EXCHANGE TRANSFUSION WITH VS -101: A NEW PEGYLATED-HB DESIGNED TO RESTORE PERFUSION AND INCREASE O 2 CARRYING CAPACITY.

ABSTRACT: Blood products are the current standard for resuscitation of hemorrhagic shock. However, logistical constraints of perishable blood limit availability and prehospital use, meaning alternatives that provide blood-like responses remain an area of active investigation and development. VS-101 is a new PEGylated human hemoglobin-based oxygen carrier that avoids the logistical hurdles of traditional blood transfusion. This study sought to determine the safety and ability of VS -101 to maintain circulatory function and capillary oxygen delivery in a severe (50%) exchange transfusion (ET) model. Anesthetized, male Sprague Dawley rats were prepared for cardiovascular monitoring and phosphorescence quenching microscopy of interstitial fluid oxygen tension (P ISFo2 ) in the spinotrapezius muscle. Fifty-percent isovolemic ET of estimated total blood volume with either lactated Ringer's solution (LRS, n = 8) or VS -101 (n = 8) at 1 mL/kg/min was performed, and animals were observed for 240 min. VS -101 maintained P ISFo2 at baseline with a transient 18 ± 4 mm Hg decrease ( P < 0.05) in mean arterial pressure (MAP). In contrast, ET with LRS decreased P ISFo2 by approximately 50% ( P < 0.05) and MAP by 74 ± 10 mm Hg ( P < 0.05). All VS -101 animals survived 240 min, the experimental endpoint, while 100% of LRS animals expired by 142 min. VS -101 animals maintained normal tissue oxygenation through 210 min, decreasing by 25% ( P < 0.05 vs. baseline) thereafter, likely from VS -101 vascular clearance. No arteriolar vasoconstriction was observed following VS -101 treatment. In this model of severe ET, VS -101 effectively maintained blood pressure, perfusion, and P ISFo2 with no vasoconstrictive effects. Further elucidation of these beneficial resuscitation effects of VS -101 is warranted to support future clinical trials.

How Nitric Oxide Hindered the Search for Hemoglobin-Based Oxygen Carriers as Human Blood Substitutes.

The search for a clinically affordable substitute of human blood for transfusion is still an unmet need of modern society. More than 50 years of research on acellular hemoglobin (Hb)-based oxygen carriers (HBOC) have not yet produced a single formulation able to carry oxygen to hemorrhage-challenged tissues without compromising the body's functions. Of the several bottlenecks encountered, the high reactivity of acellular Hb with circulating nitric oxide (NO) is particularly arduous to overcome because of the NO-scavenging effect, which causes life-threatening side effects as vasoconstriction, inflammation, coagulopathies, and redox imbalance. The purpose of this manuscript is not to add a review of candidate HBOC formulations but to focus on the biochemical and physiological events that underly NO scavenging by acellular Hb. To this purpose, we examine the differential chemistry of the reaction of NO with erythrocyte and acellular Hb, the NO signaling paths in physiological and HBOC-challenged situations, and the protein engineering tools that are predicted to modulate the NO-scavenging effect. A better understanding of two mechanisms linked to the NO reactivity of acellular Hb, the nitrosylated Hb and the nitrite reductase hypotheses, may become essential to focus HBOC research toward clinical targets.

Systemic and microvascular comparison of Lactated Ringer's solution, VIR-HBOC, and alpha-alpha crosslinked haemoglobin-based oxygen carrier in a rat 10% topload model.

Medical support for traumatic haemorrhage is lacking for far-forward combat units. VIR-HBOC (haemoglobin-based oxygen carrier) is a novel biological therapeutic under development as a field-stable resuscitation fluid. HBOCs have a long history of complications, chief among them is vasoconstrictive hypertension, which must be resolved before efficacy testing. As such, VIR-HBOC was compared against Lactated Ringers (LRS; vehicle) and a cross-linked haemoglobin (ααHb; a known vasoactive HBOC) in a rat topload model. Twenty-three male, Sprague Dawley rats were randomly assigned to receive a 10% infusion (estimated total blood volume) of one test article while normotensive and under anaesthesia. Cardiovascular, blood chemistry and oximetry, microvascular arteriolar diameters, and interstitial tissue oxygenation parameters were measured. Circulatory half-life was calculated by plasma total haemoglobin. Treatment with ααHb caused immediate increases in mean arterial pressure compared to LRS and VIR-HBOC groups, and corresponding arteriolar vasoconstriction (p < .05), which did not occur for LRS or VIR-HBOC. Circulatory half-lives for VIR-HBOC and ααHb were calculated as 340 and 157 min, respectively. This first report of VIR-HBOC showed no evidence of a hypertensive or vasoactive effect. It was well-tolerated over the eight-hour time course of this topload model, which warrants further investigation in studies of haemorrhagic shock.

