Biochemical and physiological studies on the safety and efficacy of blood products and their...
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• Biochemical and physiological studies on the safety and efficacy of blood products and their interactions with the vascular system
• Albumin, PPF, Dextrans, HESs, A1PI, r-Apolipoprotein A1, C1 Esterase Inhibitor, Butyrylcholinesterase, Hb and PFC-based blood substitutes, and heme-based products
• Develop policy and guidance documents
Research
Regulatory Review
Laboratory of Biochemistry and Vascular Biology
Physical Protein ChemistryAndrew Shrake, Ph.D. (PI)
(Ewa Marszal, Ph.D.)
Vascular BiologyFelice D’Agnillo, Ph.D. (PI)
Biochemistry of Blood SubstitutesAbdu I. Alayash, Ph.D. (PI)
Yiping Jia, Ph.D.
Biochemistry of Blood Substitutes
Abdu Alayash, Ph.D.Yiping Jia, Ph.D.
Laboratory of Biochemistry andVascular Biology
BPAC March ‘06
Oxygen Therapeutics, “Blood Substitutes”
2,3 DPGPFC
PFC
PFC
RBC PFC Emulsion
Tetramer Conjugated Tetramer
Polymer Encapsulated Hb
HBOC Associated Pre-Clinical andClinical Side Effects
(Mackenzie C.F. and Bucci E. Hosp. Med. 65:582, 2004)
• Vasoactivity/hypertension
• Gastrointestinal side effects
• Pancreatic and liver enzymes elevation
• Oxidative stress
• Cardiac involvement
• Proinflammatory activity
• Neurotoxicity
ROS = Reactive Oxygen; RNS = Reactive Nitrogen Species
HBOCs: Redox Challenges Outside RBCs!
• Free Hb is inherently toxic:
generates ROS, and reacts with ROS & RNS (i.e. NO) vascular “injury”
• Nature of chemical modification: Non-site specificity conformational & heme instability
O-R-PolyHbA0 (HbA0 cross-linked and polymerized with O-raffinose)
Hallmarks of Functional Abnormality
• Non-sigmoidal oxygen equilibrium curve
• Non-saturating • Non-cooperative (Hill
coefficient = 1.0 vs. 2.5)• pH insensitivity
log PO2
-0.5 0.0 0.5 1.0 1.5 2.0
Sa
tura
tio
n
0.00
0.25
0.50
0.75
1.00
BloodO-R-PolyHbA0
HbA0
Biochemistry (2002)
Biochemistry (2002), Biochemical J. (2004)
O-R-PolyHbA0
Identification of the Origin of Altered Function
(1) Heme Destabilization
(2) Protein Destabilization (locked T state)
O2 O2
O2
O2 O2
O2 O2
Tense (T) Deoxy
Tense (T) Oxy
Locked (T) State
O2 O2
O2 O2
Relaxed (R) Oxy
Normal Conformational Change
Tetragonal Heme Fe
HbA0
Rhombic Heme Fe
O-R-PolyHbA0
O-R-PolyHbA0:Actual Chemical Modification
(MALDI-MS)
Proteins (2005)
•Non-uniform O-raffinose oxidation products
•Non-specific cross-links•Modified cysteines
Destabilization of the protein!
Research Significance
Our studies on the complex chemical and recombinant modifications associated with HBOCs provide an invaluable pre-clinical tool in predicting product stability, functionality and potential toxicity.
Physical Protein Chemistry
Andrew Shrake, Ph.D.(Ewa Marszal, Ph.D.)
Laboratory of Biochemistry andVascular Biology
BPAC March ‘06
1-PI Projects
● Investigation of the structure of 1-PI polymer
● Characterization of differences in isoelectric focusing behavior of licensed 1-PI products
● Development of WHO 1-PI reference standard
● Expression of human 1-PI in E. coli and A. niger
● Assay development (ELISA and potency assay)
LungsEmphysema
Liver Disease
1-PI
Neutrophil elastase
Neutrophils
Bone marrow
Pathogenesis of 1-PI deficiency
Adapted from Crystal et al., 1997
Structure of 1-PI Polymer
Relevance of investigation of the structure of 1-PI polymer
protein polymers form in vivo
mechanisms underlying conformational diseasesprevention
protein polymers are present in products
safety issue related to long-time use
A sheet
C sheet
RCL
C232
The structure of 1-PI
PDB 1QLP
Loop-A sheet model of 1-PI polymer
Nature Cell Biol. 2, E207 (2000)
C232
A -sheet
Head-to-head model of 1-PI polymer
Marszal et al. JBC 2003
Listed as one of the models of 1-PI polymers in a review “A protein family under “stress” – Serpin stability, folding, and misfolding”by Devlin & Bottomley, Frontiers in Bioscience 2005
head-to-tailhead-to-head
Research Significance
• Expertise in plasma-derived product characterization, comparability, safety, and follow-on biologics
• Impact on potential new treatment modalities
Vascular Biology
Felice D’Agnillo, Ph.D.
Laboratory of Biochemistry andVascular Biology
BPAC March ‘06
Vascular Endothelial Responses to Biologics and Pathogens
• Hemoglobin-based oxygen carriers
• Counterterrorism – Anthrax
• Blood-derived products
Redox Active Hb Induces Endothelial Cytotoxicity
Medium
Redox activeHb
H2O2
Fe2+ Fe4+ Fe3+
H2O2
D’Agnillo, Am J Physiol. 287, 2004.
● Hemorrhages
● Vasculitis
● Vascular leakage
Vascular Endothelial Responsesto Anthrax Toxin
Anthrax Lethal Toxin Induces Endothelial Barrier Dysfunction
Time (h)
*
*
*
*
TE
ER
(% r
ela
tiv
e t
o c
on
tro
l)
0
20
40
60
80
100
120
0 20 40 60 80
Medium
LT
Warfel et al., Am J Pathol. 166, 2005.
LT
Medium
Phase F-actin/nuclei VE-cadherin
Anthrax Lethal Toxin Alters Endothelial Adherens Junctions
Research Significance
Contribute to the safety and efficacy evaluation of current and anticipated blood-derived biologics by examining preclinical in vitro and in vivo models and relevant vascular biomarkers