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Laboratory of Biochemistry and Vascular Biology. Biochemistry of Blood Substitutes Abdu I. Alayash, Ph.D. (PI) Yiping Jia, Ph.D. Physical Protein Chemistry Andrew Shrake, Ph.D. (PI) (Ewa Marszal, Ph.D.). Vascular Biology Felice D’Agnillo, Ph.D. (PI). Research.

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Biochemical and physiological studies on the safety and efficacy of blood products and their interactions with the vascu


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slide1

Laboratory of Biochemistry and Vascular Biology

Biochemistry of Blood Substitutes

Abdu I. Alayash, Ph.D. (PI)

Yiping Jia, Ph.D.

Physical Protein Chemistry

Andrew Shrake, Ph.D. (PI)

(Ewa Marszal, Ph.D.)

Vascular Biology

Felice D’Agnillo, Ph.D. (PI)

Research

  • 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

Regulatory Review

slide2

Laboratory of Biochemistry and

Vascular Biology

Biochemistry of Blood Substitutes

Abdu Alayash, Ph.D.

Yiping Jia, Ph.D.

BPAC March ‘06

slide3

a

PFC

b

b

2,3 DPG

PFC

a

PFC

Oxygen Therapeutics, “Blood Substitutes”

RBC

PFC Emulsion

Tetramer

Conjugated Tetramer

Polymer

Encapsulated Hb

slide4
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
slide5

HBOCs: Redox Challenges

Outside RBCs!

Free Hb is inherently toxic

:

generates ROS, and reacts with ROS &

Þ

RNS (i.e. NO)

vascular

injury

Nature of chemical modification:

Þ

Non

-

site specificity

conformational &

heme instability

ROS = Reactive Oxygen; RNS = Reactive Nitrogen Species

slide6

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

Biochemistry (2002)

slide7

O-R-PolyHbA0Identification of the Origin of Altered Function

O2

O2

Locked (T) State

O2

O2

O2

O2

Tense (T) Oxy

O2

O2

O2

Normal Conformational Change

O2

O2

Tense (T) Deoxy

Relaxed (R) Oxy

Tetragonal Heme Fe

Rhombic Heme Fe

(1) Heme Destabilization

O-R-PolyHbA0

HbA0

(2) Protein Destabilization (locked T state)

Biochemistry (2002), Biochemical J. (2004)

o r polyhba 0 actual chemical modification maldi ms
O-R-PolyHbA0:Actual Chemical Modification (MALDI-MS)
  • Non-uniform O-raffinose oxidation products
  • Non-specific cross-links
  • Modified cysteines

Destabilization of the protein!

Proteins (2005)

slide9

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.

slide10

Laboratory of Biochemistry and

Vascular Biology

Physical Protein Chemistry

Andrew Shrake, Ph.D.

(Ewa Marszal, Ph.D.)

BPAC March ‘06

slide11

a1-PI Projects

● Investigation of the structure of a1-PI polymer

● Characterization of differences in isoelectric focusing behavior of licensed a1-PI products

● Development of WHO a1-PI reference standard

● Expression of human a1-PI in E. coli and A. niger

● Assay development (ELISA and potency assay)

slide12

Pathogenesis of a1-PI deficiency

Lungs

Emphysema

Liver

Disease

a1-PI

Neutrophil

elastase

Neutrophils

Bone

marrow

Adapted from Crystal et al., 1997

slide13

Structure of a1-PI Polymer

  • Relevance of investigation of the structure of a1-PI polymer
  • protein polymers form in vivo

mechanisms underlying conformational diseases

prevention

  • protein polymers are present in products

safety issue related to long-time use

slide14

The structure of a1-PI

A sheet

C sheet

RCL

C232

PDB 1QLP

slide15

Loop-A sheet model ofa1-PI polymer

Nature Cell Biol. 2, E207 (2000)

slide16

Head-to-head model of a1-PI polymer

head-to-tail

head-to-head

A b-sheet

C232

Marszal et al. JBC 2003

Listed as one of the models of a1-PI polymers in a review

“A protein family under “stress” – Serpin stability, folding, and misfolding”

by Devlin & Bottomley, Frontiers in Bioscience 2005

research significance
Research Significance
  • Expertise in plasma-derived product characterization, comparability, safety, and follow-on biologics
  • Impact on potential new treatment modalities
slide18

Laboratory of Biochemistry and

Vascular Biology

Vascular Biology

Felice D’Agnillo, Ph.D.

BPAC March ‘06

slide19

Vascular Endothelial Responses to Biologics and Pathogens

  • Hemoglobin-based oxygen carriers
  • Counterterrorism – Anthrax
  • Blood-derived products
slide20

H2O2

Fe2+

Fe4+

Fe3+

H2O2

Medium

Redox active

aaHb

Redox Active Hb Induces Endothelial Cytotoxicity

D’Agnillo, Am J Physiol. 287, 2004.

slide21

Vascular Endothelial Responses

to Anthrax Toxin

●Hemorrhages

● Vasculitis

● Vascular leakage

slide22

*

*

*

*

Anthrax Lethal Toxin Induces Endothelial Barrier Dysfunction

Warfel et al., Am J Pathol. 166, 2005.

120

100

80

TEER

(% relative to control)

60

Medium

40

LT

20

0

0

20

40

60

80

Time (h)

slide24

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