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  5. Evaluating the Safety and Efficacy of Hemoglobin-based Blood Substitutes
  1. Biologics Research Projects

Evaluating the Safety and Efficacy of Hemoglobin-based Blood Substitutes

Principal Investigator: A.I. Alayash, PhD
Office / Division / Lab: OBRR / DBCD / LBVB

General Overview

The development of a safe and effective blood substitute would greatly improve the emergency treatment of accident victims and wounded soldiers, as well as patients undergoing cardiac surgery, especially when whole blood is in short supply.

One type of artificial blood substitute that scientists have studied extensively is called a "hemoglobin-based oxygen carrier" (HBOC). HBOCs use the natural oxygen-carrying molecule called hemoglobin (Hb) to carry oxygen throughout the body. However, because the Hb used for HBOCs is not inside red blood cells it tends to accumulate to toxic levels in the blood. This cell free Hb can cause high blood pressure; Hb can also escape the blood vessels and damage the kidneys and other organs. Therefore, FDA has not approved any HBOCs for use in the United States, and the regulatory agencies of most other countries also have not approved HBOCs.

Our laboratory is trying to overcome the problem of Hb toxicity in order to enable industry to manufacture safe and effective HBOCs. We are studying how free Hb causes its toxic effects and are developing ways to prevent these effects.

We previously published results of a study in animal models showing that a molecule called haptoglobin found in blood can bind to Hb, preventing it from causing damage. Haptoglobin had this beneficial effect either when it was directly infused into animals or when animals were treated with a drug that increased the body's production of haptoglobin. These findings suggest that it might be possible to design safer HBOCs, since Hb bound to haptoglobin can still carry oxygen and release it to tissues.

Our work is contributing to the regulatory and research efforts of CBER to support development of safe and effective products that improve public health in the US and worldwide.

Scientific Overview

HBOCs have many potential advantages over human blood, including availability compatibility, and long-term storage. However, they also raise a number of concerns, including toxicity. Several major manufacturers recently terminated their clinical trials in the US because of significantly increased adverse events in patients who were infused with currently available investigational HBOC products.

The focus of research of the Biochemistry of Hemoglobin (Hb)-based Substitutes Section is on the structural-functional characterization of modified Hb in relation to its redox (reduction-oxidation) chemistry and toxicity. Specifically, we study the potential contributions of Hb-based reactive intermediates to oxidative and signaling cascades both in vitro and in vivo. We are also investigating several potential molecular interventions for directly or indirectly overcoming Hb toxicity in vitro and in vivo.

The basis of HBOC toxicity is poorly understood; since most research done by industry is proprietary, and there is only minimal exchange of information among investigators occurs. Our major contributions to the field of HBOCs include, 1) defining toxicological pathways that arise from and are driven by the heme prosthetic group of the molecule; 2) designing protective molecular strategies to suppress or control Hb oxidative side reactions; 3) correlating Hb and its various redox and oxygenation states with the expression of hypoxia-inducible factor (HIF-1alpha), an "oxygen sensor," and other hypoxia-sensitive genes in a model of exchange transfusion; and 4) defining the site-specific nature of the interaction between haptoglobin (Hp) and Hb, which is the basis of the Hb clearance pathway in human macrophages. More recently we discovered functions of Hp in controlling blood pressure effects that not only represent a new paradigm for blood-substitutes research but might also have clinical use in the treatment of hemolytic anemias.

Our mission-oriented laboratory research on the safety and efficacy evaluation of HBOCs has been published in major peer-reviewed journals and presented at national and international meetings.


