First Report of Compound Heterozygosity for Hb S (HBB: c.20A>T) and Hb Haringey (HBB: c.131A>G).

Sickle cell disease variants include hemoglobinopathies that result from inheritance of the sickle cell globin mutation with another globin mutation. The most common variants include the homozygous disease state (Hb SS disease), Hb S (HBB: c.20A>T)/Hb C (HBB: c.19G>A) disease and Hb S/ß-thalassemia (Hb S/ß-thal). Other rare/less common variants such as Hb S/Hb E (HBB: c.79G>A) and Hb S/HPFH [hereditary persistence of fetal hemoglobin (Hb)] disease exist. We report the first case of compound heterozygosity for Hb S and Hb Haringey (HBB: c.131A>G) in a 35-year-old male following a positive sickle screen test on hospital admission for pancreatitis. Ion exchange high performance liquid chromatography (HPLC), Hb electrophoresis and genetic sequencing were utilized to identify a new sickle Hb variant: Hb S/Hb Haringey. Hb S/Hb Haringey is a newly discovered sickle cell variant which seems to portray a mild/benign clinical phenotype of sickle cell disease.

Brain neurochemical and hemodynamic findings in the NY1DD mouse model of mild sickle cell disease.

To characterize the cerebral profile associated with sickle cell disease (SCD), we used in vivo proton MRI and MRS to quantify hemodynamics and neurochemicals in the thalamus of NY1DD mice, a mild model of SCD, and compared them with wild-type (WT) control mice. Compared with WT mice, NY1DD mice at steady state had elevated cerebral blood flow (CBF) and concentrations of N-acetylaspartate (NAA), glutamate (Glu), alanine, total creatine and N-acetylaspartylglutamate. Concentrations of glutathione (GSH) at steady state showed a negative correlation with BOLD signal change in response to 100% oxygen, a marker for oxidative stress, and mean diffusivity assessed using diffusion-tensor imaging, a marker for edematous inflammation. In NY1DD mice, elevated basal CBF was correlated negatively with [NAA], but positively with concentration of glutamine ([Gln]). Immediately after experimental hypoxia (at reoxygenation after 18 hours of 8% O2 ), concentrations of NAA, Glu, GSH, Gln and taurine (Tau) increased only in NY1DD mice. [NAA], [Glu], [GSH] and [Tau] all returned to baseline levels two weeks after the hypoxic episode. The altered neurochemical profile in the NY1DD mouse model of SCD at steady state and following experimental hypoxia/reoxygenation suggests a state of chronic oxidative stress leading to compensatory cerebral metabolic adjustments.

Plasma expander and blood storage effects on capillary perfusion in transfusion after hemorrhage.

BACKGROUND: Treating hemorrhage with blood transfusions in subjects previously hemodiluted with different colloidal plasma expanders, using fresh autologous blood or blood that has been stored for 2 weeks, allows identifying the interaction between type of plasma expander and differences in blood storage. STUDY DESIGN AND METHODS: Studies used the hamster window chamber model. Fresh autologous plasma, 130-kDa starch-based plasma expander (hydroxyethyl starch [HES]), or 4% polyethylene glycol-conjugated albumin (PEG-Alb) was used for 20% of blood volume (BV) hemodilution. Hemodilution was followed by a 55% by BV 40-minute hemorrhagic shock period, treated with transfusion of fresh or blood that was stored for 2 weeks. Outcome was evaluated 1 hour after blood transfusion in terms of microvascular and systemic variables. RESULTS: Results were principally dependent on the type of colloidal solution used during hemodilution, 4% PEG-Alb yielding the best microvascular recovery evaluated in terms of the functional capillary density. This result was consistent whether fresh blood or stored blood was used in treating the subsequent shock period. Fresh blood results were significantly better in systemic and microvascular terms relative to stored blood. HES and fresh plasma hemodilution yielded less favorable results, a difference that was enhanced when fresh versus stored blood was compared in their efficacy of correcting the subsequent hemorrhage. CONCLUSION: The type of plasma expander used for hemodilution influences the short-term outcome of subsequent volume resuscitation using blood transfusion, 4% PEG-Alb providing the most favorable outcome by comparison to HES or fresh plasma.

