Dissociation of local nitric oxide concentration and vasoconstriction in the presence of cell-free hemoglobin oxygen carriers

Cell-free hemoglobin's (CFH) high affinity for nitric oxide (NO) could limit CFH's use as an oxygen-carrying blood replacement fluid because it scavenges NO, causing vasoconstriction and hypertension. However, the extent to which perivascular NO levels change following intravascular administration of hemoglobin (Hb) with different molecular dimensions correlates with vasoconstrictive responses in the microcirculation is unknown. The study objective was to determine vasoconstrictive effects following bolus infusions of (1) αα cross-linked Hb; (2) polymerized bovine Hb; or (3) polyethylene glycol-decorated Hb (PEG-Hb), by measurements of in vivo microvessel diameter, blood flow, perivascular NO concentration, and systemic hemodynamic parameters. All CFHs caused reductions in perivascular NO levels, not correlated to microvascular responses. PEG-Hb (largest molecular volume) maintained blood flow, while the others caused vasoconstriction and reduced perfusion. All solutions increased mean arterial pressure due to vasoconstriction and blood volume expansion, except for PEG-Hb, which increased blood pressure due to blood volume expansion and maintenance of cardiac output. In conclusion, perivascular NO reduction is similar for all Hb solutions because NO binding affinities are similar; however, effects on vascular resistance are related to the type of molecular modification, molecular volume, and oxygen affinity

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 αα-fumaryl cross-link at Lys-99(α). The propylated hexaPEGylated cross-linked haemoglobin, (propyl-PEG5K)(6)-αα-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 α1β2 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)-αα-Hb sediment as stable dimeric and tetrameric molecules, respectively. The ββ-succinimidophenyl PEG-2000 cross-link at Cys-93(β) outside the central cavity also influences the molecular properties of haemoglobin, comparable to that by the αα-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. αα-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