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  1. Biologics Research Projects

Red Blood Cell Toxicokinetics and Proteomics

Principal Investigator: Paul W. Buehler, PhD
Office / Division / Lab: OBRR / DBCD / LBVB

General Overview

In certain medical situations, such as sickle cell disease, thalassemia, malaria, exposure to toxins, medical assist device use and during transfusion red blood cells can undergo hemolysis (disintegrate) over time and release free hemoglobin (or its components: heme and iron) and other cellular material (membrane lipids). Understanding the effects of red blood cell damage allows us to better address disease states and medical interventions where hemolysis occurs. As a result our scientific efforts are designed to improve public health by first understanding the toxic effects of hemolysis and then investigate methods to prevent or limit the effects of hemolysis. Public funding of these research efforts help gather and assemble information on the effects of red blood cell destruction in the body. Data is then made accessible to help researchers further pursue the findings and facilitate therapeutic development, allow for patients to understand the effects of hemolysis in various medical conditions and provide a basis for FDA's regulatory decision making regarding therapeutics that treat or cause red blood cell destruction.

On one hand FDA is faced with regulating therapeutic interventions that cause red blood cell destruction as a side effect of their intended treatment. On the other hand FDA is faced with regulating therapeutic interventions to prevent or decrease the toxic effects of red blood cell destruction. Our program in the Laboratory of Biochemisty and Vascular Biology studies how red blood cells disintegrate and cause damage to the body during various medical interventions. Important to this work is that we focus on the identity and quantity of red blood cell components required to cause toxicity and ways to counteract the effects. This data is used to help FDA regulatory efforts set acceptable limits of red blood cell destruction in medical intervention and disease treatment. The knowledge gained from this work will also support the regulatory review work of FDA and ability of the agency to provide effective guidance to industry in preventing toxic effects of hemoglobin (or its components: heme and iron) and other cellular material (membrane lipids)

In order to obtain this knowledge we are studying the mechanisms that trigger red blood cell hemolysis, by doing protein and tissue analysis to evaluate toxicity induced by the processes of hemolysis, and developing appropriate animal models and biomarkers to investigate the underlying hemolysis in transfusion medicine and hemolytic diseases (both acquired and genetic). (A biomarker is a substance that can be measured and used to indicate the state of normal biological processes, diseases, or response to therapy.) This work also includes, cell based studies, computer simulation, and when required, animal studies under normal conditions and in animals exhibiting diseases of red blood cell destruction.

Our laboratory is also currently studying several protein-based therapeutic product candidates in order to gain insights into their tissue protetive effects during red blood cell hemolysis.

Scientific Overview

Our laboratory is currently pursuing biochemical characterization of a number of protein-based products that interact with the vascular system, with a special focus on hemoglobin-based oxygen carriers (HBOCs), some plasma proteins, and anthrax toxin therapies.

We established a program in red cell toxicokinetics and proteomics to evaluate concepts central to red blood cell (RBC) destruction, with a special emphasis on understanding the toxicity of free hemoglobin, heme, iron and lipids released from damaged RBC. The overall objective of our research is to understand the underlying mechanisms of RBC destruction and the toxic effects of red blood cellular components under normal conditions and in models of disease. Some examples include animal models of vascular dysfunction (atherosclerosis), sickle cell anemia and thalassemia. Additionally we evaluate therapeutic proteins, to promote the design of safer and more effective treatments, and to establish useful biomarkers. This approach provides a strategy for minimizing RBC-induced toxicity in animals that could provide insight into human response and predict clinical risks.

The scientific approach and aims of this program are as follows:

  1. Develop and study appropriate animal models to investigate the underlying pathophysiolgic mechanisms of RBC hemolysis.
  2. Use proteomic analysis of blood and tissue protein modifications as biomarkers of injury caused by extracellular Hb oxidation and degradation arising from RBC hemolysis resulting from disease and transfusions and othe medical interventions such as drug and medical device induced hemolysis.
  3. Evaluate mechanisms contributing to the loss of RBC deformability to improve our understanding of the effects of RBC morphology and in vivo hemolysis during disease and medical interventions.
  4. Design protective strategies by enhancing clearance mechanisms of extracellular hemoglobin, heme and iron by either co-administration of natural or synthetic binding proteins/peptides. This would allow for improved outcome in disease and following medical interventions.
  5. Determine blood levels of RBC components (hemoglobin, heme, iron and lipid) released after hemolysis that are associated with toxicity (toxicokinetics) to determine acceptable limits of RBC destruction. This data may initially be obtained from animals and humans and further used to construct computer simulated models that predict safe as well as unsafe levels of RBC destruction.