PEG-albumin supraplasma expansion is due to increased vessel wall shear stress induced by blood viscosity shear thinning.

We studied the extreme hemodilution to a hematocrit of 11% induced by three plasma expanders: polyethylene glycol (PEG)-conjugated albumin (PEG-Alb), 6% 70-kDa dextran, and 6% 500-kDa dextran. The experimental component of our study relied on microelectrodes and cardiac output to measure both the rheological properties of plasma-expander blood mixtures and nitric oxide (NO) bioavailability in vessel walls. The modeling component consisted of an analysis of the distribution of wall shear stress (WSS) in the microvessels. Our experiments demonstrated that plasma expansion with PEG-Alb caused a state of supraperfusion with cardiac output 40% above baseline, significantly increased NO vessel wall bioavailability, and lowered peripheral vascular resistance. We attributed this behavior to the shear thinning nature of blood and PEG-Alb mixtures. To substantiate this hypothesis, we developed a mathematical model of non-Newtonian blood flow in a vessel. Our model used the Quemada rheological constitutive relationship to express blood viscosity in terms of both hematocrit and shear rate. The model revealed that the net effect of the hemodilution induced by relatively low-viscosity shear thinning PEG-Alb plasma expanders is to reduce overall blood viscosity and to increase the WSS, thus intensifying endothelial NO production. These changes act synergistically, significantly increasing cardiac output and perfusion due to lowered overall peripheral vascular resistance.

Packing density of the PEG-shell in PEG-albumins: PEGylation induced viscosity and COP are inverse correlate of packing density.

PEG-Alb represents a new class of low viscogenic plasma expanders that achieve super perfusion in vivo by mimicking the vasodilatory influence of high viscogenic plasma expanders. PEGylation-engineered structure of PEG albumin can be envisaged as a deformable molecular domain around the rigid central protein core. The correlation between the structure of PEG-shell in terms of packing of the PEG inside the PEG shell and PEGylation induced plasma expander (PE)-like properties of albumin has been investigated as a function of the number and length of the PEG-chain. The increase in molecular radius of albumin on PEGylation is non-linear as a function of the number of PEG chains conjugated. The packing density of PEG within the PEG-shell is an inverse correlate of PEG-chain size; i.e. the shorter chains pack more compactly than the longer ones. The PEGylation induced increase in the viscosity and COP of albumin is an exponential correlation of the number of ethylene oxide units (-CH(2)-CH(2)-O-) conjugated and is also a function of the PEG-chain length. At equivalence of PEG mass conjugated, the viscosity and COP of PEG-albumin adducts correlate inversely with packing density of PEG. All PEGylated albumins are not equivalent on the basis of total PEG mass conjugated. Accordingly, the structure of PEG albumin and its solution properties can be engineered to optimize a given total PEG mass for the application of PEG albumin as a resuscitation fluid. The extension arms minimize the influence of PEG shell on the structure of the protein core. We speculate that EAF-PEGylation is a preferable platform for PEGylation of protein therapeutics and is expected to generate products with better therapeutic efficacy.

Increased inter dimeric interaction of oxy hemoglobin is necessary for attenuation of reductive pegylation promoted dissociation of tetramer.

The propensity of site-specific carboxymethylation and propylation of Val-1(α) of Hb to attenuate the reductive hexaPEGylation-induced dissociation of tetramers has been investigated. Only reductive propylation of Val-1(α), which increases the stability of oxy Hb, attenuates the reductive hexaPEGylation-induced dissociation. Increasing the stability of the oxy conformation of Hb by chemical or genetic approaches is a strategy to generate PEGylated Hbs with native-like tetramer stability using direct PEGylation platforms. This new approach and EAF-PEGylation are the only two alternate PEGylation strategies available to design stable second-generation vasoinactive uncrosslinked PEGylated Hbs with native-like tetramer stability.

