New Strategy for the Preparation of NO-Treated Red Blood Cells as a Blood Substitute
Antonio Tsuneshige and Takashi Yonetani
Summary.
Our previous studies on the a-nitrosyl derivative of human adult hemoglobin (HbA), tetrameric Hb in which only the two a-subunits are ligated with nitric oxide (NO) [Yonetani et al., (1998) J Biol Chem 273: 20323], have shown that the a-nitrosyl HbA is a cooperative, low-affinity oxygen carrier. We have developed a simple method to convert all the intra-human red blood cells (RBCs) to a 50% saturation of hemes with NO exclusively bound to the a-subunits. Oxygen equilibrium measurements showed that the a-NO RBCs also exhibit reduced oxygen affinity (increased P
50) and dimin- ished Bohr effect (i.e., the pH dependence of P
50). Despite the fact that its oxygen-carrying capacity is reduced by 50%, since two a-subunits are already ligated with NO, and only two b-subunits are capable of oxygen binding, a-NO RBCs can efficiently deliver oxygen to tissues under normal physiolog- ical conditions, making this material an excellent candidate for blood trans- fusion. Crucial steps in our protocol for the preparation of a-NO Hb-containing RBCs were revised and technical adjustments were made in order to improve the quality and integrity of RBCs during and after treat- ment, while avoiding the use of potentially noxious chemicals that could entail harmful effects. We were also able to reduce the preparation time. All of which will make viable an increased production volume of quality NO-treated RBCs for practical use.
Key words.
Hemoglobin, Nitrosyl hemoglobin, Red blood cells, Blood substi- tute, Rejuvenation, Erythrocytes
186
Department of Biochemistry and Biophysics and the Johnson Foundation, University of Pennsylvania Medical Center, Philadelphia, PA 19104-6059, USA
Introduction
The main component in human red blood cells (RBCs) is hemoglobin (Hb), a tetrameric heme protein composed of two heterodimers of the form ab. Its major physiological function is the transport of oxygen from lungs to periph- eral tissues, and carbon dioxide from tissues to lungs. Each subunit of the tetramer has a heme group to which oxygen binds reversibly and altogether in a cooperative manner. However, Hb as such cannot perform its physiolog- ical function as its oxygen affinity is rather high, thus hampering the release of oxygen at tissue levels.
The major metabolic process carried out in RBCs is glycolysis. Among the metabolites produced inside RBCs from the consumption of glucose is 2,3- diphosphoglycerate (DPG). This compound is the principal functional mod- ulator of Hb in vivo. When DPG interacts with Hb, the oxygen affinity for oxygen is lowered to optimal levels so that gas transport can be accomplished effectively within the reduced gradient in partial pressures of oxygen between lungs and tissues.
Despite all the efforts aimed at designing alternative blood substitutes, and all the potential risks of infection the use of blood for transfusion entails if pathogens remain undetected, stored blood is the only reasonable and feasible alternative for transfusion available to date. However, great impro- vements have been accomplished recently on detecting infectious threats present in stored blood, all of which ensure a relatively safer use of this blood for transfusions.
One of the main problems with stored blood is that intracellular levels of DPG decay with time as glycolytic activity ceases in the absence of nutrients.
As a consequence, the oxygen affinity of stored RBCs increases with time, hampering the optimal delivery of oxygen from lungs to tissues, and making it less suitable for transfusion. On the other hand, DPG cannot be infused into RBCs to restore the optimal levels found in fresh RBCs. This fact imposes a limit to the shelf lifetime of stored blood for practical use. This period can vary from 3 to 5 weeks, depending on the country. The amount of expired blood that is discarded from blood banks constitutes from 5 to 20% of the total volume of collected blood; a number that is far from insignificant.