  1. Shock 2019 Oct;52(1S Suppl. 1):41-9
    Mechanisms of toxicity and modulation of hemoglobin-based oxygen carriers (HBOCs).
    Alayash AI
  2. Free Radic Biol Med 2019 Sep;141:348-61
    Redox states of hemoglobin determine left ventricle pressure recovery and activity of mitochondrial complex IV in hypoxic rat hearts.
    Edmondson M, Jana S, Meng F, Strader MB, Baek JH, Gao Y, Buehler PW, Alayash AI
  3. Front Physiol 2019 Jul 24;10:931
    Antisickling drugs targeting betaCys93 reduce iron oxidation and oxidative changes in sickle cell hemoglobin.
    Kassa T, Wood F, Strader MB, Alayash AI
  4. Am J Hematol 2019 Apr;94(4):E88-90
    Voxelotor treatment of a patient with sickle cell disease and very severe anemia.
    Shet AS, Mendelsohn L, Harper J, Ostrowski D, Henry ER, Gwaabe E, Nichols J, Alayash AI, Eaton WA, Thein SL
  5. J Biol Chem 2019 Mar 15;294(11):4145-59
    Substitutions in the beta subunits of sickle-cell hemoglobin improve oxidative stability and increase the delay time of sickle-cell fiber formation.
    Meng F, Kassa T, Strader MB, Soman J, Olson JS, Alayash AI
  6. Bioconjug Chem 2019 Mar 20;30(3):568-71
    Interactions of an anti-sickling drug with hemoglobin in red blood cells from a patient with sickle cell anemia.
    Strader MB, Liang H, Meng F, Harper J, Ostrowski DA, Henry ER, Shet AS, Eaton WA, Thein SL, Alayash AI
  7. JCI Insight 2018 Nov 2;3(21):e120451
    Hemoglobin oxidation-dependent reactions promote interactions with band 3 and oxidative changes in sickle cell-derived microparticles.
    Jana S, Strader MB, Meng F, Hicks W, Kassa T, Tarandovskiy I, De Paoli S, Simak J, Heaven MR, Belcher JD, Vercellotti GM, Alayash AI
  8. Cell Mol Life Sci 2018 Oct;75(20):3781-801
    Dissecting the biochemical architecture and morphological release pathways of the human platelet extracellular vesiculome.
    De Paoli SH, Tegegn TZ, Elhelu OK, Strader MB, Patel M, Diduch LL, Tarandovskiy ID, Wu Y, Zheng J, Ovanesov MV, Alayash A, Simak J
  9. Redox Biol 2018 Aug 22;19:218-25
    Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin.
    Kettisen K, Strader MB, Wood F, Alayash AI, Bülow L
  10. Biosci Rep 2018 Jul 2;38(4):BSR20180370
    Comparison of the oxidative reactivity of recombinant fetal and adult human hemoglobin: implications for the design of hemoglobin-based oxygen carriers.
    Simons M, Gretton S, Silkstone GGA, Rajagopal BS, Allen-Baume V, Syrett N, Shaik T, Leiva-Eriksson N, Ronda L, Mozzarelli A, Strader MB, Alayash AI, Reeder BJ, Cooper CE
  11. Blood Cells Mol Dis 2018 May;70:78-86
    Oxidative pathways in the sickle cell and beyond.
    Alayash AI
  12. Bioconjug Chem 2018 May 16;29(5):1560-75
    Comprehensive biochemical and biophysical characterization of hemoglobin-based oxygen carrier therapeutics: all HBOCs are not created equally.
    Meng F, Kassa T, Jana S, Wood F, Zhang X, Jia Y, D'Agnillo F, Alayash AI
  13. Transfusion 2018 Jan;58(1):255-66
    Proceedings of the Food and Drug Administration's public workshop on new red blood cell product regulatory science 2016.
    Vostal JG, Buehler PW, Gelderman MP, Alayash AI, Doctor A, Zimring JC, Glynn SA, Hess JR, Klein H, Acker JP, Spinella PC, D'Alessandro A, Palsson B, Raife TJ, Busch MP, McMahon TJ, Intaglietta M, Swartz HM, Dubick MA, Cardin S, Patel RP, Natanson C, Weisel JW, Muszynski JA, Norris PJ, Ness PM
  14. Biochem J 2017 Dec 11;474(24):4171-92
    Engineering oxidative stability in human hemoglobin based on the Hb providence (betaK82D) mutation and genetic crosslinking.