Quercetin Completely Ameliorates Hypoxia-Reoxygenation-Induced Pathophysiology Severity in NY1DD Transgenic Sickle Mice: Intrinsic Mild Steady State Pathophysiology of the Disease in NY1DD Is Also Reversed.

The vaso-occlusive crisis (VOC) is a major complication of sickle cell disease (SCD); thus, strategies to ameliorate vaso-occlusive episodes are greatly needed. We evaluated the therapeutic benefits of quercetin in a SCD transgenic sickle mouse model. This disease model exhibited very mild disease pathophysiology in the steady state. The severity of the disease in the NY1DD mouse was amplified by subjecting mice to 18 h of hypoxia followed by 3 h of reoxygenation. Quercetin (200 mg/kg body weight) administered to hypoxia challenged NY1DD mice in a single intraperitoneal (i.p.) dose at the onset of reoxygenation completely ameliorated all hypoxia reoxygenation (H/R)-induced pathophysiology. Additionally, it ameliorated the mild intrinsic steady state pathophysiology. These results are comparable with those seen with semisynthetic supra plasma expanders. In control mice, C57BL/6J, hypoxia reoxygenation-induced vaso-occlusion was at significantly lower levels than in NY1DD mice, reflecting the role of sickle hemoglobin (HbS) in inducing vaso-occlusion; however, the therapeutic benefits from quercetin were significantly muted. We suggest that these findings represent a unique genotype of the NY1DD mice, i.e., the presence of high oxygen affinity red blood cells (RBCs) with chimeric HbS, composed of mouse α-chain and human ßS-chain, as well as human α-chain and mouse ß-chain (besides HbS). The anti-anemia therapeutic benefits from high oxygen affinity RBCs in these mice exert disease severity modifications that synergize with the therapeutic benefits of quercetin. Combining the therapeutic benefits of high oxygen affinity RBCs generated in situ by chemical or genetic manipulation with the therapeutic benefits of antiadhesive therapies is a novel approach to treat sickle cell patients with severe pathophysiology.

INI1/SMARCB1 Rpt1 domain mimics TAR RNA in binding to integrase to facilitate HIV-1 replication.

INI1/SMARCB1 binds to HIV-1 integrase (IN) through its Rpt1 domain and exhibits multifaceted role in HIV-1 replication. Determining the NMR structure of INI1-Rpt1 and modeling its interaction with the IN-C-terminal domain (IN-CTD) reveal that INI1-Rpt1/IN-CTD interface residues overlap with those required for IN/RNA interaction. Mutational analyses validate our model and indicate that the same IN residues are involved in both INI1 and RNA binding. INI1-Rpt1 and TAR RNA compete with each other for IN binding with similar IC50 values. INI1-interaction-defective IN mutant viruses are impaired for incorporation of INI1 into virions and for particle morphogenesis. Computational modeling of IN-CTD/TAR complex indicates that the TAR interface phosphates overlap with negatively charged surface residues of INI1-Rpt1 in three-dimensional space, suggesting that INI1-Rpt1 domain structurally mimics TAR. This possible mimicry between INI1-Rpt1 and TAR explains the mechanism by which INI1/SMARCB1 influences HIV-1 late events and suggests additional strategies to inhibit HIV-1 replication.

PEGylation of Val-1(alpha) destabilizes the tetrameric structure of hemoglobin.