Hemoglobin extravasation in the brain of rats exchange-transfused with hemoglobin-based oxygen carriers.

Haemoglobin (Hb)-based oxygen carriers are under consideration as oxygen therapeutics. Their effect on apoptosis is critical, because the onset of pro-apoptotic pathways may lead to tissue damage. MP4OX, a polyethylene glycol-conjugated human Hb preserves the baseline level of neuron apoptosis with respect to sham. Here we develop a method for measuring Hb extravasation in brain. We exchange transfused rats by haemorrhaging 50% of their blood with simultaneous, isovolemic replacement with Hextend (negative control), MP4OX, or αα-cross-linked Hb. Animals were sacrificed 2 h after transfusion, brain tissue was harvested and processed for double-staining immunofluorescence, whereby Hb ? chain and NeuN (a neuron protein) were stained and quantitated. Whereas Hextend did not induce Hb extravasation, in both MP4OX and ??Hb brains Hb molecules were detected outside neurons. The level of extravasated Hb chains was > 3-fold higher in Hb compared to MP4OX. Western blot analysis revealed that the expression levels of protein related to redox imbalance (e.g., Nrf2, iNOS and ERK phosphorylation) were higher in ααHb than MP4OX. In conclusions, higher Hb extravasation in ααHb than MP4OX induces redox imbalance, which causes higher anti-oxidant response. Whereas Nrf2 response may be considered protective, iNOS response appears damaging.

Impact of acellular hemoglobin-based oxygen carriers on brain apoptosis in rats.

BACKGROUND: Extracellular hemoglobin (Hb)-based oxygen carriers (HBOCs) are under extensive consideration as oxygen therapeutics. Their effects on cellular mechanisms related to apoptosis are of particular interest, because the onset of proapoptotic pathways may give rise to tissue damage. STUDY DESIGN AND METHODS: The objective was to assess whether the properties of the Hb that replaces blood during an isovolemic hemodilution would modulate apoptotic-response mechanisms in rat brain and whether such signaling favors cytoprotection or damage. We exposed rats to exchange transfusion (ET; 50% blood volume and isovolemic replacement with Hextend [negative colloid control], MP4OX [PEGylated HBOC with high oxygen affinity], and ααHb [αα-cross-linked HBOC with low oxygen affinity; n=4-6/group]). Sham rats acted as control. Animals were euthanized at 2, 6, and 12 hours after ET; brain tissue was harvested and processed for analysis. RESULTS: In MP4OX animals, the number of neurons that overexpressed the hypoxia-inducible factor (HIF)-1α was higher than in ααHb, particularly at the early time points. In addition, MP4OX was associated with greater phosphorylation of protein kinase B (Akt), a well-known cytoprotective factor. Indeed, the degree of apoptosis, measured as terminal deoxynucleotidyl transferase-positive neurons and caspase-3 cleavage, ranked in order of MP4OX < Hextend < ααHb. CONCLUSION: Even though both HBOCs showed increased levels of HIF-1α compared to shams or Hextend-treated animals, differences in signaling events resulted in very different outcomes for the two HBOCs. ααHb-treated brain tissue showed significant neuronal damage, measured as apoptosis. This was in stark contrast to the protection seen with MP4OX, apparently due to recruitment of Akt and neuronal specific HIF-1α pathways.

MP4, a vasodilatory PEGylated hemoglobin.

