The purified vepoloxamer prevents haemolysis in 42-day stored, DEHP/PVC-free red blood cell units

Abstract

Background

Use of the plasticiser di(2-ethylhexyl) phthalate (DEHP) in polyvinyl chloride (PVC) blood bags poses a potential dilemma. The presence of DEHP in blood bags has been shown to be beneficial to red blood cells during storage by diminishing haemolysis. However, DEHP use in PVC may be carcinogenic or estrogenising. Vepoloxamer is a poloxamer with rheological and cytoprotective rheological properties and a favourable toxicity profile in clinical trials. We hypothesised that vepoloxamer may be sufficient to replace the plasticiser DEHP to prevent elevated haemolysis while conserving the biochemical and redox potential++ in RBCs stored for up to 42 days.

Materials and methods

Paired analyses of aliquots from pooled RBC suspensions of ABO identical donors were aseptically split into test storage containers (DEHP/PVC or DEHP-free/ethylene vinyl acetate [EVA]) supplemented with or without vepoloxamer (at concentrations of 0.1, 1, 5 or 7.89 mg/mL) and cold stored for up to 42 days.

Results

Vepoloxamer significantly prevented the increased haemolysis induced by the absence of DEHP in EVA bags in a dose-dependent manner by days 28 and 42 of storage (approx. 50% reduction of the maximum concentration of vepoloxamer; p<0.001). There was an inverse correlation between the concentration of vepoloxamer used and the haemolysis rate (r²=0.27, p<0.001) and a direct correlation between haemolysis and phosphatidylserine (PS) exposure (r²=0.42; p<0.01). Increased osmotic fragility and shear induced deformability of 42-day stored RBC in EVA bags was significantly corrected by the addition of vepoloxamer.

Discussion

Vepoloxamer, in a concentration-dependent fashion, is able to partly rescue the increased haemolysis and PS exposure induced by the absence of the commonly used plasticiser DEHP. These results provide initial but strong evidence to support vepoloxamer use to replace DEHP in long-term storage of RBC.

Introduction

Di-(2-ethylhexyl) phthalate (DEHP) is a plasticiser produced commercially to provide flexibility to an otherwise rigid polyvinyl chloride (PVC)¹ with concentrations in PVC of approximately 30%, although these can be up to 80%^2,3. Since DEHP is non-covalently bound to the PVC polymer, its lipophilic nature means it can leach into the storage content of a container, especially when the surface comes into contact with lipid-containing fluids. The concentration of DEHP in whole blood (WB) or blood components can be efficiently measured⁴ and increases upon storage^5,6, although absolute amounts depend on component composition and storage temperature. While most DEHP is found in fluid phase, especially bound to lipoproteins⁷, a small proportion can be associated with red blood cells (RBCs)⁸, resulting in intravenous exposure and circulating DEHP in blood^9,10. The value of DEHP in RBC bags is not only related to the increased flexibility of the bag but also because it improves RBC survival during storage and after transfusion¹¹. DEHP affects RBC integrity by inhibiting the deterioration of the RBC membrane, which prevents the haemolysis, microvesicle formation, and morphological changes that occur during refrigerated storage¹². DEHP is associated with the RBC membrane and cytosol, and this is a potential mechanism for the increased RBC stability¹³. While the molecular mechanism of this protective role is not completely understood, some studies have demonstrated that DEHP protects against membrane damage by altering the interaction between phospholipids and pro-oxidant adenine nucleotide translocators in the cytoplasmic or mitochondrial membrane of cells¹⁴.

Although human urinary excretion levels are usually below the exposure limits of the US Environmental Protection Agency (EPA) and other worldwide environmental agencies, extensive laboratory studies have been done into the long-term effect on health due to the cumulative exposure in massive transfusion protocols and/or chronic exposure to DEHP and possible synergistic endocrine effects. DEHP undergoes metabolic degradation resulting in the formation of the monoester, mono(2-ethylhexyl)phthalate (MEHP), and further oxidative forms¹⁵. MEHP has been shown to be carcinogenic via the activation of two nuclear transcription factors, PPARα and PPARγ, important to cell differentiation¹⁶. In addition, DEHP is a developmental and reproductive toxicant suspected of having endocrine disrupting or modulating effects¹⁷ and promoting inflammation¹⁸.