JCI Insight 2017 May 4;2(9):e93577
Iron accelerates hemoglobin oxidation increasing mortality in vascular diseased guinea pigs following transfusion of stored blood.
Baek JH, Yalamanoglu A, Gao Y, Guenster R, Spahn DR, Schaer DJ, Buehler PW

Blood 2017 Feb 16;129(7):896-905
A mechanistic investigation of thrombotic microangiopathy associated with intravenous abuse of Opana ER.
Hunt R, Yalamanoglu A, Tumlin J, Schiller T, Baek JH, Wu A, Fogo AB, Yang H, Wong E, Miller P, Buehler PW, Kimchi-Sarfaty C

PLoS One 2017 Feb 2;12(2):e0171219
Hemoglobin induced cell trauma indirectly influences endothelial TLR9 activity resulting in pulmonary vascular smooth muscle cell activation.
Loomis Z, Eigenberger P, Redinius K, Lisk C, Karoor V, Nozik-Grayck E, Ferguson SK, Hassell K, Nuss R, Stenmark K, Buehler P, Irwin DC

Am J Respir Crit Care Med 2016 May 15;193(10):1111-22
Haptoglobin preserves vascular nitric oxide signaling during hemolysis.
Schaer CA, Deuel JW, Schildknecht D, Mahmoudi L, Garcia-Rubio I, Owczarek C, Schauer S, Kissner R, Banerjee U, Palmer AF, Spahn DR, Irwin DC, Vallelian F, Buehler PW, Schaer DJ

Toxics 2016 Mar;4(1):4010006
Transcriptional suppression of renal antioxidant enzyme systems in guinea pigs exposed to polymerized cell-free hemoglobin.
Rentsendorj O, Zhang X, Williams MC, Buehler PW, D'Agnillo F

Cell Death Dis 2016 Jan 21;7:e2064
Hemoglobinuria-related acute kidney injury is driven by intrarenal oxidative reactions triggering a heme toxicity response.
Deuel JW, Schaer CA, Boretti FS, Opitz L, Garcia-Rubio I, Baek JH, Spahn DR, Buehler PW, Schaer DJ

Free Radic Biol Med 2015 Dec;89:931-43
Different target specificities of haptoglobin and hemopexin define a sequential protection system against vascular hemoglobin toxicity.
Deuel JW, Vallelian F, Schaer CA, Puglia M, Buehler PW, Schaer DJ

Free Radic Biol Med 2015 Aug;85:259-68
Spin trapping combined with quantitative mass spectrometry defines free radical redistribution within the oxidized hemoglobin:haptoglobin complex.
Vallelian F, Garcia-Rubio I, Puglia M, Kahraman A, Deuel JW, Engelsberger WR, Mason RP, Buehler PW, Schaer DJ

Toxicology 2015 Jul 3;333:89-99
Sodium nitrite potentiates renal oxidative stress and injury in hemoglobin exposed guinea pigs.
Baek JH, Zhang X, Williams MC, Hicks W, Buehler PW, Felice D

Haematologica 2015 May;100(5):611-22
Reversal of hemochromatosis by apo-transferrin in non-transfused and transfused Hbbth3/+ (heterozygous b1/ b2 globin gene deletion) mice.
Gelderman MP, Baek JH, Yalamanoglu A, Puglia M, Vallelian F, Burla B, Vostal J, Schaer DJ, Buehler PW

Free Radic Biol Med 2015 May;82:50-62
Hemoglobin induced lung vascular oxidation, inflammation, and remodeling contributes to the progression of hypoxic pulmonary hypertension and is attenuated in rats with repeat dose haptoglobin administration.
Irwin DC, Baek JH, Hassell K, Nuss R, Eigenberger P, Lisk C, Loomis Z, Maltzahn J, Stenmark KR, Nozik-Grayck E, Buehler PW