A hexaPEGylated hemoglobin (Hb), (Propyl-PEG5K)(6)-Hb, is essentially in alphabeta dimers (Hu et al. (2007) Biochem. J. 402, 143-151). In order to provide a biochemical insight into the tetramer-dimer dissociation of this PEGylated Hb, we prepared and characterized two PEGylated Hbs site-specifically modified at Val-1(alpha) and at Val-1(beta), respectively. PEGylation at Val-1(alpha) and at Val-1(beta) increase the tetramer-dimer dissociation constant (K(d)) of Hb by 2 and 1 order of magnitude, respectively. Accordingly, the sites of PEGylation can determine the tetramer stability of the PEGylated Hb. In order to determine the role of the polyethylene glycol (PEG) chains on the tetramer stability of Hb, we prepared a propylated Hb site-specifically modified at Val-1(alpha). Interestingly, site-specific propylation of Hb at Val-l(alpha) stabilizes the Hb tetramer by 1 order of magnitude. Therefore, conjugation of the PEG chains at Val-1(alpha) can greatly destabilize the tetramer stability of Hb. On the structural aspects, the PEG chains conjugated at Va-1(alpha) unfavorably alter the heme environment and quaternary structure and destabilize the alpha1beta2 interface of Hb. On the functional aspects, the PEG chains conjugated at Val-1(alpha) decrease the Hill coefficient, the Bohr effect of Hb and the sensitization to the presence of the allosteric effectors. In contrast, PEGylation of Hb at Val-1(beta) gives rise to less pronounced structural alteration and different functional change.

Non-conservative surface decoration of hemoglobin: influence of neutralization of positive charges at PEGylation sites on molecular and functional properties of PEGylated hemoglobin.

High hydrodynamic volume, high viscosity and high colloidal osmotic pressure (COP) of PEGylated hemoglobin (Hb) have been suggested to neutralize the vasoactivity of acellular Hb. Consequences of non-conservative PEGylation (positive charge of the amino groups at the PEGylation sites is neutralized) using succinimidyl-ester of propionic acid PEG5K on the properties of PEGylated Hb have now been investigated. Non-conservative PEGylation of Hb leads to a much higher increase in the COP and viscosity of Hb than conservative extension arm facilitated (EAF) PEGylation of Hb. Introduction of alphaalpha-fumaryl crosslinking decreased the COP of non-conservative PEGylated Hb by stabilization of interdimeric interactions. Compared to the EAF-PEGylated alphaalpha-fumaryl Hb, non-conservative PEGylated product shows a comparable COP and higher viscosity. Conservative PEGylation of alphaalpha-fumaryl Hb by reductive alkylation chemistry does not increase the COP to this level, but enhanced the molecular volume and viscosity comparable to EAF-PEGylated product. Thus, the molecular properties of PEGylated Hb can be fine tuned using different PEGylation platforms and provide a unique opportunity for the design of second generation PEGylated Hbs.

Extension arm facilitated pegylation of alphaalpha-hemoglobin with modifications targeted exclusively to amino groups: functional and structural advantages of free Cys-93(beta) in the PEG-Hb adduct.

Cys-93(beta) of hemoglobin (Hb) was reversibly protected as a mixed disulfide with thiopyridine during extension arm facilitated (EAF) PEGylation and its influence on the structural and functional properties of the EAF-PEG-Hb has been investigated. Avoiding PEGylation of Cys-93(beta) in the EAF-PEG-Hb lowers the level of perturbation of heme pocket, alpha1beta2 interface, autoxidation, heme loss, and the O(2) affinity, as compared to the EAF-PEG-Hb with PEGylation of Cys-93(beta).The structural and functional advantages of reversible protection of Cys-93(beta) during EAF PEGylation of oxy-Hb has been compared with Euro PEG-Hb generated by EAF PEGylation of deoxy Hb where Cys-93(beta) is free in the final product. The alphaalpha-fumaryl cross-linking and EAF PEGylation targeted exclusively to Lys residues has been combined together for generation of second-generation EAF-PEG-Hb with lower oxygen affinity. The PEG chains engineered on Lys as well as PEGylation of Cys-93(beta) independently contribute to the stabilization of oxy conformation of Hb and hence increase the oxygen affinity of Hb. However, oxygen affinity of the EAF-PEG-alphaalpha-Hb is more sensitive to the presence of PEGylation on Cys-93(beta) than that of the EAF-PEG-Hb. The present modified EAF PEGylation platform is expected to facilitate the design of novel versions of the EAF-PEG-Hbs that can now integrate the advantages of avoiding PEGylation of Cys-93(beta).