As a consequence of the harmful effects of DEHP, alternative methods to replace the protective effect of DEHP on haemolysis are warranted. A possible alternative is the use of a rheological agent with a favourable toxicity profile that mimics the beneficial effects of DEHP. Vepoloxamer is a highly purified form of the linear non-ionic amphiphilic copolymer poloxamer 188 (Figure 1). It is comprised of an internal hydrophobic polyoxypropylene chain flanked at either end by hydrophilic polyoxyethylene blocks. It exhibits rheologic, anti-thrombotic and cyto-protective properties in vitro and in vivo^19–31. Its basic structure confers surface-active properties that enable the modulation of the biophysical properties of the cell membrane, including stability, hydration repair, flexibility, and adhesive properties, all of which serve crucial roles in biological responses. Vepoloxamer inhibits polymer-induced RBC aggregation and adhesion to endothelial cells^32,33. Substantial research has demonstrated that vepoloxamer has cytoprotective and haemorheologic properties, and inhibits inflammatory processes and thrombosis^19,22,29,34. The drug has been used in clinical trials of sickle cell patients with vaso-occlusive disease including a Phase III, double blind, placebo-controlled trial (registered as NCT01737814). In this last trial, although vepoloxamer was found not to reduce the duration of the vaso-occlusive disease in sickle cell patients when compared with a placebo control, the drug was found to be well tolerated and safe when administered in healthy and seriously ill humans.

Figure 1.

Chemical formula for vepoloxamer.

Vepoloxamer consists of repeated ethylene and propylene oxide groups. With n=80 and m=27, Vepoloxamer has a calculated molecular weight of 8,624 Daltons.

This study was designed to determine whether vepoloxamer could be used in RBC storage additive solutions to replace the effect of DEHP in reducing the haemolysis rate after long-term storage, and, if it proved to be protective, to explore the mechanisms involved in improving RBC viability in vitro.

Materials and methods

Blood collection and processing

Whole blood (500 mL ±10%) from 30 consenting donors aged 18–70 years old who fulfilled American Association of Blood Banks (AABB) and US Food and Drug Administration (FDA) criteria for donation (except for travel) was collected into a collection set with CPD anticoagulant (Code 4R3329, Fenwal Inc., Lake Zurich, IL, USA). Units were leucoreduced using the integral RS-2000 filter and stored in additive solution AS-1 within 8 hours of collection. Pools of two RBC suspensions from ABO identical donors aseptically split into five aliquots containing 95 mL of RBC suspension, and stored either DEHP/PVC or DEHP-free/ethylene vinyl acetate (EVA) bags (Evolve EV-12+F-M12, Origen Biomedical, Austin, TX, USA), supplemented with or without 5 mL of GMP-grade vepoloxamer (Figure 1) or saline control and mixed before storage. Groups of paired analysis consisted of DEHP/PVC with no vepoloxamer (with added physiological saline solution, n=15), EVA with no vepoloxamer (with added physiological saline solution, n=15) and EVA with vepoloxamer at concentrations of 0.1 (n=9), 1 (n=15), 5 (n=15) or 7.89 (n=6) mg/mL in physiological saline solutions. RBC units were cold stored (1–6 °C) for 42 days.

