Effects of the molecular mass of tense-state polymerized bovine hemoglobin on blood pressure and vasoconstriction

Abstract

Despite recent advances in the design of hemoglobin (Hb)-based oxygen carriers (HBOCs), vasoconstriction, presumably caused by nitric oxide (NO) scavenging, vessel wall hyperoxygenation, and/or extravasation, has been identified as the principal road block hampering commercial development of HBOCs. This study was designed to analyze systemic and microvascular responses to the molecular mass and plasma concentration of tense (T)-state polymerized bovine Hb (PolybHb) solutions. Experiments were performed using the hamster window chamber model subjected to successive hypervolemic infusions of T-state PolybHb solutions. PolybHb plasma concentrations were evaluated, namely, 0.5, 1.0 and 1.5 g/dl, respectively. Infusion of PolybHb solutions with molecular mass >500 kDa elicited hypertension and vasoconstriction proportional to the plasma concentration and inversely proportional to the PolybHb cross-link density. However, two high-molecular mass PolybHb solutions, PolybHb(40:1)(high) PolybHb(50:1)(high), did not elicit vasoconstriction at all concentrations studied, whereas PolybHb(50:1)(high) only elicited moderate hypertension at the highest concentration studied. In contrast, infusion of PolybHb solutions with molecular mass <500 kDa elicited significant hypertension and vasoconstriction compared with PolybHb solutions with molecular mass >500 kDa that was proportional to the plasma concentration and inversely proportional to the PolybHb cross-link density. We present promising results for highly cross-linked T-state PolybHb solutions with molecular mass >500 kDa [PolybHb(40:1)(high) PolybHb(50:1)(high)], which supports the concept that HBOC size/molecular mass influences its proximity to the vascular endothelium and molecular diffusivity. The hemodynamics of HBOC within the plasma layer surrounding the abluminal side endothelium regulates NO production and consumption, vessel oxygen flux, and extravasation. Although mechanistically attractive, neither of these hypotheses can be directly tested in vivo and will require further investigation.

Fig. 1.

Molecular mass distribution (A) and SDS-PAGE (B) of low- and high-molecular-mass T-state polymerized bovine Hb (PolybHb) fractions. A: molecular mass distribution of low- and high-molecular-mass PolybHb fractions. Dotted lines represent PolybHb fractions <500 kDa in molecular mass; solid lines denote PolybHb fractions >500 kDa in molecular mass. B: SDS-PAGE of fractionated PolybHb solutions. The first and last lanes represent protein molecular mass markers, and eight sample lanes were loaded with PolybHb. The left lanes were loaded with PolybHb fractions <500 kDa in molecular mass, whereas the right lanes were loaded with PolybHb fractions >500 kDa in molecular mass. Cross-link densities ranged from 20:1 to 50:1.

Fig. 2.

Relative changes in mean arterial pressure (MAP) from baseline after infusion of PolybHb. A: PolybHb fractions with molecular mass <500 kDa. Baseline MAP values (mmHg) for each group were as follows: PolybHb(20:1), 110 ± 9; PolybHb(30:1), 113 ± 7; PolybHb(40:1), 107 ± 8; PolybHb(50:1), 111 ± 9. B: PolybHb fractions with molecular mass >500 kDa. Baseline MAP values (mmHg) for each group were as follows: PolybHb(20:1), 107 ± 8; PolybHb(30:1), 110 ± 8; PolybHb(40:1), 108 ± 10; PolybHb(50:1), 112 ± 8. BL, baseline level (broken line). Values are means ± SD. †P < 0.05 relative to baseline. #P < 0.05 compared with PolybHb(50:1).

Fig. 3.

Relative changes from baseline in heart rate (HR) after infusion of PolybHb. A: PolybHb fractions with molecular mass <500 kDa. Baseline HR values (beats/min) for each group were as follows: PolybHb(20:1), 424 ± 22; PolybHb(30:1), 427 ± 18; PolybHb(40:1), 431 ± 23; PolybHb(50:1), 423 ± 19. B: PolybHb fractions with molecular mass >500 kDa. Baseline HR values (beats/min) for each group were as follows: PolybHb(20:1), 430 ± 24; PolybHb(30:1), 437 ± 25; PolybHb(40:1), 422 ± 24; PolybHb(50:1), 440 ± 24. Broken line represents baseline level. Values are means ± SD. †P < 0.05 relative to baseline. #P < 0.05 compared with PolybHb(50:1).

Fig. 4.

Relative changes from baseline in arteriolar diameter after infusion of PolybHb. A: PolybHb fractions with molecular mass <500 kDa. Baseline arteriolar diameters (μm) for each group were as follows: PolybHb(20:1), 68 ± 12 (n = 28); PolybHb(30:1), 66 ± 10 (n = 31); PolybHb(40:1), 65 ± 11 (n = 26); PolybHb(50:1), 64 ± 8 (n = 25). B: PolybHb fractions with molecular mass >500 kDa. Baseline arteriolar diameters (μm) for each group were as follows: PolybHb(20:1), 67 ± 9 (n = 26); PolybHb(30:1), 68 ± 10 (n = 24); PolybHb(40:1), 64 ± 11 (n = 28); PolybHb(50:1), 66 ± 7 (n = 23). Broken line represents baseline level. Values are means ± SD; n = no. of examined vessels. †P < 0.05 relative to baseline. #P < 0.05 compared with PolybHb(50:1).

Fig. 5.

Relative changes from baseline in arteriolar blood flow after infusion of PolybHb. A: PolybHb fractions with molecular mass <500 kDa. Baseline arteriolar blood flow values (nl/s) for each group were as follows: PolybHb(20:1), 8.9 ± 3.2; PolybHb(30:1), 8.2 ± 3.8; PolybHb(40:1), 8.0 ± 4.2; PolybHb(50:1), 7.7 ± 2.9. B: PolybHb fractions with molecular mass >500 kDa. Baseline arteriolar blood flow values (nl/s) for each group were as follows: PolybHb(20:1), 8.5 ± 3.9; PolybHb(30:1), 8.5 ± 4.5; PolybHb(40:1), 7.1 ± 3.6; PolybHb(50:1), 8.3 ± 3.4. Broken line represents baseline level. Values are means ± SD. †P < 0.05 relative to baseline. #P < 0.05 compared with PolybHb(50:1).

Fig. 6.

Relative changes from baseline in functional capillary density (FCD) after infusion of PolybHb. A: PolybHb fractions with molecular mass <500 kDa. Baseline FCD values (cm⁻¹) for each group were as follows: PolybHb(20:1), 112 ± 10; PolybHb(30:1), 104 ± 7; PolybHb(40:1), 98 ± 8; PolybHb(50:1), 102 ± 11. B: PolybHb fractions with molecular mass >500 kDa. Baseline FCD values (cm⁻¹) for each group were as follows: PolybHb(20:1), 102 ± 11; PolybHb(30:1), 112 ± 9; PolybHb(40:1), 116 ± 11; PolybHb(50:1), 109 ± 14. Broken line represents the baseline level. †P < 0.05 relative to baseline. #P < 0.05 compared with PolybHb(50:1).

Fig. 7.

Relative changes from baseline in calculated peripheral vascular resistance (PVR) after infusion of PolybHb. A: PolybHb fractions with molecular mass <500 kDa. B: PolybHb fractions with molecular mass >500 kDa. Broken line represents baseline level. †P < 0.05 relative to baseline. #P < 0.05 compared with PolybHb(50:1).