Evaluating the Safety and Efficacy of Hemoglobin-based Blood Substitutes
Abdu I. Alayash, Ph.D., D.Sc.
Office of Blood Research and Review
Division of Blood Components and Devices
Laboratory of Biochemistry and Vascular Biology
Biosketch
Dr. Alayash joined CBER in 1989, beginning as a Senior Staff Fellow in the Office of Blood Research and Review (OBRR). Dr. Alayash is the Chief of the Laboratory of Biochemistry and Vascular Biology (LBVB), which he established in 2004. Dr. Alayash obtained his PhD in Biochemistry (1978) as well as a DSc in Biochemistry (2011) from the University of Essex, Colchester, United Kingdom. In 2013, Dr. Alayash was awarded an honorary doctorate from Lund University, Sweden for research accomplishments in the field of blood substitutes. He is a member of the FDA Senior Biomedical Research and Biomedical Product Assessment Service (SBRBPAS).
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 elective surgeries, especially when blood is not available. These products, also known as "hemoglobin-based oxygen carriers" (HBOCs), have undergone extensive testing in animals and in humans over the last 3 decades. HBOCs use the natural oxygen-carrying molecule, hemoglobin (Hb), to carry oxygen throughout the body. However, because the Hb used for HBOCs is no longer inside red blood cells (RBCs), it tends to be toxic in the blood. This cell-free Hb can cause high blood pressure; Hb can also escape blood vessels and damage the kidneys and other organs. Because there was no scientific and/or regulatory historical precedent, understanding how free Hb behaves outside the protective environment of an RBC becomes a very challenging problem facing industrial and the scientific communities. Our laboratory is trying to a) understand the complexities associated with the interaction of Hb with the vascular system and b) attempt to overcome toxicities.
We have successfully used the body's own defense mechanisms, such as plasma-derived haptoglobin (oxidized Hb scavenger) and hemopexin (heme scavenger) against Hb oxidative toxicity in animal models. Further, we have embarked on a two-prong strategy. First, we explored naturally occurring mutants of human Hb, which are considered experiments in nature that have evolved over the years to resist oxidation or have developed into a full hematological disorder. Second, we utilized this knowledge to design approaches to increase or decrease these reactions in acellular oxygen therapeutics that will result in oxidatively stable and safe protein. For example, we have recently discovered that Hb Providence is a very stable protein. We have introduced the Providence mutation into a newly designed HBOC, which shows a remarkable resistance to oxidation. This is a critical element in the design of safe and effective oxygen therapeutics.
Several manufacturers and research institutions are now designing second-generation blood substitutes that are oxidatively stable and could potentially be effective lifesaving oxygen therapeutics. Our work therefore is contributing to the regulatory and research efforts of CBER to support development of safe and effective products that improve public health in the U.S. and worldwide. Using our extensive expertise with Hb oxygen transport and reduction-oxidation (redox) chemistry, we have begun a pilot study on the effects of COVID-19 infection on oxygen homeostasis and other signaling pathways.
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. The basis of HBOC toxicity is poorly understood; most research done by industry is proprietary, and there is only minimal exchange of information among investigators. The focus of research in this program is on the structural-functional characterization of modified Hb in relation to its redox 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 have also investigated several potential molecular interventions to directly or indirectly control Hb toxicity in vitro and in vivo.
To date, our major contributions to the field of HBOCs include: 1) fully characterizing (biochemical and biophysical) all HBOCs that have been tested in humans; 2) defining toxicological pathways that arise from and are driven by the heme prosthetic group of the molecule; we have indeed recently confirmed that heme acts as Damage Associated Molecular Pattern (DAMP) molecule that triggers inflammatory events; 3) correlating various redox and oxygenation states of free Hb and Hb-laden microparticles from sickle cell blood with mitochondrial respiration and dysfunction; 4) designing protective molecular strategies to suppress or control Hb oxidative side reactions, including the use of oxidatively stable mutant Hb as a potential HBOC prototype; 5) defining the impact of SAR-COV-2 spike protein binding on cellular and subcellular oxygen sensing and mitochondrial functions in human pulmonary arterial endothelial cells. 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.
Important Links
- Innovation and Regulatory Science- Research Summary: FDA scientists discover key chemical steps in formation of microparticles carrying abnormal hemoglobin in sickle cell disease
- Innovation and Regulatory Science- Research Summary: FDA comparison of characteristics of hemoglobin-based oxygen carriers could guide development of safe and effective artificial blood products
- Innovation and Regulatory Science- Scientific Poster: Microparticles from sickle cell disease red blood cells
- ORCID ID: 0000-0002-2899-6757
Publications
- Curr Med Chem 2022 Jan;29(3):467-88
C1-inhibitor: structure, functional diversity and therapeutic development.
Karnaukhova E - FEBS Open Bio 2021 Dec;11(12):3293-303
Caffeic acid: an antioxidant with novel antisickling properties.
Kassa T, Whalin JG, Richards MP, Alayash AI - Front Physiol 2021 Sep 27;12:711976
The impact of COVID-19 infection on oxygen homeostasis: a molecular perspective.
Alayash AI - Lab Invest 2021 Jan;101(1):4-11
Beta-cysteine 93 in human hemoglobin: a gateway to oxidative stability in health and disease.
Alayash AI - Haematologica 2021 Jan 1;106(1):9-11
Targeting the red cell enzyme pyruvate kinase with a small allosteric molecule AG-348 may correct underlying pathology of a glycolytic enzymopathy.
Alayash AI - Int J Mol Sci 2021 Jan 26;22(3):1199
Human plasma and recombinant hemopexins: heme binding revisited.
Karnaukhova E, Owczarek C, Schmidt P, Schaer DJ, Buehler PW - Redox Rep 2020 Dec;25(1):95-103
Effects of α subunit substitutions on the oxidation of betaCys93 and the stability of sickle cell hemoglobin.
Hicks W, Meng F, Kassa T, Alayash AI - Int J Mol Sci 2020 Dec 11;21(24):E9453
The Providence mutation (betaK82D) in human hemoglobin substantially reduces betaCysteine 93 oxidation and oxidative stress in endothelial cells.
Jana S, Strader MB, Alayash AI - Sci Rep 2020 Aug 26;10(1):14218
Post-translational modification as a response to cellular stress induced by hemoglobin oxidation in sickle cell disease.
Strader MB, Jana S, Meng F, Heaven MR, Shet AS, Thein SL, Alayash AI - Shock 2019 Oct;52(1S Suppl. 1):41-9
Mechanisms of toxicity and modulation of hemoglobin-based oxygen carriers (HBOCs).
Alayash AI - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - Blood Cells Mol Dis 2018 May;70:78-86
Oxidative pathways in the sickle cell and beyond.
Alayash AI - 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 - 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 - 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 - 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 - 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 - 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 - Antioxid Redox Signal 2017 May 10;26(14):745-7
Redox chemistry of hemoglobin-associated disorders.
Bulow L, Alayash AI - 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 - Anal Biochem 2017 Mar 15;521:11-9
Determination of extinction coefficients of human hemoglobin in various redox states.
Meng F, Alayash AI - Biomolecules 2017 Jan 4;7(1):7010002
Hemoglobin-based blood substitutes and the treatment of sickle cell disease: more harm than help?
Alayash AI - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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