Evaluation of Invertebrate Hemoglobins As Novel Blood Substitutes

While donated blood is the preferred treatment for hemorrhagic shock, it has a relatively short shelf life (42 days) and it must be constantly refrigerated at 4^oC. Consequently, donated blood is often unavailable in remote areas where it is needed most (e.g. military battlefields). One way to address this problem is by developing “blood substitutes” that mimic the most important functions of blood (e.g. O₂ transport) but are much more stable than red blood cells. Most previous blood substitutes have been based on human or bovine hemoglobin, since hemoglobin is an excellent O₂ transporter and it can easily be modified to increase its stability. Unfortunately, while these hemoglobin-based oxygen carriers (HBOCs) showed early promise, they all eventually failed clinical trials due to an increased risk of severe adverse events including hypertension, stroke, and heart attack. We suggest that all of these problems can be attributed to removing the naturally intracellular human hemoglobin from the protective environment of the red blood cell and exposing it to the harsh conditions in the bloodstream. Therefore, our hypothesis is that the naturally extracellular hemoglobins (also known as erythrocruorins or Ec’s) found in some invertebrates (e.g. earthworms) may be much better blood substitutes. In this study, we have purified and compared Ec’s from a variety of organisms, including worms (e.g. Lumbricus terrestris), leeches (e.g. Nephelopsis obscura), clams (e.g. Astarte castanea), and a snail (Biomphalaria glabrata). Our results show that these erythrocruorin-based O₂ carriers (EBOCs) are highly stable (T_m = 50-60^oC), resistant to oxidation during storage and in vivo, and possess a wide range of O₂ transport properties (e.g. P₅₀ = 2-20 mm Hg at 25^oC). In addition, we have also covalently modified earthworm hemoglobin in two ways: (1) glutaraldehyde cross-linking and (2) conjugation to polyacrylic acid. The cross-linked hemoglobin is highly stable (T_m = 70^oC), while the PAA-conjugated hemoglobin is actually able to resist denaturation in the autoclave at 121^oC for 30 minutes. Finally, preliminary animal studies by our group and others also show that Ecs can be transfused into mice, rats, and hamsters without any of the harmful side effects observed with previous HBOCs. Overall, these results suggest that EBOCs may be safe and effective blood substitutes that are stable enough to be deployed in areas where donated blood is unavailable.