PAR-1 on the Astrocyte End Foot: A New
Target for Pharmacological Intervention in
Neurological Diseases
‫התערבות פרמקולוגית‬
‫ייחודית במסלול הפעלת‬
‫הרצפטור לתרומבין – בסיס‬
‫חדש לטיפול בפגיעה עצבית‬
Joab Chapman, Efrat Shavit.
PAR: Protease-Activated Receptor
2
‫המרכז הרפואי המשולב ע"ש חיים שיבא‬
Development of novel PAR-1-based therapeutic compounds for
neuro-inflammatory and malignant diseases:
diabetic neuropathy and glioblastoma multiforme
‫המרכז הרפואי המשולב ע"ש חיים שיבא‬
The Neuron
The functional unit of the nervous system
Scheme of Astrocyte with Neuron
Motor Neuron in Cell Culture
Astrocyte in Brain with Neurons
Glial endfeet at synapse and node of Ranvier
‫המרכז הרפואי המשולב ע"ש חיים שיבא‬
Development of novel PAR-1-based therapeutic compounds for
neuro-inflammatory and malignant diseases:
diabetic neuropathy and glio-blastoma multiforme
• The thrombin receptor PAR-1 (protease activated receptor) is
found mainly on astrocytes
• We have found PAR-1 both at the synapse and node of
Ranvier
Shavit et al. J neurochem 2011
Shavit
et‫חיים‬
al.‫ש‬Brain
2008
‫שיבא‬
"‫המשולב ע‬
‫המרכז הרפואי‬
Thrombin receptor PAR-1 on myelin at the node
of Ranvier: a new anatomy and physiology of
conduction block
Shavit et al. Brain.2008
‫המרכז הרפואי המשולב ע"ש חיים שיבא‬
PAR1 activation by independent proteases
PAR-1 physiological/pathophysiological role
‫המרכז הרפואי המשולב ע"ש חיים שיבא‬
Thrombin-like activity in brain
‫המרכז הרפואי המשולב ע"ש חיים שיבא‬
Thrombin Activity in Ischemic vs.
Healthy Brains
40
Control R
Control L
Stroke R
Stroke L
Slice #3
Thrombin Activity [mUnits/ml]
35
30
25
20
15
10
5
0
3
Slice #11
4
5
6
7
8
Slice #
Slice #
9
10
11

24h following MCAo

Each group n=6
16
Thrombin like activity in brain slices
1800
1600
1400
1200
1000
Sod1R
Sod L
FI/Min 800
WT R
600
WTL
400
200
0
3
4
5
6
7
-200
Slices
8
9
10
11
12
Significant elevated thrombin-like activity in disease
models involving the peripheral nervous system
30
0.006
**
0.005
0.004
0.003
0.002
0.001
Thrombin-like activity (mu/ml)
Thrombin-like activity (U/ml)
0.007
W.T
SOD-1
25
20
15
10
5
0
0
Controls
Diabetics
3
4
5
6
7
8
slice
9
10
Aronovich et al. (submitted) Abu Rahal et al. (submitted)
‫המרכז הרפואי המשולב ע"ש חיים שיבא‬
11
12
PAR-1 (red) Astrocytes (green) in cerebellum of Wild
Type Mice
PAR-1
GFAP
PAR-1/GFAP
GFAP
PAR-1/GFAP
GFAP
PAR-1/GFAP
GFAP
PAR-1/GFAP
GFAP
PAR-1/GFAP
GFAP
PAR-1/GFAP
250µm
W.T
PAR-1
25µm
PAR-1
SOD
250µm
PAR-1
25µm
PAR-1
SOD treated with
FTS 40mg/kg
250µm
PAR-1
25µm
PAR-1 in Frontal Brain of SOD ALS Mouse Model
PAR1 activation by independent proteases
PAR-1 physiological/pathophysiological role
‫המרכז הרפואי המשולב ע"ש חיים שיבא‬
Thrombin inhibition (specific and general) significantly
improves nerve-conduction and survival in neuroinflammationinvolved diseases
SOD-1 (ALS animal120
model) survival
precent survival
100
80
60
SOD-1-‫בלי טיפול‬
SOD-1,PAR-1-antagonist
25 µg/kg
TLCK 4.4mg/kg
40
20
0
130
150
age(days)
170
‫המרכז הרפואי המשולב ע"ש חיים שיבא‬
Increased PAR-1 level in glioma cells
FCS free
FCS
‫המרכז הרפואי המשולב ע"ש חיים שיבא‬
Development of novel PAR-1-based therapeutic compounds for
neuro-inflammatory and malignant diseases:
diabetic neuropathy and glioblastoma multiforme
Thrombin-like activity
is generated by
glioma cell-lines
PAR-1 pathway modulation inhibits
glioma-cells proliferation
‫המרכז הרפואי המשולב ע"ש חיים שיבא‬
PAR1 activation by independent proteases
PAR-1 physiological/pathophysiological role
‫המרכז הרפואי המשולב ע"ש חיים שיבא‬
Existing drugs may not be ideal






Do not cross blood brain barrier
Toxic to normal brain function
Intrinsically inhibit coagulation
Block one pathway only (e.g. PAR-1/Par-4)
May miss specific brain disease targets
Pose complex IP issues
General Strategy Outline
Diagnosis
Identify targets in
neurological
disease
Whole animal
experiment
Tox, Phase 1
Identify targets in
PAR-1 activation
Rapid, high
throughput
screening
Disease targets: Outline
Excess
activation of
PAR-1
Internal
NeuroTrypsins
External
NeuroThrombin
Serum
derived
Thrombin
Targeting central and peripheral
nervous system diseases
Internal
External
 Autoimmune
 Neoplastic
(GBS)
 Trauma
(GBM)
 Neurodegenerative
 ICH/Stroke
(ALS)
 Infection
 Diabetic
neuropathy
 Metabolic
 Epilepsy
(Generation)
 Epilepsy
(Diabetes)
(Symptom)
Targets in the PAR-1 activation pathway:
protease based approaches






Identity of protease
(thrombin/trypsin)
Synthesis (Vit K)
Release
Activation (cascade)
Endogenous inhibitors
Docking at PAR-1
‫שרשרת הקרישה‬
‫מה שמקובל‬
‫‪The group‬‬
‫המרכז הרפואי המשולב ע"ש חיים שיבא‬