A vasodilatory hemoglobin (Hb)-based O(2) carrier (HBOC) has been developed by surface conjugation polyethylene glycol to tetrameric human Hb (MP4, Sangart, San Diego). Because the NO-binding kinetics of MP4 are similar to vasoconstrictive HBOCs, we propose that the decoupling of NO scavenging from vascular response is a consequence of MP4's high O(2) affinity (p50 = 5 mmHg) and unique surface chemistry. The release of ATP from erythrocytes is vasodilatory and the application of a high O(2) affinity HBOC minimizes ATP interference with intravascular ATP signaling. A second potential mechanism of action for MP4 involves the surface conjugation of polyethylene glycol (PEG) to tetrameric human Hb. It has been shown that the addition of unconjugated high molecular weight (Mw) PEG to cultured lung endothelial cells causes an immediate and significant reduction in endothelial permeability; an effect opposite to that of endothelial agonists such as cell-free Hb. It appears that some of the benefits of the PEG-endothelium interaction are carried onto molecules such as PEGylated Hb and PEGylated albumin, as demonstrated by favorable hemodynamic responses in vivo. PEGylation of ß93 cysteine residues, as in MP4, has also been reported to increase the nitrite reductase activity of Hb and enhance conversion of endogenous nitrite to bioactive NO.

Erythrocytic ATP release in the presence of modified cell-free hemoglobin.

The red blood cell (RBC) has been proposed as an O(2) sensor through a direct link between the desaturation of intracellular hemoglobin (Hb) and ATP release, leading to vasodilation. We hypothesized that the addition of cell-free Hb to the extracellular space provides a supplementary O(2) source that reduces RBC desaturation and, consequently, ATP release. In this study, the saturation of RBC suspensions was lowered by additions of deoxygenated hemoglobin-based oxygen carrier (HBOC) and then assayed for extracellular ATP. When an acellular human Hb intramolecularly cross-linked between alpha subunits (alphaalphaHb, p50 = 33 mmHg) was added to the red cell suspension, ATP production was significantly less than that in the presence of a lower p50 HBOC (Hb cross-linked between beta subunits, betabetaHb, p50 = 8 mmHg). These results provide a potential mechanism for the O(2) affinity of HBOCs to interfere with a vasodilatory signal.

Hemospan: design principles for a new class of oxygen therapeutic.

Hemoglobin-based oxygen carriers have been under development for decades, but safety concerns have prevented commercial approval. Early designs for modified hemoglobins by polymerization or intramolecular cross-linking reactions increased molecular size and decreased oxygen affinity, but all exhibited side effects of vasoconstriction and reduced blood flow. A new strategy has been established by applying principles of oxygen transport to cell-free hemoglobin. Sangart has developed a new oxygen therapeutic, Hemospan, using site-specific, poly(ethylene) glycol conjugation chemistry designed on two principles: (i) increased macromolecular size to prolong intravascular retention time, and (ii) increased oxygen affinity to prevent premature oxygen offloading in arterioles. In contrast to early-generation products, Hemospan infusion maintains normal arteriolar vascular tone and capillary flow. Phase I and Phase II clinical trials have been completed, showing that Hemospan is well-tolerated in humans, with evidence of efficacy to impart hemodynamic stability in surgical patients under anesthesia. Phase III trials in orthopedic surgery have recently completed enrollment in Europe.

Hemospan improves outcome in a model of perioperative hemodilution and blood loss in the rat: comparison with hydroxyethyl starch.

