Apohemoglobin Haptoglobin Complexes Attenuate the Hypertensive Response to Low Molecular Weight Polymerized Hemoglobin

Background: The presence of low molecular weight (MW) hemoglobin (Hb) species in previous generations of Hb-based oxygen (O₂) carriers (HBOCs) restricted their application as red blood cell (RBC) substitutes. Among the myriad of strategies to produce Hb, using glutaraldehyde crosslinking to form polymerized Hb (PolyHb) remains popular due to its low cost and excellent scalability. However, the commercially developed PolyHbs, HBOC-201® (Biopure Corp, Cambridge, MA, USA) and PolyHeme® (Northfield Laboratories Inc., Northfield, IL, USA), primarily contained fractions of material at or below 250 kDa in MW. These low MW PolyHb and unpolymerized Hb are able to extravasate into the interstitial space where they scavenge nitric oxide (NO), resulting in vasoconstriction, systemic hypertension and oxidative tissue injury. Recent strategies to mitigate hypertension have used adenosine and nitroglycerine to attenuate the hypertensive response of HBOC-201®. While effective at controlling systemic hypertension, administration of adenosine and nitroglycerine must be carefully controlled to prevent hypotension and cannot address HBOC extravasation into the tissue space. Instead, we propose that employing the naturally occurring mechanism of Hb detoxification is a potential strategy to mitigate systemice hypertension resulting from administration of low MW PolyHb. The naturally occurring Hb scavenging protein, haptoglobin (Hp), plays a pivotal role in detoxifying stroma free Hb in the blood. Recently, we determined that Hp preferentially binds to low MW PolyHb (MW < 256 kDa). By binding to Hp, the molecular diameter of low MW PolyhHb is effectively increased. This increase in size may reduce tissue extravasation and subsequent NO scavenging of PolyHb.

Methodology: In this study, we synthesized a low MW PolyHb. The hydrodynamic diameter, O₂ affinity, and size distribution were measured. The Hp used for this study was purified from human Cohn Fraction IV derived from pooled human plasma. The resulting Hp contained both Hp2-1 and Hp2-2 phenotypes. Hp binding to PolyHb was confirmed in vitro with stop-flow fluorescence spectrometry and size exclusion chromatography. Hp was stabilized by binding it to heme-free Hb (apoHb) in the form of an apoHb-Hp complex. Systemic parameters, including heart rate (HR) and mean arterial pressure (MAP), were used as indicators of reduced hypertension. Additionally, intravital microscopy was used to examine how co-administration of apoHb-Hp influences functional capillary density (FCD), vascular tone, and blood flow.

Results and Conclusions: Polymerization of Hb resulted in decreased O₂ affinity and increased MW. PolyHb contained a significant fraction of low MW species (< 250 kDa). Hp was able to bind low MW PolyHb. Hp binding resulted in a significant increase in the average size of PolyHb. In the apoHp-Hb group, there was negligible change in MAP, FCD, HR, and microhemodynamics compared to baseline conditions. When compared to the systemic and microcirculatory changes observed in the apoHb and saline groups, the relatively small changes in the apoHb-Hp group indicate that Hp-based species may serve as materials to counteract the pressor effects of low MW HBOCs that are capable of binding to Hp. Thus, the administration of a Hb scavenging apoHb-Hp solution maintained physiologic hemodynamics during the transfusion of PolyHb.