Methods

For all aliquots, a complete blood count was analysed (Coulter Ac.T5 Diff CP analyzer, Coulter Corp., Miami, FL, USA). Spun haematocrit was determined using a microhaematocrit centrifuge, as previously described³⁵. Supernatant haemoglobin and haemolysis rates were calculated as previously described³⁶. pH, pO₂, pCO₂, extracellular glucose, sodium, potassium and lactate concentrations were measured using ABL805 FLEX Analyzer (Radiometer, Copenhagen, Denmark) following the manufacturer’s instructions. Intracellular adenosine-5′-triphospate (ATP) levels were determined as previously described³⁷. Osmotic fragility was analysed as previously described³⁸ and deformability under shear stress conditions was assessed by ektacytometry at a maximum pressure of 60 Pa³⁹. The above listed analyses were performed on days 28 and 42 of storage.

Also on day 42 of storage, oxidative stress was assessed by determination of the oxidised glutathione/reduced glutathione (GSSG/GSH) ratio⁴⁰. Oxidised peroxiredoxin-2 (PRX2) levels were analysed by Western blot as previously described⁴¹, and band density analysed by the Image J software⁴² in relation to the loading control developed with anti-β-actin (Sigma, St. Louis, MO, USA) and normalised to the values of the DEHP group. Eryptosis was analysed by determination of the percentage of cells with exposed extracellular phosphatidylserine (PS) residues as assessed by annexin-V binding and flow cytometry analysis. Briefly, RBCs were washed in annexin-V binding buffer containing (in mM) 125 NaCl, 10 HEPES, pH 7.4, and 5 CaCl₂ (BD Biosciences, San Jose, CA, USA). Erythrocytes were stained with annexin-FITC (BD Biosciences) at a 1:10 dilution. After 15 min, samples were washed with annexin-V binding buffer and measured by flow cytometry (FACSCalibur; Becton Dickinson). Annexin-V binding was analysed on a gate of appropriate forward and sideward scatter (logarithmic transformation) and annexin-V fluorescence intensity was measured in FL1.

Data are presented as average ±1 standard deviation (SD). Comparative statistical analysis was performed by ANOVA test with Bonferroni correction. p<0.05 was considered statistically significant.

Results

As expected, the absence of DEHP in EVA bags results in increased haemolysis by days 28 and 42 of storage (Figure 2A and B). Addition of vepoloxamer significantly reverses the increased haemolysis induced by removal of DEHP in EVA bags in a dose-dependent manner by day 28 and day 42 of storage (Figure 2A and B). Addition of vepoloxamer at the highest concentration tested (7.89 mg/mL) resulted in reduction of haemolysis at levels similar to the levels observed in the PVC/DEHP control. The haemolysis rate was inversely correlated with increasing concentrations of vepoloxamer (r²=0.27, p<0.001) indicating an association between both parameters. Similarly, the lack of a plateau phase suggests that the highest concentration of vepoloxamer may have not reached the peak of its biological effect on haemolysis prevention. A similar effect was observed by day 28 and especially by day 42 of storage on RBC osmotic fragility (Figure 2C and D) and deformability under shear stress at 60 Pa (Figure 2E and F), with a significant dose-dependent effect of vepoloxamer on the reversal of the increased osmotic fragility of day 42 stored RBC (Figure 2D) and shear-stress deformability of RBC stored in EVA bags (Figure 2E and F). These changes may have resulted in a reversal of the eryptosis of RBC when stored in EVA, as assessed by analysis of PS exposure through the determination of the percentage of RBC with the ability to bind annexin-V in units containing increasing concentrations of vepoloxamer by day 42 of storage (Figure 3A), with a direct correlation between haemolysis and PS exposure (r²=0.42; p<0.001). These results indicate that vepoloxamer prevents the RBC storage lesion associated with replacement of PVC/DEHP for EVA as constituent of the storage bags and are consistent with the hypothesised role of vepoloxamer as an RBC membrane intercalator that interferes with the process of eryptosis and in vitro haemolysis.

Figure 2.

Addition of vepoloxamer results in a dose-dependent reduction in haemolysis and fragility of red blood cells (RBC) stored in ethylene vinyl acetate (EVA) bags.