OBJECTIVES: Hemospan (Sangart Inc, San Diego, CA) (MP4) is a hemoglobin-based oxygen carrier consisting of human hemoglobin modified with polyethylene glycol. This study evaluated the effects of MP4 on blood volume, hemodynamics, and metabolic stability in a rat model of hemodilution and hemorrhage. MP4 was compared with hydroxyethyl starch solutions of differing concentrations (ie, HES 260/0.45 and HES 130/0.4). DESIGN: An open-label, randomized comparison of treatments. SETTING: Pharmaceutical industry. PARTICIPANTS: Sprague Dawley rats. INTERVENTIONS: Rats underwent 50% hemodilution with one of the solutions. Control rats were not hemodiluted. Blood volume was determined at baseline and 0, 60, and 120 minutes after exchange. In separate groups, hemodilution and subsequent 60% hemorrhage were examined to determine effectiveness of hemodilution. MEASUREMENTS AND MAIN RESULTS: Endpoints were blood volume after hemodilution and survival, hemodynamics, and acid-base status during hemorrhage. Volume expansion was similar with MP4 (159% of infused volume) and HES 260/0.45 (145%) and less with HES 130/0.4 (104%). The duration of expansion was longest with MP4 (1-2 hours). In the hemorrhage studies, 2-hour survival was 90% with MP4, 50% with controls, and 10% and 0% with HES 260/0.45 and HES 130/0.4, respectively. The severity of hemodynamic and acid-base changes paralleled the survival, with the least disturbance observed in MP4-treated animals. CONCLUSIONS: Hemodilution with MP4 was more effective in maintaining hemodynamic and metabolic stability than starch solutions or no hemodilution before simulated intraoperative hemorrhage. The benefit of MP4 is not ascribed solely to volume expansion. The results suggest that perioperative administration of MP4 may improve outcomes in surgical settings.

Sites of modification of hemospan, a poly(ethylene glycol)-modified human hemoglobin for use as an oxygen therapeutic.

Hemospan is an acellular hemoglobin-based oxygen therapeutic in clinical trials in Europe and the United States. The product is prepared by site-specific conjugation of maleimide-activated poly(ethylene) glycol (PEG, MW approximately 5500) to human oxyhemoglobin through maleimidation reactions either (1) directly to reactive Cys thiols or (2) at surface Lys groups following thiolation using 2-iminothiolane. The thiolation/maleimidation reactions lead to the addition of approximately 8 PEGs per hemoglobin tetramer. Identification of PEG modified globins by SDS-PAGE and MALDI-TOF reveals a small percentage of protein migrating at the position for unmodified globin chains and the remaining as separate bands representing globin chains conjugated with 1 to 4 PEGs per chain. Identification of PEG modification sites on individual alpha and beta globins was made using reverse-phase HPLC, showing a series of alpha globins conjugated with 0 to 3 PEGs and a series of beta globins conjugated with 0 to 4 PEGs per globin. Mass analysis of tryptic peptides from hemoglobin thiolated and maleimidated with N-ethyl maleimide showed the same potential sites of modification regardless of thiolation reaction ratio, with seven sites identified on beta globins at beta8, beta17, beta59, beta66, beta93, beta95, and beta132 and three sites identified on alpha globins at alpha7, alpha16, and alpha40.

Targeted O2 delivery by blood substitutes: in vitro arteriolar simulations of first- and second-generation products.

The O(2) transport from mixtures of commercially produced hemoglobin-based O(2) carriers (HBOCs) and red blood cells (RBCs) flowing through arteriolar-sized (25-mum) conduits is simulated. A generalized treatment of extraluminal O(2) transport processes is used to reflect variations in physiological conditions, such as increased O(2) consumption. Of the HBOCs considered, polymerized bovine hemoglobin (PolyBvHb, p50=54 mmHg), tetrameric cross-linked human hemoglobin (alphaalphaHb, p50=33 mmHg), and PEGylated human hemoglobin (MP4, p50=5 mmHg), only MP4 does not increase O(2) extraction ratios when compared to RBC suspensions alone. A reduction in arteriolar O(2) extraction is likely to be beneficial for HBOCs by preventing O(2)-induced vasoactivity and maximizing the supply of O(2) available to the capillaries. Results from in vivo HBOC transfusion experiments cannot be predicted by the model, unless PolyBvHb has a significant decrease in extraluminal O(2) transport resistance as compared to MP4. This result is consistent with the literature that shows arteriolar O(2) consumption to increase with intravascular pO(2).

Solution structure of poly(ethylene) glycol-conjugated hemoglobin revealed by small-angle X-ray scattering: implications for a new oxygen therapeutic.