Induced plasma expander-like properties as a function of PEG-chains on extension arm facilitated PEGylation of albumin: "mushroom to brush-like" conformational transition of the PEG-albumin conjugate.

Plasma expander-like properties of albumin induced on hexa as well as dodecacPEGylation using Extension Arm Facilitated PEGylation platform make it an excellent resuscitation fluid. PEGylation induced changes in the structure, drug binding, and plasma expander-like properties of bovine serum albumin has been now investigated as a function of PEGylation. The molecular volume of albumin increases on PEGylation nearly linearly; in the beginning up to about six PEG chains are conjugated, then plateau off, while the viscosity and colloidal osmotic pressure change very little initially and then increase exponentially as a function of PEG chains conjugated. PEGylation has essentially no influence on the secondary structure or drug properties of albumin. Tryphtophyl fluorescence of albumin is quenched on PEGylation as a direct correlate of the changes in molecular radius of PEG-albumin. It is concluded that hexaPEGylated and dodecaPEGylated albumin belong to two different configurational states of PEG-albumin in terms of packing of PEG-chains on the molecular surface of the protein. The results suggest a transition of PEGylated albumin from the initial mushroom-like conformation to brush conformation as the PEGylation increases. The therapeutic efficacy of the two PEGylated species is needed to establish the optimum level of PEGylation to function as resuscitation fluids.

Semisynthetic supra plasma expanders: a new class of therapeutics to improve microcircualtion in sickle cell anaemia.

Compromised microcirculation and endothelial dysfunction are hallmarks of sickle cell disease (SCD). EAF PEG Haemoglobin (Hb) and EAF PEG Albumin (Alb) represent a novel class of semisynthetic colloidal supra plasma expanders that improve microcirculation. The therapeutic activity of supra plasma expanders to attenuate vaso-occlusion and restore the haemodynamic functions in patients with SCD has been investigated using NY1DD, a transgenic mouse model of mild SCD without anaemia. Vaso-occlusion and perturbation of haemodynamics are amplified in NY1DD by hypoxia-reoxygenation protocol. EAF P5K6 Alb and Alb T12 (Alb conjugated with 12 copies of antioxidant tempo) attenuate vaso-occlusion when infused at the start of reoxygenation. However, only EAF PEG Alb restores haemodynamics close to levels in control C57BL. EAF P5K6 Alb-T12, active plasma expander conjugated with antioxidant, completely clears vaso-occlusion and restores normal haemodynamics. EAF PEG Hb also completely clears vaso-occlusion and restores normal haemodynamics. Pretreating NY1DD with EAF PEG Hb protects it from hypoxia reoxygenation-induced damages. EAF P5K6 Alb T12 attenuates the endothelial dysfunction in S + S Antilles mice as reflected by the vasodilatory response of its arteries and arterioles to vaso-dilators. Active plasma expanders are novel therapeutics to restore normal haemodynamics in SCD patients to improve tissue oxygenation during episodes of painful vaso-occlusive crisis. Abbreviations: 2-IT: 2-immothiolane; Mal-T: 4-Maleimido tempo; Alb: human serum albumin (HSA); Alb-T12: human albumin conjugated with 12 copies of tempo; EAF: extension arm facilitated; EAF PEG Hb: extension arm facilitated PEGylated haemoglobin; EAF PEG Alb: extension arm facilitated PEGylated albumin; EAF P3K6 Hb: extension arm facilitated PEGylated haemoglobin conjugated with 6 copies of PEG3K; EAF P5K6 Alb T12: extension arm facilitated PEGylated albumin conjugated with 6 copies of PEG5K and 12 copies of tempo; Hb: haemoglobin; HAS: human serum albumin (Alb); PEG: polyethylene glycol; MP4: MalPEG Hb, is formulated at 4.2 g/dL in lactated Ringer's solution, a product of Sangart; SCD: sickle cell disease; NO: nitric oxide; SEC: size exclusive chromatography; Vrbc: red cell velocity; Q: volumetric flow rates, Q; SNP: sodium nitroprusside.

