: Research Funding. Vercellotti: CSL Behring: Research Funding; Mitobridge, an Astellas Company: Consultancy, Research Funding. Liu: Glycan Therapeutics: Current Employment.
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