Developing protein therapeutics has posed challenges due to short circulating times and toxicities. Recent advances using poly(ethylene) glycol (PEG) conjugation have improved their performance. A PEG-conjugated hemoglobin (Hb), Hemospan, is in clinical trials as an oxygen therapeutic. Solutions of PEG-hemoglobin with two (P5K2) or six to seven strands of 5-kD PEG (P5K6) were studied by small-angle x-ray scattering. PEGylation elongates the dimensions (Hb < P5K2 < P5K6) and leaves the tertiary hemoglobin structure unchanged but compacts its quaternary structure. The major part of the PEG chains visualized by ab initio reconstruction protrudes away from hemoglobin, whereas the rest interacts with the protein. PEGylation introduces intermolecular repulsion, increasing with conjugated PEG amount. These results demonstrate how PEG surface shielding and intermolecular repulsion may prolong intravascular retention and lack of reactivity of PEG-Hb, possibly by inhibiting binding to the macrophage CD163 hemoglobin-scavenger receptor. The proposed methodology for assessment of low-resolution structures and interactions is a powerful means for rational design of PEGylated therapeutic agents.

Toxicity and hemodynamic effects after single dose administration of MalPEG-hemoglobin (MP4) in rhesus monkeys.

Maleimide-polyethylene glycol-modified (MalPEG) hemoglobin, 4.3 g/dL (MP4; Hemospan), is a hemoglobin-based oxygen carrier consisting of human hemoglobin (Hb) modified with maleimide polyethylene glycol. This study evaluates the potential toxicity and hemodynamic actions of a single dose of MP4 administered by exchange transfusion to rhesus monkeys. Monkeys were administered MP4 (21 mL/kg, or approximately 30% of estimated blood volume) or an equivalent volume of lactated Ringer's solution (LR). In the toxicity study, blood samples were obtained predose and 3, 7, and 13 days after dosing for clinical chemistry and hematology. Animals were euthanized for complete necropsy and histopathology on day 3 or day 13. A separate group of animals was used for evaluation of arterial pressure, core temperature, and electrocardiogram, by telemetry, for 7 days after dosing with MP4. The results demonstrate no significant toxicity, with only modest, transient elevation of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) on day 3. Mild anemia caused by hemodilution was observed at each time point in both groups, but to a slightly greater degree in the MP4-treated animals. Histologic observations included foamy or vacuolated macrophages in the spleen and marrow of the sternum, rib, and femur, representing the accumulation of test article or a metabolite. In the telemetry study, no changes occurred in arterial pressure, heart rate, or electrocardiogram attributable to administration of MP4 at any time for 7 days after administration. These results demonstrate that MP4 is safe and is without hemodynamic effects when administered as an exchange transfusion of 30% of estimated blood volume.

A quantitative framework for the design of acellular hemoglobins as blood substitutes: implications of dynamic flow conditions.

The delivery of oxygen to tissue by cell-free carriers eliminates intraluminal barriers associated with red blood cells. This is important in arterioles, since arteriolar tone controls capillary perfusion. We describe a mathematical model for O(2) transport by hemoglobin solutions and red blood cells flowing through arteriolar-sized tubes to optimize values of p50, Hill number, hemoglobin molecular diffusivity and concentration. Oxygen release is evaluated by including an extra-luminal resistance term to reflect tissue oxygen consumption. For low consumption (i.e., high resistance to O(2) release) a hemoglobin solution with p50=15 mmHg, n=1, D(HBO2)=3 x 10(-7) cm(2)/s delivers O(2) at a rate similar to that of red blood cells. For high consumption, the p50 must be decreased to 5 mmHg. The model predicts that regardless of size, hemoglobin solutions with higher p50 will present excess O(2) to arteriolar walls. Oversupply of O(2) to arteriolar walls may cause constriction and paradoxically reduced capillary perfusion.

Oxidation and haem loss kinetics of poly(ethylene glycol)-conjugated haemoglobin (MP4): dissociation between in vitro and in vivo oxidation rates.

