MP4 appears to be free of a pressor effect, a major limitation to the development of a safe and effective RBC substitutes in the past.

We investigated the effect of nitric oxide (NO) on the induction of the stress protein heme oxygenase and its protective role in vascular endothelial cells exposed to hydrogen peroxide. Treatment of porcine aortic endothelial cells for 6 h with the NO-releasing compounds (0.1-1 mM) sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosydnonimine (SIN-1) resulted in a concentration-dependent increase in heme oxygenase activity. At 1 mM, the activity of heme oxygenase was augmented 8.5-fold with SNP, 5.8-fold with SNAP, and 5.7-fold with SIN-1 over the control value. In contrast, endothelial cells exposed to 100 microM S-bromoguanosine 3',5'-cyclic monophosphate, a tissue-permeable analogue that mimics the action of guanosine 3',5'-cyclic monophosphate, did not show any change in heme oxygenase activity. Activation of the inducible NO synthase by the synergistic action of bacterial lipopolysaccharide (250 ng/ml) and interferon-gamma (100 U/ml) also increased endothelial heme oxygenase activity by 3.2-fold (P < 0.05 vs control). Methylene blue (1 microM), an inhibitor of both NO synthase and guanylate cyclase activities, completely abolished this effect. Cells previously exposed to SNAP and SIN-1 exhibited a significant protection against the cytotoxicity mediated by hydrogen peroxide (250 microM) (P < 0.05). Conversely, SNP did not show any protective effects, possibly because of catalytic iron released during its chemical decomposition. In fact, the iron chelator deferoxamine (5 mM) completely suppressed the SNP-mediated cytotoxicity and partially attenuated the activity of heme oxygenase to a level equal to that mediated by SIN-1 and SNAP. These results indicate that NO is a determinant in the modulation of the activity of heme oxygenase leading to a major resistance of the endothelium to oxidative stress.

Changes in mean arterial pressure were monitored in rats following 50% isovolemic exchange transfusion with solutions of chemically modified hemoglobins. Blood pressure responses fall into three categories: 1) an immediate and sustained increase, 2) an immediate yet transient increase, or 3) no significant change either during or subsequent to exchange transfusion. The reactivities of these hemoglobins with nitric monoxide ( ⅐ NO) were measured to test the hypothesis that different blood pressure responses to these solutions result from differences in ⅐ NO scavenging reactions. All hemoglobins studied exhibited a value of 30 M ؊1 s ؊1 for both ⅐ NO bimolecular association rate constants and the rate constants for ⅐ NO-induced oxidation in vitro. Only the ⅐ NO dissociation rate constants and, thus, the equilibrium dissociation constants varied. Values of equilibrium dissociation constants ranged from 2 to 14 pM and varied inversely with vasopressor response. Hemoglobin solutions that exhibited either transient or no significant increase in blood pressure showed tighter ⅐ NO binding affinities than hemoglobin solutions that exhibited sustained increases. These results suggest that blood pressure increases observed upon exchange transfusion with cell-free hemoglobin solutions can not be the result of ⅐ NO scavenging reactions at the heme, but rather must be due to alternative physiologic mechanisms.Control of blood pressure and resistance to blood flow is achieved by a dynamic constriction and relaxation of smooth muscle tissue which surrounds all blood vessels except capillaries. Vascular smooth muscle tension is continually adjusted by a complex system that causes either vasoconstriction or vasodilation, depending on metabolic need (1). Research performed over the last decade has established that endotheliumderived nitric oxide ( ⅐ NO) 1 can cause vasodilation. ⅐ NO is produced by endothelial cells that lie between the intravascular space and the surrounding smooth muscle. Among the findings was the demonstration that ⅐ NO donors (e.g. nitroprusside, nitroglycerin) lead to vasorelaxation through activation of guanylate cyclase, whereas inhibitors of ⅐ NO synthesis (e.g. N Gmonomethyl-L-arginine) or scavengers (e.g. hemoglobin) cause vasoconstriction (for reviews, see Refs. 2 and 3).Since cell-free hemoglobin is being developed as a red cell substitute (4), reactions between hemoglobin and ⅐ NO are of potential importance in maintenance of microvascular blood flow and O 2 delivery. Despite the wide variation that exists in the physical properties (O 2 affinity, molecular mass, and solution properties) of different cell-free hemoglobins, it appears vasoconstriction is a feature common to many hemoglobin solutions (for reviews, see Refs. 2, 3, and 5). It is tempting to conclude that ⅐ NO scavenging is the principal, if not sole mechanism for vasoconstriction associated with cell-free hemoglobin. However, it is well established that multiple factors contribute to the physiological control of vascular smooth muscle to...

Increasing the molecular size of acellular hemoglobin (Hb) has been proposed as an approach to reduce its undesirable vasoactive properties. The finding that bovine Hb surface decorated with about 10 copies of PEG5K per tetramer is vasoactive provides support for this concept. The PEGylated bovine Hb has a strikingly larger molecular radius than HbA (1). The colligative properties of the PEGylated bovine Hb are distinct from those of HbA and even polymerized Hb, suggesting a role for the colligative properties of PEGylated Hb in neutralizing the vasoactivity of acellular Hb. To correlate the colligative properties of surface-decorated Hb with the mass of the PEG attached and also its vasoactivity, we have developed a new maleimide-based protocol for the site-specific conjugation of PEG to Hb, taking advantage of the unusually high reactivity of Cys-93(beta) of oxy HbA and the high reactivity of the maleimide to protein thiols. PEG chains of 5, 10, and 20 kDa have been functionalized at one of their hydroxyl groups with a maleidophenyl moiety through a carbamate linkage and used to conjugate the PEG chains at the beta-93 Cys of HbA to generate PEGylated Hbs carrying two copies of PEG (of varying chain length) per tetramer. Homogeneous preparations of (SP-PEG5K)(2)-HbA, (SP-PEG10K)(2)-HbA, and (SP-PEG20K)(2)-HbA have been isolated by ion exchange chromatography. The oxygen affinity of Hb is increased slightly on PEGylation, but the length of the PEG-chain had very little additional influence on the O(2) affinity. Both the hydrodynamic volume and the molecular radius of the Hb increased on surface decoration with PEG and exhibited a linear correlation with the mass of the PEG chain attached. On the other hand, both the viscosity and the colloidal osmotic pressure (COP) of the PEGylated Hbs exhibited an exponential increase with the increase in PEG chain length. In contrast to the molecular volume, viscosity, and COP, the vasoactivity of the PEGylated Hbs was not a direct correlate of the PEG chain length. There appeared to be a threshold for the PEG chain length beyond which the protection against vasoactivity is decreased. These results suggest that the modulation of the vasoactivity of Hb by PEG could be a function of the surface shielding afforded by the PEG, the latter being a function of the disposition of the PEG chain on the protein surface, which in turn is a function of the length of the PEG chain. Thus, the biochemically homogeneous PEGylated Hbs described in the present study, surface-decorated with PEG chains of appropriate size, could serve as potential candidates for Hb-based oxygen carriers.