Autoxidation of the site-specifically PEGylated hemoglobins: role of the PEG chains and the sites of PEGylation in the autoxidation.

The PEGylated hemoglobin (Hb) has been evaluated as a potential blood substitute. In an attempt to understand the autoxidation of the PEGylated Hb, we have studied the autoxidation of the PEGylated Hb site-specifically modified at Cys-93(beta) or at Val-1(beta). PEGylation of Hb at Cys-93(beta) perturbed the heme environment and increased the autoxidation rate of Hb, which is at a higher level than that caused by PEGylation at Val-1(beta). The perturbation of the heme environment of Hb is attributed to the maleimide modification at Cys-93(beta) and not due to conjugation of the PEG chains. However, the PEG chains enhance the autoxidation and the H 2O 2 mediated oxidation of Hb. Accordingly, the PEG chains are assumed to increase the water molecules in the hydration layer of Hb and enhance the autoxidation by promoting the nucleophilic attack of heme. The autoxidation rate of the PEGylated Hb does not show an inverse correlation with the oxygen affinity. The H 2O 2 mediated structural loss and the heme loss of Hb are increased by maleimide modification at Cys-93(beta) and further decreased by conjugation of the PEG chains. The autoxidation of the PEGylated Hbs is attenuated significantly in the plasma, possibly due to the presence of the antioxidant species in the plasma. This result is consistent with the recent suggestion that there is no direct correlation between the in vitro and in vivo autoxidation of the PEGylated Hb. Therefore, the pattern of PEGylation can be manipulated for the design of the PEGylated Hb with minimal autoxidation.

PEGylation of human serum albumin: reaction of PEG-phenyl-isothiocyanate with protein.

Successful and cost-effective PEGylation protocols require pure functionalized PEG reagents, which can be synthesized by simple and efficient procedures, exhibit high stability against hydrolysis, and maintain a level of reactivity with protein functional groups under mild reaction conditions. PEG-phenyl-isothiocyanate (PIT-PEG) is a new functionalized PEG having these characteristics, and has been synthesized by condensation of the bifunctional reagent 4-isothiocyanato phenyl isocyanate with monomethoxy PEG (mPEG). The data of (1)H NMR and colormetric analysis of the new PEG reagent establish that the mPEG has been quantitatively functionalized. The t 1/4 values for the hydrolysis of PIT-PEG5K in 100 mM phosphate solution at pH 6.5 and 9.2 are about 95 and 40 h, respectively. Incubation of human serum albumin (HSA, 0.5 mM) with a 10-fold molar excess of PIT-PEG (3K or 5K) at pH 6.5 and 9.2 generated PEG-HSA conjugates with average of 3.5 and 6.0 PEG chains per HSA molecule, respectively. The circular dichroism spectra of the conjugates showed that PEGylation of HSA has little influence on the secondary structure of HSA. The hexaPEGylated HSA, (TCP-PEG5K) 6-HSA, exhibited very high hydrodynamic volume, and the molecular radius of HSA increased from 3.95 to 6.57 nm on hexaPEGylation. The hexaPEGylation also increased the viscosity of 4% HSA from 1.05 to 2.10 cP, and the colloid osmotic pressure from 15.2 to 48.0 mmHg. The large increase in the hydrodynamic volume and the solution properties of (TCP-PEG5K) 6-HSA suggest that it could be a potential candidate as a plasma volume expander. PIT-PEG is a useful addition to the spectrum of functionalized PEG reagents available for surface decoration of proteins with PEG.

Analysis of functionalization of methoxy-PEG as maleimide-PEG.