Our previous studies on the a-nitrosyl derivative of Hb, that is, a tetrameric Hb in which only the heme groups in the a-subunits are ligated with nitric oxide (NO), have demonstrated that under acidic conditions and/or the presence of allosteric effectors, such as DPG, this derivative shows strikingly reduced affinity for oxygen in the b-subunits while exhibiting cooperativity, despite the fact that both the a-subunits are already ligated [1]. Based on this observation, we attempted to produce this derivative within intact RBCs [2–4]
and the results were extremely promising. For the present study, we tried to
optimize previous pilot preparation protocols in order to make the entire process applicable to large scale production, while reducing dramatically the preparation time without compromising the quality of the final product. At the same time, we concentrated on avoiding the use of reagents that can be potentially noxious, and replaced them with reagents similar in functionality but exerting protective action over the integrity of RBCs. We also discuss the requirements blood substitutes should meet to make them suitable for practical use in transfusion.
Material and Methods
Expired packed RBCs were obtained from the local branch of the American Red Cross and were processed as previously described [4] with modifications.
RBCs were washed three times with 0.15 M sodium phosphate buffer, pH 5.5, containing 2 mM adenine, 45 mM glucose, and 29 mM mannitol (Sigma Aldrich, St. Louis, MO USA), which is a modification of the SAGM solution (sodium, adenine, glucose, mannitol), by repeated resuspension and centri- fugation at 3,000 rpm for 10 min. RBCs were then suspended in a 4-fold volume of the same buffer. Deoxygenation was achieved at room temperature by gently bubbling washed pure Ar into the suspension at room temperature in the presence of egg yolk lecithin (Sigma Aldrich) to prevent foam formation.
Nitric oxide was provided in two alternative ways. In the first method, we used an NO donor, FK409 (Fujisawa Pharmaceuticals, Osaka, Japan), also knows as NOR-3. This compound was dissolved in a minimal amount of dimethyl sulfoxide and added to a thoroughly deoxygenated RBC suspension.
The amount of NO spontaneously released was detected by EPR as nitrosyl heme, at determined time intervals. No reducing agent was added in the collected samples. The total NO releasing capacity was determined by EPR spectroscopy in the presence of sodium dithionite for the complete release of NO and its quenching by binding to the hemes, by a titration similar to that done with glutathione-SNO (GSNO), as previously described [4]. The second method was carried out with GSNO as NO donor. However, different from our previous protocols, NO was released by spontaneous decomposition of the reagent, rather by reduction in the presence of sodium dithionite.
Results and Discussion
In the present work, special care was taken to improving the previous pilot
preparation protocols towards the accomplishment of three main goals: (i)
feasibility to upscale the production, (ii) reduction of preparation time while
improving the quality of the final product, and (iii) avoidance of potentially noxious chemicals that might compromise biocompatibility.
From earlier experience, it was noted that prolonged shaking of RBC suspensions during the deoxygenation process resulted in a considerable breakage of cells, likely due to mechanical sheer forces created between the vessel wall surface and the cells. Cell membrane fragility to mechanical stress can be improved by the presence of mannitol [5] and adenine [6]. Our mod- ified formulation of the RBC suspension solution containing these two com- ponents fulfilled several objectives, one of them being the preservation of integrity if the RBC membranes after extensive nitrogen gas bubbling into the suspension at room temperature. The complete removal of oxygen from the RBC suspension is crucial before the addition of the NO releasing agent in order to prevent the formation of harmful nitrogen oxides that might oxidize Hb and damage the RBC membrane. Oxygen removal was accomplished in a much shorter period of time (3–4 h) by direct bubbling of nitrogen into the suspension at room temperature. We also noticed that despite the exposure of RBCs to physical stress and NO, a reduced degree of hemolysis was pro- duced when compared to our previous protocols. It seems indeed that the presence of adenine and mannitol in the suspension solution helped to pre- serve the integrity of the RBC membrane along the rejuvenation treatment.
Moreover, with the complete removal of oxygen, the presence of sodium dithionite was not necessary and with this, the addition of this potentially noxious agent was prevented.