J Proteome Res 2015 Feb 6;14(2):1089-100
Integrative proteome and transcriptome analysis of extramedullary erythropoiesis and its reversal by transferrin treatment in a mouse model of beta-thalassemia.
Vallelian F, Gelderman MP, Schaer CA, Puglia M, Opitz L, Baek JH, Vostal J, Buehler P, Schaer DJ

Cell Death Differ 2015 Apr;22(4):597-611
Proteasome inhibition and oxidative reactions disrupt cellular homeostasis during heme stress.
Vallelian F, Deuel JW, Opitz L, Schaer CA, Puglia M, Lonn M, Engelsberger W, Schauer S, Karnaukhova E, Spahn DR, Stocker R, Buehler PW, Schaer DJ

Front Physiol 2014 Oct 9;5:385
Modeling hemoglobin and hemoglobin:haptoglobin complex clearance in a non-rodent species-pharmacokinetic and therapeutic implications.
Boretti FS, Baek JH, Palmer AF, Schaer DJ, Buehler PW

Front Physiol 2014 Oct 28;5:415
Haptoglobin, hemopexin, and related defense pathways-basic science, clinical perspectives, and drug development.
Schaer DJ, Vinchi F, Ingoglia G, Tolosano E, Buehler PW

Toxins 2014 Mar 31;6(4):1244-59
Extracellular hb enhances cardiac toxicity in endotoxemic Guinea pigs: protective role of haptoglobin.
Baek JH, Zhang X, Williams MC, Schaer DJ, Buehler PW, D'Agnillo F

Antioxid Redox Signal 2013 Nov 10;19(14):1619-33
Human hp1-1 and hp2-2 phenotype-specific haptoglobin therapeutics are both effective in vitro and in Guinea pigs to attenuate hemoglobin toxicity.
Lipiski M, Deuel JW, Baek JH, Engelsberger WR, Buehler PW, Schaer DJ

Antioxid Redox Signal 2013 Jun 10;18(17):2264-73
Haptoglobin Binding Stabilizes Hemoglobin Ferryl Iron and the Globin Radical on Tyrosine beta145.
Cooper CE, Schaer DJ, Buehler PW, Wilson MT, Reeder BJ, Silkstone G, Svistunenko DA, Bulow L, Alayash AI

Cold Spring Harb Perspect Med 2013 Jun 1;3(6):a013433
Cell-Free Hemoglobin and Its Scavenger Proteins: New Disease Models Leading the Way to Targeted Therapies.
Schaer DJ, Buehler PW

PLoS One 2013;8(3):e59841
Haptoglobin preferentially binds beta but not alpha subunits cross-linked hemoglobin tetramers with minimal effects on ligand and redox reactions.
Jia Y, Wood F, Buehler PW, Alayash AI

Blood 2013 Feb 21;121(8):1276-84
Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins.
Schaer DJ, Buehler PW, Alayash AI, Belcher JD, Vercellotti GM

Am J Physiol Lung Cell Mol Physiol 2012 Aug;303(4):L312-26
Free hemoglobin induction of pulmonary vascular disease: evidence for an inflammatory mechanism.
Buehler PW, Baek JH, Lisk C, Connor I, Sullivan T, Kominsky DJ, Majka SM, Stenmark KR, Nozik-Grayck E, Bonaventura J, Irwin DC

Toxicol Sci 2012 Jun;127(2):567-81
Down selection of polymerized bovine hemoglobins for use as oxygen releasing therapeutics in a guinea pig model.
Baek JH, Zhou Y, Harris DR, Dominik SJ, Palmer AF, Buehler PW

J Clin Invest 2012 Apr 2;122(4):1444-58
Hemoglobin-driven pathophysiology is an in vivo consequence of the red blood cell storage lesion that can be attenuated in guinea pigs by haptoglobin therapy.
Baek JH, D'Agnillo F, Vallelian F, Pereira CP, Williams MC, Jia Y, Schaer DJ, Buehler PW