The design of the extension arm-facilitated PEGylation (EAFP) of proteins takes advantage of the high selective and quantitative aspects of the thiol-maleimide reaction. However, the efficiency of EAFP with hemoglobin varied with the batches of maleimide-PEG. The low level of functionalization of monomethoxy-PEG (mPEG) as maleimide-PEG has been now investigated as the potential source of this variation. New chemical approaches for the estimation of the functionalization of mPEG using the reaction of the thiol groups of glutathione, dithiothreitol, and hemoglobin with maleimide-PEG have been developed. The single-step modular approach to the synthesis of maleimidophenyl-PEG (MPPEG) that involved the condensation of p-maleimidophenyl isocyanate with mPEG has been optimized to generate a product with an overall purity of 80%. The NMR approach correlates well with the estimates made by the new chemical approaches. Commercial maleimide-PEG reagents synthesized using multiple steps exhibited a lower level of functionalization as reflected by these chemical estimations. The better functionalization of MPPEG increases the efficiency of EAFP as reflected by the generation of hexaPEGylated Hb and the masking of the D antigen of RBCs. This new EAFP protocol is expected to improve the cost effectiveness of the generation of hexaPEGylated Hb, PEGylated albumin, and PEGylated RBCs as new PEGylated therapeutics.

Volume resuscitation from hemorrhagic shock with albumin and hexaPEGylated human serum albumin.

The effect of restoring intravascular volume with polyethylene glycol (PEG) conjugated to human serum albumin (PEG-Alb) on systemic parameters and microvascular hemodynamics after hemorrhagic shock resuscitation was studied in the hamster window chamber model. Moderate hemorrhagic shock was induced by controlled arterial bleeding of 50% of blood volume, and hypovolemia was maintained for 1h. Fluid resuscitation was accomplished by infusion of 25% of blood volume and recovery was followed over 90 min. The PEG-Alb (six chains of maleimide phenyl PEG conjugated human serum albumin at 4%) resuscitation group was compared human serum albumin (HSA) at 5% (HSA5) and 10% (HSA10) protein concentrations. Systemic parameters, microvascular perfusion and capillary perfusion (functional capillary density, FCD) were measured by noninvasive methods. Hyperoncotic solutions provided rapid restoration of blood pressure, blood gas parameters and microvascular perfusion. Systemic and microvascular recovery was best and most rapid with PEG-Alb and followed by HSA10 and HSA5. Only recovery with PEG-Alb was sustained beyond 90 min. Hemodynamic functional benefits of PEG-Alb and the potential disadvantages associated with HSA, suggest PEG-Alb as better resuscitation solution.

Survival time in severe hemorrhagic shock after perioperative hemodilution is longer with PEG-conjugated human serum albumin than with HES 130/0.4: a microvascular perspective.

INTRODUCTION: Preoperative hemodilution is an established practice that is applied to reduce surgical blood loss. It has been proposed that polyethylene glycol (PEG) surface decorated proteins such as PEG-conjugated human serum albumin may be used as non-oxygen-carrying plasma expanders. The purpose of this study was to determine whether there is any difference in survival time after severe hemorrhagic shock following extreme hemodilution using a conventional hydroxyethyl starch (HES)-based plasma expander or PEG-albumin. METHODS: Experiments were performed using the hamster skinfold window preparation. Human serum albumin that was surface decorated with PEG was compared with Voluven 6% (Fresenius Kabi, Austria; a starch solution that is of low molecular weight and has a low degree of substitution; HES). These plasma expanders were used for a 50% (blood volume) exchange transfusion to simulate preoperative hemodilution. Exchange transfusion was followed by a 60% (blood volume) hemorrhage to reproduce a severe surgical bleed over a 1 hour period. Observation of the animal was continued for another hour during the shock phase. RESULTS: The PEG-albumin group exhibited significantly greater survival rate than did the HES group, in which none of the animals survived the hemorrhage phase of the experiment. Among the treatment groups there were no changes in mean arterial pressure and heart rate from baseline after hemodilution. Both groups experienced gradual increases in arterial oxygen tension and disturbance in acid-base balance, but this response was more pronounced in the HES group during the shock period. Mean arterial pressure remained elevated after the initial hemorrhage period in the PEG-albumin group but not in the HES group. Maintenance of a greater mean arterial pressure during the initial stages of hemorrhage is proposed to be in part due to the improved volume expansion with PEG-albumin, as indicated by the significant decrease in systemic hematocrit compared with the HES group. PEG-albumin treatment yielded higher functional capillary density during the initial stages of hemorrhage as compared with HES treatment. CONCLUSION: The ability of PEG-albumin to prolong maintenance of microvascular function better than HES is a finding that would be significant in a clinical setting involving preoperative blood management and extreme blood loss.

Influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated haemoglobin.

The influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated {PEG [poly(ethylene glycol)]-conjugated} haemoglobin has been investigated. The sites and the extent of PEGylation of haemoglobin by reductive alkylation are not influenced by the presence of an alphaalpha-fumaryl cross-link at Lys-99(alpha). The propylated hexaPEGylated cross-linked haemoglobin, (propyl-PEG5K)(6)-alphaalpha-Hb, exhibits a larger molecular radius and lower colloidal osmotic pressure than propylated hexaPEGylated non-cross-linked haemoglobin, (propyl-PEG5K)(6)-Hb. Perturbation of the haem microenvironment and the alpha1beta2 interface by PEGylation of haemoglobin is reduced by intramolecular cross-linking. Sedimentation velocity analysis established that PEGylation destabilizes the tetrameric structure of haemoglobin. (Propyl-PEG5K)(6)-Hb and (propyl-PEG5K)(6)-alphaalpha-Hb sediment as stable dimeric and tetrameric molecules, respectively. The betabeta-succinimidophenyl PEG-2000 cross-link at Cys-93(beta) outside the central cavity also influences the molecular properties of haemoglobin, comparable to that by the alphaalpha-fumaryl cross-link within the central cavity. However, the influence of the two cross-links on the oxygen affinity of PEGylated haemoglobin are very distinct, indicating that the high oxygen affinity of PEGylated haemoglobin is not a direct consequence of the dissociation of the haemoglobin tetramers into dimers. alphaalpha-Fumaryl cross-linking is preferred to modulate both oxygen affinity and molecular properties of PEGylated haemoglobin, and cross-linking outside the central cavity could only modulate molecular properties of PEGylated haemoglobin. It is suggested that PEGylation induces a hydrodynamic drag on haemoglobin and this plays a role in the microcirculatory properties of PEGylated haemoglobin.

Non-hypertensive tetraPEGylated canine haemoglobin: correlation between PEGylation, O2 affinity and tissue oxygenation.

TetraPEGylated canine Hb, [SP (succinimidophenyl)-PEG5K]4-canine-Hb, with PEGylation at its four reactive cysteine residues (a111 and b93) has been prepared and characterized. The hydrodynamic volume and the molecular radius of (SP-PEG5K)4-canine-Hb are intermediate to those of di- and hexaPEGylated human Hb as expected. However, the COP (colloidal osmotic pressure) of tetraPEGylated canine Hb is closer to that of hexaPEGylated human Hb than to that of diPEGylated human Hb. The O2 affinity of tetraPEGylated canine Hb is higher than that of canine Hb and comparable with that of hexaPEGylated Hb. The O2 affinity of tetraPEGylated canine Hb is not responsive to the presence of DPG (diphosphoglycerate) or chloride, but it retains almost full response to L-35, an allosteric effector that interacts at the aa-end of the central cavity. The tetraPEGylated canine Hb is vasoinactive in hamster in 10% top load infusion studies. It is also essentially non-hypertensive in an extreme exchange haemodilution protocol in hamster just as di- and hexaPEGylated human Hb. The O2 delivery by tetraPEGylated canine Hb is comparable with that of hexaPEGylated Hb but not as efficient as diPEGylated Hb. These results demonstrate that PEGylation-induced solution properties of PEG [poly(ethylene glycol)]-Hb conjugates are dictated by the level and chemistry of PEGylation and the interplay of these plays a critical role in tissue oxygenation. The studies imply the need to establish the right level (and/or pattern) of PEGylation and O2 affinity of Hb-PEG adducts in designing O2-carrying plasma volume expanders, and this remains the primary challenge in the design of PEGylated Hb as blood substitutes.