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O. Abdulmalik et al.
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Discussion
We designed S-NACH, a modified low molecular weight heparin, to be devoid of anticoagulant properties, while acquiring new direct anti-sickling properties. In vitro, S-NACH directly modified intracellular HbS, increased oxygen affinity, and inhibited sickling of RBC under hypoxia. Additionally, S-NACH reduced the levels of cir- culating sickled cells in Townes SCD mice. We confirmed the in vitro release of endothelial-TFPI by S-NACH30 in C57/B mice, as demonstrated by a significant increase in plasma TFPI. Based on this, we speculate that S-NACH might exert local antithrombotic activity by increasing the concentration of endothelial TFPI in the vascular area. An in vivo increase in plasma TFPI levels after S-NACH administration confirms our earlier reported findings.26 According to Kemme et al., TFPI release increased by 3-fold (from 62.9 ng/mL to 237 ng/mL) after infusion of heparin.42 Kouta et al. reported a marked increase of TFPI release (~2.5-fold) within 20 min after intravenous admin- istration of different types of heparins (bovine, ovine, and porcine) to non-human primates.43 Additionally, our observation with different species, including mice, rats, and rabbits (unpublished data) are consistent with these results.
We demonstrated in vitro that S-NACH permeated RBC membranes to modify HbS and exert a significant anti- sickling effect by maintaining normal RBC morphology, protecting against the typical changes in RBC morphology
that occur in untreated samples from individuals with SCD. Although there was no prior evidence to indicate a relationship between RBC morphology and inflammatory mediators, the effect of both on decreased sickling and blood viscosity cannot be ruled out.6 Based on our previ- ous studies, the observed reduction in the levels of pro- inflammatory cytokines was not unexpected. For exam- ple, in one previous study in an asthma-induced mouse model, S-NACH caused a robust reduction in airway eosinophilia, mucus production, and airway hyperrespon- siveness even after chronic repeated challenges with aller- gen (ovalbumin).44 These effects were linked to suppres- sion of Th2 cytokines IL-4/IL-5/IL-13/GM-CSF and upreg- ulation of IL-10. The levels of these inflammatory cytokines increased around 2- to 8-fold (in both serum and bronchoalveolar lavage fluid) after induction with the allergen and decreased again to baseline after treatment with S-NACH. Similar observations were made for total white blood cell count, as well as eosinophil, macrophage, and lymphocyte counts, which were markedly elevated in the asthma-induced mouse model (6-, 4-, 1.5-, 1.5-, and 4-fold, respectively) after exposure to an allergen. S- NACH also reduced lung fibrosis in mice that were chron- ically exposed to the allergen. As we showed in that study, the protective effects of S-NACH were attributable to modulation of the IL-4/JAK1 signal transduction pathway through inhibition of STAT6 phosphorylation and subse- quent inhibition of GATA-3 and inducible nitric oxide syn-
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Figure 3. Effects of S-NACH under normoxia. (A) A sulfated non-anticoagulant heparin derivative (S-NACH) increases the release of endogenous tissue factor pathway inhibitor (TFPI). C57/B mice were treated with 100 and 300 mg/kg of free S-NACH, and plasma was obtained after 2 h. TFPI in plasma was measured in duplicate. TFPI levels were compared between S-NACH-treated samples and phosphate-buffered saline (PBS)-treated control samples, (n=4) (*P<0.05). (B) S-NACH treatment decreases sickling of red blood cells (RBC) in Townes sickle cell disease (SCD) mice. Blood smears were made from tail snips before and after subcutaneous injection of S-NACH (10 mg/kg) at the time points shown. 5-hydroxymethyl-2-furfural (5-MF) was used as a positive control (*P<0.05). (C) Morphology of the RBC from Townes SCD mice was examined in stained blood smears and expressed in percentage. RBC from four different fields or 120 cells were analyzed to calculate the percentage of sickled RBC. Blood from untreated samples contained higher percentages of sickled and distorted RBC (shown by arrows). S-NACH treatment decreased the pres- ence of sickled RBC for up to 4 h with the greatest decrease seen at 2 h (n=6) (*P<0.05). SD: standard deviation.
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haematologica | 2022; 107(2)