One of the difficulties encountered in our earlier preparations was the tuning of the rate at which NO was administered into the RBC suspension. As NO is added in small quantities, the chances of one NO molecule to bind to either a- or b-subunits on a deoxyHb tetramer are equal. This distinct process does not trigger the transition from a low to a high affinity conformation as long as most of the other binding sites in the Hb tetramer remain unoccu- pied. When NO binds to an a-subunit, the axial coordination of the heme iron with the proximal histidine is prone to break and by doing so the NO mole- cule remains attached to the a-heme, converting the Hb tetramer into a species with extremely low affinity for ligands [1]. On the other hand, binding of NO to a b-subunit is a reversible process and does not convert the tetramer into a high affinity species as long as the saturation level of the other subunits remains low. Subsequently, NO is released and continues redistributing among other binding sites with a preferential binding to the a-subunits.
However, when NO is added in bulk amounts, the chances to saturate all
hemes at once in a tetramer are high. A fully NO-ligated Hb exhibits high
affinity and as a result the release of NO from the b-subunits is hindered, and
so is truncated the redistribution of NO that leads to the enrichment of Hb
molecules containing NO-ligated a-hemes.
In this work, we studied the use of two substances that release NO over time. As shown in Fig. 1, FK409 displays a half-life time of NO release of 70 min, at 15°C, pH 7.4, following a first order reaction scheme. This rate is slow enough to prevent the “choking” of hemes with NO within a Hb tetramer and allows the redistribution of NO. This observation led us to consider GSNO as an NO donor. Figure 2 shows the time profile of NO-Hb formation within RBCs, carried out at room temperature. It can be noticed that the rate of NO release is much faster than that for FK409, even after temperature corrections from 22°C to 15°C are made. Nevertheless, as revealed in the inset, the NO released binds preferentially to the a-subunits, as detected by the formation of the characteristic hyperfine structure around g ~ 2, which originates from a 5-coordinated nitrosyl-heme. It is clear that the complete deoxygenation of RBCs can be achieved in 3–4 h at room temperature, and that the redistribu- tion of NO after addition of the NO donor is completed in about 3 h.
With the present improvements, it was feasible to carry out the preparation of NO-treated RBCs at room temperature and in large volumes. Using a
Fig. 1. Time profile of NO-Hb formation within deoxygenated RBCs after incubation with FK409 at 15°C, pH 7.4. A suspension of 10% RBCs in the modified SAGM solution was thor- oughly deoxygenated with argon. Then an undersaturating amount over hemes of FK409 dissolved in a minimum amount of dimethyl sulfoxide was added into the suspension.
Aliquots were taken anaerobically at discrete times, placed inside EPR tubes and frozen in liquid nitrogen. The concentration of nitrosyl heme was determined by EPR spectroscopy as previously described [2–4]. The inset shows the kinetic behavior of the decomposition of FK409, following a first order reaction with a half lifetime of 70 min
modified suspension solution and the bubbling of argon into the suspension, complete removal of oxygen from RBCs was achieved without the use of sodium dithionite in a relatively short period of time, while preserving the integrity of cell membranes. The spontaneous and gradual release of NO from NO donors (FK409 or GSNO) facilitated the redistribution of NO with pref- erential binding to the a-subunits, and avoided the problems involved with the bulk addition of NO into the RBC suspension. Subsequent studies will focus on the control of NO release from several NO donors and the effect tem- perature exerts on the redistribution of NO throughout vacant hemes.
Knowing these parameters, it should be possible to predict the distribution of NO inside RBCs without the need for EPR monitoring.
Acknowledgments.
We thank Dr. Makoto Suematsu, Keio University Medical School, Tokyo, Japan, and Dr. Toshiro Inubushi, Shiga University of Medical Science, Ohtsu, Japan, for their continuous support and invaluable critical input to these studies. We also thank Dr. Yoshihiko Oyanagui, Fujisawa Pharmaceuticals, Osaka, Japan, for kindly providing the NO donor FK409 used in the present study. This study was supported by NIH grant HL 14508.
Fig. 2. Time profile of NO-Hb formation within deoxygenated RBCs after incubation with GSNO at room temperature, pH 5.5. The final concentration corresponds approximately to an amount of 50% over heme concentration. Packed RBCs were suspended in a volume about 30 times of the modified SAGM solution. The figure in the inset shows the gradual formation of NO-Hb as detected over time by EPR. Most of the formation of NO-Hb within RBCs was completed after 3 h and did not vary after overnight incubation at 4°C
References
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