Haemoglobin-based oxygen carriers can undergo oxidation of ferrous haemoglobin into a non-functional ferric form with enhanced rates of haem loss. A recently developed human haemoglobin conjugated to maleimide-activated poly(ethylene glycol), termed MP4, has unique physicochemical properties (increased molecular radius, high oxygen affinity and low cooperativity) and lacks the typical hypertensive response observed with most cell-free haemoglobin solutions. The rate of in vitro MP4 autoxidation is higher compared with the rate for unmodified SFHb (stroma-free haemoglobin), both at room temperature (20-22 degrees C) and at 37 degrees C (P<0.001). This appears to be attributable to residual catalase activity in SFHb but not MP4. In contrast, MP4 and SFHb showed the same susceptibility to oxidation by reactive oxygen species generated by a xanthine-xanthine oxidase system. Once fully oxidized to methaemoglobin, the rate of in vitro haem loss was five times higher in MP4 compared with SFHb in the fast phase, which we assign to the beta subunits, whereas the slow phase (i.e. haem loss from alpha chains) showed similar rates for the two haemoglobins. Formation of MP4 methaemoglobin in vivo following transfusion in rats and humans was slower than predicted by its first-order in vitro autoxidation rate, and there was no appreciable accumulation of MP4 methaemoglobin in plasma before disappearing from the circulation. These results show that MP4 oxidation and haem loss characteristics observed in vitro provide information regarding the effect of poly(ethylene glycol) conjugation on the stability of the haemoglobin molecule, but do not correspond to the oxidation behaviour of MP4 in vivo.

Enhanced molecular volume of conservatively pegylated Hb: (SP-PEG5K)6-HbA is non-hypertensive.

Recent studies have suggested that the "pressor effect" of acellular Hb is a consequence of perturbation of the macro-and microcirculatory system in multiple ways, and that PEGylation is an effective approach for controlling the same. In an attempt to confirm this concept, a new and simple thiolation mediated, maleimide chemistry-based conservative PEGylation protocol has been developed to conjugate multiple copies of PEG-chains to Hb. This approach combines the high reactivity of maleimides towards thiols with the propensity of iminothiolane to derivatize the epsilon-amino groups of proteins into reactive thiol groups, with conservation of their positive charge. One of the PEGylated products, namely (SP-PEG5K)6-HbA, that carries on an average six copies of PEG5000 chains per Hb, is non-hypertensive in hamster top load and in rat 50% exchange transfusion models. This hexa-PEGylated-Hb has (i) a hydrodynamic volume corresponding to that of an oligomerized Hb of 256kDa, (ii) a molecular radius of approximately 6.8 nm, (iii) high oxygen affinity, (iv) lowered Bohr effect, and (v) increased viscosity and colloidal osmotic pressure. These properties of (SP-PEG5K)6-HbA are consistent with the emerging new paradigms for the design of Hb based oxygen carriers and confirm the concept that the "pressor effect" of Hb is a multifactorial event. The thiolation mediated maleimide chemistry-based PEGylation protocol described here for the generation of (SP-PEG5K)6-Hb is simple, highly efficient, and is carried out under oxy conditions. The results demonstrate that a non-hypertensive PEG-Hb can be generated by conjugation of a lower number of PEG chains than previously reported.

Effect of acute hypoxia on microcirculatory and tissue oxygen levels in rat cremaster muscle.

Repeated exposure to brief periods of hypoxia leads to pathophysiological changes in experimental animals similar to those seen in sleep apnea. To determine the effects of such exposure on oxygen levels in vivo, we used an optical method to measure PO2 in microcirculatory vessels and tissue of the rat cremaster muscle during a 1-min step reduction of inspired oxygen fraction from 0.21 to 0.07. Under control conditions, PO2 was 98.1 +/- 1.9 Torr in arterial blood, 52.2 +/- 2.8 Torr in 29.0 +/- 2.7-microm arterioles, 26.8 +/- 1.7 Torr in the tissue interstitium near venous capillaries, and 35.1 +/- 2.6 Torr in 29.7 +/- 1.9-microm venules. The initial fall in PO2 during hypoxia was significantly greater in arterial blood, being 93% complete in the first 10 s, whereas it was 68% complete in arterioles, 47% at the tissue sites, and 38% in venules. In the 10- to 30-s period, the fall in normalized tissue and venular PO2 was significantly greater than in arterial PO2. At the end of hypoxic exposure, PO2 at all measurement sites had fallen very nearly in proportion to that in the inspired gas, but tissue oxygen levels did not reach critical PO2. Significant differences in oxyhemoglobin desaturation rate were also observed between arterial and microcirculatory vessels during hypoxia. In conclusion, the fall in microcirculatory and tissue oxygen levels in resting skeletal muscle is significantly slower than in arterial blood during a step reduction to an inspired oxygen fraction of 0.07, and tissue PO2 does not reach anaerobic levels.