Biochem Biophys Res Commun 2011 Dec 16;416(3-4):421-6
Inactivation of prolyl hydroxylase domain (PHD) protein by epigallocatechin (EGCG) stabilizes hypoxia-inducible factor (HIF-1alpha) and induces hepcidin (Hamp) in rat kidney.
Manalo DJ, Baek JH, Buehler PW, Struble E, Abraham B, Alayash AI

Biotechnol Prog 2011 Jul;27(4):1172-84
Synthesis, biophysical properties, and oxygenation potential of variable molecular weight glutaraldehyde-polymerized bovine hemoglobins with low and high oxygen affinity.
Zhou Y, Jia Y, Buehler PW, Chen G, Cabrales P, Palmer AF

Biochem Biophys Res Commun 2011 Jun 10;409(3):412-7
Sodium nitrite induces acute central nervous system toxicity in guinea pigs exposed to systemic cell-free hemoglobin.
Buehler PW, Butt OI, D'Agnillo F

Antioxid Redox Signal 2011 May 1;14(9):1713-28
Blood aging, safety and transfusion: capturing the "radical" menace.
Buehler PW, Karnaukhova E, Gelderman MP, Alayash AI

Biochim Biophys Acta 2011 Apr-Jun;1809(4-6):262-8
Induction of hypoxia inducible factor (HIF-1α) in the rat kidneys by iron chelation with the hydroxypyridinone, CP94.
Baek JH, Reiter CE, Manalo DJ, Buehler PW, Hider RC, Alayash AI

Am J Pathol 2011 Mar;178(3):1316-28
Blood-brain barrier disruption and oxidative stress in Guinea pig after systemic exposure to modified cell-free hemoglobin.
Butt OI, Buehler PW, D'Agnillo F

Trends Mol Med 2010 Aug 12;16(10):447-57
Hemoglobin-based oxygen carriers: from mechanisms of toxicity and clearance to rational drug design.
Buehler PW, D'Agnillo F, Schaer DJ

J Proteome Res 2010 Aug 6;9(8):4061-70
Quantitative mass spectrometry defines an oxidative hotspot in hemoglobin that is specifically protected by haptoglobin.
Pimenova T, Pereira CP, Gehrig P, Buehler PW, Schaer DJ, Zenobi R

Biotechnol Bioeng 2010 May 1;106(1):76-85
Functional comparison of hemoglobin purified by different methods and their biophysical implications.
Elmer J, Buehler PW, Jia Y, Wood F, Harris DR, Alayash AI, Palmer AF

Biomaterials 2010 May;31(13):3723-35
Synthesis, biophysical properties and pharmacokinetics of ultrahigh molecular weight tense and relaxed state polymerized bovine hemoglobins.
Buehler PW, Zhou Y, Cabrales P, Jia Y, Guoyong Sun, Harris DR, Tsai AG, Intaglietta M, Palmer AF

Antioxid Redox Signal 2010 Feb;12(2):185-98
Hemoglobin can attenuate hydrogen peroxide-induced oxidative stress by acting as an antioxidative peroxidase.
Widmer CC, Pereira CP, Gehrig P, Vallelian F, Schoedon G, Buehler PW, Schaer DJ

Am J Respir Cell Mol Biol 2010 Feb;42(2):200-9
Mixed S-nitrosylated polymerized bovine hemoglobin species moderate hemodynamic effects in acutely hypoxic rats.
Irwin D, Buehler PW, Alayash AI, Jia Y, Bonventura J, Foreman B, White M, Jacobs R, Piteo B, TissotvanPatot MC, Hamilton KL, Gotshall RW

Antioxid Redox Signal 2010 Feb;12(2):275-91
Toxicological Consequences of Extracellular Hemoglobin: Biochemical and Physiological Perspectives.
Buehler PW, D'Agnillo F

Antioxid Redox Signal 2010 Feb;12(2):199-208
Differential induction of renal heme oxygenase and ferritin in ascorbate and non-ascorbate producing species transfused with modified cell-free hemoglobin.
Butt OI, Buehler PW, D'Agnillo F

Ann Pharmacother 2009 Oct;43(10):1583-97
Comparing Generic and Innovator Drugs: A Review of 12 Years of Bioequivalence Data from the United States Food and Drug Administration(October).
Davit BM, Nwakama PE, Buehler GJ, Conner DP, Haidar SH, Patel DT, Yang Y, Yu LX, Woodcock J