(A and B) Haemolysis. (C and D) Osmotic fragility. (E and F) Deformability index (ektacytometry at 60 Pa). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (ANOVA test with Bonferroni correction). DEHP: polyvinyl chloride bag with di(2-ethylhexyl) phthalate (DEHP) plasticiser.

Figure 3.

Addition of vepoloxamer results in a dose-dependent reduction of eryptosis with no changes in glycolytic flux, potassium leakage or redox potential by day 42 of storage.

(A) Percentage of red blood cell (RBC) binding annexin-V. (B) Intracellular adenosine-5′-triphospate (ATP) concentration levels. Extracellular concentration of (C) glucose and (D) lactate. (E) Extracellular potassium concentration. (F) Intracellular level of oxidised PRX2. Intracellular levels of (G) reduced glutathione (GSH) and (H) oxidised glutathione (GSSG). * p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (ANOVA test with Bonferroni correction). DEHP: polyvinyl chloride bag with di(2-ethylhexyl) phthalate (DEHP) plasticiser.

Interestingly, the amelioration of eryptosis and in vitro haemolysis is not associated with modifications in the concentrations of different biochemical parameters associated with metabolic, transmembrane potential or redox stress. There were no significant changes in ATP (Figure 3B), glycolytic flux as assessed by changes in the extracellular concentrations of glucose (Figure 3C) or lactate (Figure 3D), or potassium leakage (Figure 3E). Similarly, despite a modest reduction in oxidative stress of EVA-stored RBC compared with DEHP/EVA stored bags (Figure 3F), vepoloxamer addition did not result in any changes in the intracellular levels of oxidised peroxiredoxin-2 (PRX2) (Figure 3F) or of the intracellular levels of GSSG (Figure 3G) or in the overall intracellular concentration of the GSH (Figure 3H).

Discussion

In this study, we analysed for the first time the possibility of replacing the effect of the DEHP plasticiser used in PVC bags by adding vepoloxamer into malleable EVA bags by adding vepoloxamer at increasing concentrations. Our data support the concept that vepoloxamer does prevent the RBC storage lesion in vitro as it reduces the haemolysis, eryptosis and osmotic fragility, and increases the erythrocyte deformability under significant shear stress of up to 60 Pa. This effect seems to be unrelated to modifications in the energy production, glycolytic flux, cat ion exchange activity or redox potential, suggesting that its actions occur by direct interactions of the polymer with lipids and lipoproteins on the red cell membrane leaflets.

Toth et al.³² demonstrated that unpurified vepoloxamer, at concentrations of 0.5–5 mg/mL, inhibited both the extent and strength of RBC aggregation in a dose-dependent manner in the presence of aggregating concentrations of Dextran-70. Vepoloxamer at a concentration of 5 mg/mL was more effective in improving the rheology of younger, less dense cells. Based upon the depletion model for polymer-induced aggregation, these authors suggest that vepoloxamer acts by penetrating the depletion layer near the glycocalyx, thereby reducing the osmotic gradient between the intercellular gap and the suspending medium. Sandor et al.³³ also demonstrated that unpurified poloxamer 188 significantly reduces blood viscosity, and RBC aggregation and adhesion to endothelial cells, possibly by acting as an intercalating agent in cell membranes. Our results further support the effect of vepoloxamer on modifications of the RBC membrane that result in stabilisation as assessed by significant reduction of their osmotic fragility and increase in their deformability upon shear stress.

Conclusions

Vepoloxamer is able to significantly rescue the increased haemolysis induced by the absence of the commonly used plasticiser DEHP in a concentration-dependent fashion. While the biochemical/biophysical mechanism of this restoration remains unclear, the improved osmotic fragility and shear-stress deformability index strongly suggests that vepoloxamer may act as an intercalating agent with the ability to increase the RBC membranes flexibility and fitness to challenging storage-dependent rheological conditions. These results provide evidence that the rheological agent vepoloxamer may provide an alternative to the current use of potentially harmful plasticisers like DEHP for long-term RBC storage.