    Strader MB, Bangle R, Parker Siburt CJ, Varnado CL, Soman J, Benitez Cardenas AS, Samuel PS, Singleton EW, Crumbliss AL, Olson JS, Alayash AI
  15. Front Physiol 2017 Dec 19;8:1082
    Oxidized mutant human Hhmoglobins S and E induce oxidative stress and bioenergetic dysfunction in human pulmonary endothelial cells.
    Jana S, Meng F, Hirsch RE, Friedman JM, Alayash AI
  16. Metallomics 2017 Sep 20;9(9):1260-70
    Targeting betaCys93 in hemoglobin S with an antisickling agent possessing dual allosteric and antioxidant effects.
    Kassa T, Brad Strader M, Nakagawa A, Zapol WM, Alayash AI
  17. Antioxid Redox Signal 2017 May 10;26(14):777-93
    Exploring oxidative reactions in hemoglobin variants using mass spectrometry: lessons for engineering oxidatively stable oxygen therapeutics.
    Strader MB, Alayash AI
  18. Antioxid Redox Signal 2017 May 10;26(14):745-7
    Redox chemistry of hemoglobin-associated disorders.
    Bulow L, Alayash AI
  19. Nat Struct Mol Biol 2017 Apr;24(4):379-86
    HIV Tat protein and amyloid-beta peptide form multifibrillar structures that cause neurotoxicity.
    Hategan A, Bianchet MA, Steiner J, Karnaukhova E, Masliah E, Fields A, Lee MH, Dickens AM, Haughey N, Dimitriadis EK, Nath A
  20. Anal Biochem 2017 Mar 15;521:11-9
    Determination of extinction coefficients of human hemoglobin in various redox states.
    Meng F, Alayash AI
  21. Biomolecules 2017 Jan 4;7(1):7010002
    Hemoglobin-based blood substitutes and the treatment of sickle cell disease: more harm than help?
    Alayash AI
  22. PLoS One 2016 Dec 13;11(12):e0166657
    Evaluation of stem cell-derived red blood cells as a transfusion product using a novel animal model.
    Shah S, Gelderman MP, Lewis MA, Farrel J, Wood F, Strader MB, Alayash AI, Vostal JG
  23. Br J Haematol 2016 Nov;175(4):714-23
    Sustained treatment of sickle cell mice with haptoglobin increases HO-1 and H-ferritin expression and decreases iron deposition in the kidney without improvement in kidney function.
    Shi PA, Choi E, Chintagari NR, Nguyen J, Guo X, Yazdanbakhsh K, Mohandas N, Alayash AI, Manci EA, Belcher JD, Vercellotti GM
  24. Am J Respir Cell Mol Biol 2016 Aug;55(2):288-98
    Oxidized ferric and ferryl forms of hemoglobin trigger mitochondrial dysfunction and injury in alveolar type I cells.
    Chintagari NR, Jana S, Alayash AI
  25. Redox Biol 2016 Aug;8:363-74
    Oxidative instability of hemoglobin E (beta26 Glu-->Lys) is increased in the presence of free alpha subunits and reversed by alpha-hemoglobin stabilizing protein (AHSP): relevance to HbE/beta-thalassemia.
    Strader MB, Kassa T, Meng F, Wood FB, Hirsch RE, Friedman JM, Alayash AI
  26. FEBS Open Bio 2016 Aug 8;6(9):876-84
    Differential heme release from various hemoglobin redox states and the upregulation of cellular heme oxygenase-1.
    Kassa T, Jana S, Meng F, Alayash AI
  27. Biochemistry 2016 Jan 12;55(1):133-45
    Tales of dihydrofolate binding to R67 dihydrofolate reductase.
    Duff MR Jr, Chopra S, Strader MB, Agarwal PK, Howell EE
  28. J Biol Chem 2015 Nov 13;290(46):27939-58
    Sickle cell hemoglobin in the ferryl state promotes betaCys93 oxidation and mitochondrial dysfunction in epithelial lung cells (E10).
    Kassa T, Jana S, Strader MB, Meng F, Jia Y, Wilson MT, Alayash AI
  29. Front Physiol 2015 Feb 20;6:39
    Dissection of the radical reactions linked to fetal hemoglobin reveals enhanced pseudoperoxidase activity.
    Ratanasopa K, Strader MB, Alayash AI, Bulow L
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