J Clin Invest 2009 Aug;119(8):2271-80
Sequestration of extracellular hemoglobin within a haptoglobin complex decreases its hypertensive and oxidative effects in dogs and guinea pigs.
Boretti FS, Buehler PW, D'Agnillo F, Kluge K, Glaus T, Butt OI, Jia Y, Goede J, Pereira CP, Maggiorini M, Schoedon G, Alayash AI, Schaer DJ

Blood 2009 Mar 12;113(11):2578-86
Haptoglobin preserves the CD163 hemoglobin scavenger pathway by shielding hemoglobin from peroxidative modification.
Buehler PW, Abraham B, Vallelian F, Linnemayr C, Pereira CP, Cipollo JF, Jia Y, Mikolajczyk M, Boretti FS, Schoedon G, Alayash AI, Schaer DJ

Biochim Biophys Acta 2008 Oct;1784(10):1415-20
Peroxidase activity of hemoglobin towards ascorbate and urate: a synergistic protective strategy against toxicity of Hemoglobin-Based Oxygen Carriers (HBOC).
Cooper CE, Silaghi-Dumitrescu R, Rukengwa M, Alayash AI, Buehler PW

Free Radic Biol Med 2008 Oct 15;45(8):1150-8
The reaction of hydrogen peroxide with hemoglobin induces extensive alpha-globin crosslinking and impairs the interaction of hemoglobin with endogenous scavenger pathways.
Vallelian F, Pimenova T, Pereira CP, Abraham B, Mikolajczyk MG, Schoedon G, Zenobi R, Alayash AI, Buehler PW, Schaer DJ

Biochim Biophys Acta 2008 Oct;1784(10):1378-81
All hemoglobin-based oxygen carriers are not created equally.
Buehler PW, Alayash AI

Biochem J 2008 Sep 15;414(3):461-9
Acellular haemoglobin attenuates hypoxia-inducible factor-1alpha (HIF-1alpha) and its target genes in haemodiluted rats.
Manalo DJ, Buehler PW, Baek JH, Butt O, D'agnillo F, Alayash AI

Antioxid Redox Signal 2008 Aug;10(8):1449-62
Structural stabilization in tetrameric or polymeric hemoglobin determines its interaction with endogenous antioxidant scavenger pathways.
Buehler PW, Vallelian F, Mikolajczyk MG, Schoedon G, Schweizer T, Alayash AI, Schaer DJ

J Pharmacol Exp Ther 2007 Oct;323(1):49-60
Effects of endogenous ascorbate on oxidation, oxygenation and toxicokinetics of cell-free modified hemoglobin after exchange transfusion in rat and guinea pig.
Buehler PW, D'Agnillo F, Hoffman V, Alayash AI

Antioxid Redox Signal 2007 Jul;9(7):991-9
Gating the radical hemoglobin to macrophages: the anti-inflammatory role of CD163, a scavenger receptor.
Schaer DJ, Alayash AI, Buehler PW

Expert Opin Biol Ther 2007 May;7(5):665-75
First-generation blood substitutes: what have we learned? Biochemical and physiological perspectives.
Alayash AI, D'Agnillo F, Buehler PW

J Biol Chem 2007 Feb 16;282(7):4894-907
Structural basis of peroxide mediated changes in human hemoglobin: A novel oxidative pathway.
Jia Y, Buehler PW, Boykins RA, Venable RM, Alayash AI

Biochem J 2006 Nov 1;399(3):513-24
Ascorbate removes key precursors to oxidative damage by cell free hemoglobin in vitro and in vivo.
Dunne J, Caron A, Menu P, Alayash AI, Buehler PW, Wilson MT, Silaghi-Dumitrescu R, Faivre B, Cooper CE

Anal Chem 2006 Jul 1;78(13):4634-4641
Chemical Characterization of Diaspirin Cross-Linked Hemoglobin Polymerized with Poly(ethylene glycol).
Buehler PW, Boykins RA, Norris S, Alayash AI

Blood 2006 Jan 1;107(1):373-80
CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in the absence of haptoglobin.
Schaer DJ, Schaer CA, Buehler PW, Boykins RA, Schoedon G, Alayash AI, Schaffner A