Comparison of PEG-modified albumin and hemoglobin in extreme hemodilution in the rat.

We have reported a new polyethylene glycol (PEG)-modified, hemoglobin-based O2 carrier (MP4) with novel properties, including a large molecular excluded volume and low PO2 necessary to obtain 50% O2 (approximately 6 Torr). To evaluate the ability of MP4 to transport O2, we compared it with PEG-modified albumin (MPA) using the identical chemistry of attachment of PEG chains. The resulting solutions were well matched with respect to all physical properties except that MP4 is an O2 carrier, whereas MPA is not. An additional solution, 10% pentastarch, was matched with the PEG-modified proteins with regard to oncotic activity and viscosity but does not contain PEG. The model used to evaluate O2 transport was continuous exchange transfusion in the rat until the hematocrit was virtually unmeasurable. Objective end points included survival and the onset of anaerobic metabolism, signaled by acid-base derangement and accumulation of lactic acid. Continuous exchange transfusion of 2.5 blood volumes in rats (n=5 in each treatment group) was carried out over 60 min, such that the final hematocrit was between 0 and 5% in all animals. Animals were observed for an additional 70 min, when survivors were killed. Overall survival for the MP4 animals was 100%; no animal that received either pentastarch or MPA survived. The hematocrit at which lactic acid began to rise was approximately 14.8% in both pentastarch and MPA animals and 7.4% in the animals that received MP4. In all groups, the total hemoglobin was approximately 5 g/dl at this point. We conclude that, despite its low PO2 necessary to obtain 50% O2, MP4 effectively substitutes for red blood cell hemoglobin in its ability to oxygenate tissues in extreme hemodilution.

WITHDRAWN: PEGylated hemoglobin: Role of surface configuration of PEG for the modulation of hemoglobin vasoactivity.

This paper has been withdrawn since all of the coauthors had not approved of its submission.

Kinetics of NO and O2 binding to a maleimide poly(ethylene glycol)-conjugated human haemoglobin.

The hypertensive effect observed with most cell-free haemoglobins has been proposed to result from NO scavenging. However, a newly developed PEG [poly(ethylene glycol)]-conjugated haemoglobin, MalPEG-Hb [maleimide-activated PEG-conjugated haemoglobin], is non-hypertensive with unique physicochemical properties: high O2 affinity, low co-operativity and large molecular radius. It is therefore of interest to compare the ligand-binding properties of MalPEG-Hb with unmodified cell-free HbA (stroma-free human haemoglobin). NO association rates for deoxy and oxyMalPEG-Hb and HbA were found to be identical. These results confirm the lack of correlation between hypertension and NO for a similar modified haemoglobin with high molecular radius and low p50 (pO2 at which haemoglobin is half-saturated with O2) [Rohlfs, Bruner, Chiu, Gonzales, Gonzales, Magde, Magde, Vandegriff and Winslow (1998) J. Biol. Chem. 273, 12128-12134]. The R-state O2 association kinetic constants were also the same for the two haemoglobins. However, even though the p50 of MalPEG-Hb is approx. half of that of HbA, the biphasic O2 dissociation rates measured at relatively high pO2 (150 Torr) were 2-fold higher, giving rise to a 2-fold lower R-state equilibrium association constant for MalPEG-Hb compared with HbA. Thus the O2 affinity of MalPEG-Hb is higher only at pO2 values lower than the intersection point of the O2 equilibrium curves for MalPEG-Hb and HbA. In summary, the present studies found similar rates of NO binding to HbA and MalPEG-Hb, eliminating the possibility that the lack of vasoactivity of MalPEG-Hb is simply the result of reduced molecular reactivity with NO. Alternatively, the unique O2-binding characteristics with low p50 and co-operativity suggest that the 'R-state' conformation of MalPEG-Hb is in a more T-state configuration and restricted from conformational change.