Page 191 - 2022_02-Haematologica-web

Sulfated non-anticoagulant heparin in SCD treatment
for the treatment of SCD in the USA. L-glutamine (Endari), approved in 2017, increases the amount of the reduced form of NADH in erythrocytes, which allows sickle RBC to maintain homeostasis more appropriately during oxidative stress, ultimately resulting in fewer painful vaso-occlusive crises and adverse events.14 Crizanlizumab (Adakveo)15 and voxelotor16 (Oxbryta, GBT440) were approved in 2019. Crizanlizumab is a monoclonal antibody that targets P-selectin to prevent pathological endothelial adhesion of sickle erythrocytes and leukocytes, leading to a reduction in the frequency of painful vaso-occlusive crises.17,18 The anti-sickling agent voxelotor is the first of a new class of aromatic aldehydes that target HbS polymerization by increasing Hb O2 affin- ity.19-21 Finally, hydroxyurea, which works by inducing the expression of fetal Hb (HbF), is the most proven therapeu- tic approach for SCD,22,23 as evidenced by its sustained clinical use for over two decades. However, a reported lack of response to hydroxyurea in up to 30% of patients, and supposed poor compliance, tend to limit its use.22 The reported limitations of hydroxyurea led to investigation of other modes of therapy, including the three more recently approved drugs. However, their true benefits will only manifest over time. Additionally, the inherently complex nature of SCD dictates the urgent need for a multimodal form of therapy.
Antiplatelet molecules, anticoagulants, and heparin have been investigated to mitigate vaso-occlusive crises.24 Although heparin is beneficial, the associated risks of internal bleeding preclude its utility as a drug25 and the need for alternatives remains critical. We developed a sul- fated non-anticoagulant heparin (S-NACH) with no to low systemic anticoagulant activity that can be safely adminis- tered in mice (at doses >300 mg/kg daily for 10 days; unpublished data) without causing internal bleeding.26,27 S-NACH does not bind antithrombin and thus does not inhibit systemic antithrombin-dependent clotting factors (activated factors X and II). Sulfation on S-NACH increas- es the drug’s affinity for endothelium to cause the release of endothelial tissue factor pathway inhibitor (TFPI).26,28 Furthermore, S-NACH interferes with P-selectin-depen- dent binding of cancer cells29 and RBC30 to endothelial cells and regulates plasma levels of adhesion biomarkers in SCD mice.30 Finally, S-NACH was further optimized to interact directly with HbS to exert desirable therapeutic benefits.
In this study we tested our hypothesis that S-NACH can bind to HbS and directly prevent sickling and decrease inflammation in SCD due to the bidirectional relationship between inflammation and coagulation31 and investigated the effects of S-NACH on RBC morphology.
Methods
Reagents
S-NACH (average molecular weight 4,000 Da) was synthesized by Suzhou Ronnsi Pharma Co. Ltd. (Jiangsu Province, China). 5- hydroxymethyl-2-furfural (5-MF) and other common reagents were purchased from Sigma (St. Louis, MO, USA).
Sickle blood samples
Leftover blood samples from individuals with homozygous SS (SCD) were obtained and used, based on an approved Institutional Review Board protocol at the Children’s Hospital of Philadelphia,
with informed consent. None of the subjects had been transfused within 4 months prior to their blood samples being used, and four of the five donors were on hydroxyurea therapy.
Anti-sickling, oxygen equilibrium and hemoglobin modification studies using human sickle blood
The morphology of hypoxic sickled RBC was evaluated using a previously reported method.22,23 Blood samples from individ- ual donors with SCD (n=5) were diluted using HEMOX buffer supplemented with glucose (10 mM) and bovine serum albumin (0.2%) to adjust the hematocrit of the suspensions to about 20%. We used standardized hematocrit for anti-sickling assays to normalize the ratio of RBC to drug for assay consistency and reproducibility. The suspensions were pre-incubated under air in the absence or presence of three concentrations (0.5, 1, and 2 mM) of S-NACH at 37°C for 1 h. The suspensions were subse- quently incubated under a 2.5% O2/97.5% N2 gas mixture at 37°C for 2 h. Aliquots (5–20 mL) of each sample were collected without exposure to air into 2% glutaraldehyde solution for immediate fixation. Fixed cell suspensions were thereafter intro- duced into glass microslides (Fiber Optic Center, New Bedford, MA, USA)34 and subjected to microscopic morphological analy- sis of bright field images (at 40x magnification) of single layer cells on an Olympus BX40 microscope fitted with an Infinity 2 camera (Olympus, Waltham, MA, USA), with the coupled Image Capture software. The percentage of sickled cells for each condition was determined using blood with a computer-assisted image analysis system, as described previously.33,35 Untreated samples, as well as samples treated with GBT440/voxelotor, were used as controls. The residual samples were washed in phosphate-buffered saline (PBS) and hemolysed in hypotonic lysis buffer for subsequent analyses.
For the oxygen equilibrium study, approximately 100 mL aliquot samples from clarified lysates obtained from the anti- sickling studies were mixed with 3 mL of 0.1 M potassium phos- phate buffer, pH 7.0, in cuvettes, and subjected to hemoximetry analysis using a HemoxTM Analyzer (TCS Scientific Corp., New Hope, PA, USA) to assess P50 shifts.36-38 Degree of P50 shift (DP50) was expressed as percentage fractions of control dimethylsulfoxide-treated samples.
Finally, for the Hb adduct formation study, clarified lysates, also from the above anti-sickling study, were subjected to cation- exchange high performance liquid chromatography (Hitachi D-7000 Series, Hitachi Instruments, Inc., San Jose, CA, USA), using a weak cation-exchange column (Poly CAT A: 30 mm x 4.6 mm, Poly LC, Inc., Columbia, MD, USA). Hemoglobin isotype peaks were eluted with a linear gradient of mobile phase B from 0% to 80% at A410nm (mobile phase A: 40 mM Bis-Tris, 5 mM EDTA, pH 6.5; mobile phase B: 40 mM Bis-Tris, 5 mM EDTA, 0.15 M sodium chloride, pH 7.5).33,36 A commercial standard con- sisting of approximately equal amounts of composite HbF, HbA, HbS, and HbC (Helena Laboratories, Beaumont, TX, USA), was used as the reference isotypes. The areas of new peaks, represent- ing HbS adducts, were obtained, calculated as percentage fractions of total Hb area, and reported as levels of modified Hb. All assays were conducted in five biological replicates on different days.
Animal studies
C57/B mice aged 5-6 weeks were purchased from Harlan Laboratories (Indianapolis, IN, USA) and acclimatized for 5 days before initiating TFPI measurements after administration of S- NACH. Townes SCD mice (stock # 013071) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA), bred, genotyped, and used in experiments between 10 and 12 weeks of age. Animal studies were conducted at the animal research facility, Albany
haematologica | 2022; 107(2)
533