plasmin
DESCRIPTION
Plasminogen activators (t-PA, u-PA ). Plasminogen activator inhibitors (PAI). Plasminogen activator inhibitors (PAI). ProMMP. ECM. Plasminogen. Plasmin. MMP. Circulation. Fibrin degradation products. Fibrin. February 19, 2004. Plasminogen activator inhibitors. - PowerPoint PPT PresentationTRANSCRIPT
Plasmin
Fibrin Fibrin degradation products
Plasminogen
Plasminogen activators (t-PA, u-PA)
Plasminogen activator inhibitors (PAI)Plasminogen activator inhibitors (PAI)
February 19, 2004
ProMMP
MMP
ECM
Circulation
Plasminogen activator inhibitors
• Plasminogen activator inhibitor 1(endothelial cell type)
• Plasminogen activator inhibitor 2(placental type)
• Plasminogen activator inhibitor 3(protein C inhibitor)
February 19, 2004
Molecular properties of PAI-1
• Member of the SERPIN superfamily P1’P1
February 19, 2004
February 19, 2004
Molecular properties of PAI-1
• Member of the SERPIN superfamily
• Labile (active latent)
February 19, 2004
P1’
Active PAI-1
P1
Latent PAI-1
P1’P1
February 19, 2004
Molecular properties of PAI-1
• Member of the SERPIN superfamily
• Labile (active latent)
• Inhibits t-PA and u-PA
• Active PAI-1 binds to vitronectin
February 19, 2004
Occurrence of PAI-1
• Plasma
• Platelets
• Extracellular matrix [vitronectin !]
• Cells
February 19, 2004
• Plasma: mainly active PAI-1
• Platelets: mainly latent PAI-1
• Extracellular matrix: only active PAI-1
Antigen
Activity
90
10
Plasma Platelets
% of total PAI-1 in blood
February 19, 2004
Pathophysiological role of PAI-1
• increased levels are associated with thrombotic disorders
• role in development and progression of atherosclerosis
• causative role in cancer is still controversial (dual effects)
• role in tissue fibrosis
• circulating vs localized !
February 19, 2004
Inactivation of PAI-1
February 19, 2004
Conformational transitions
I l
I s + E EI s EI s’ E +I c
Ia + E EIa EIa’ EIc
Active
Substrate
Latent
February 19, 2004
Mechanism of inactivaction
• Direct interaction with reactive site
• Active latent
• Active substrate
• Active substrate latent
P1’P1
P1’P1
February 19, 2004
Inactivation of PAI- 1
• Polyclonal antibodies
• Monoclonal antibodies
• “Low molecular weight” inhibitors
February 19, 2004
Monoclonal Antibodies
• Detailed characterization
• Epitope localizaton
• Cloning of smaller fragments ( scFv)
February 19, 2004
PAI-1 neutralizing properties ofmonoclonal antibodies
molar ratio (MA:PAI-1)
% n
eutr
aliz
atio
n o
f P
AI-
1 ac
tivi
ty
5 10 150
50
100
MA-8H9D4MA-33H1MA-55F4MA-33B8MA-37H7
t-PA
February 19, 2004
MA-31C9
MA-56A7C10
MA-55F4
MA
NoMA
t-PA
Complex PAI-1/t-PA
non-reactivecleaved PAI-1
kD
14
20
30
43
67
94
PAI-1 neutralizing effect
February 19, 2004
Monoclonal Antibodies
• Detailed characterization
• Epitope localizaton
• Cloning of smaller fragments ( scFv)
February 19, 2004
Epitopes
ConformationalLinear
February 19, 2004
MA-33H1F7 and MA-55F4C12
neutralizing monoclonal antibodies
epitope between Glu128 and Ala156
February 19, 2004
February 19, 2004
Species cross-reactivity
Human +Porcine -Rabbit -Rat +Murine +
154
KQEKK
1 379Human PAI-1
128 156
128-131
E V E RE M D RD V Q RE V E RE V E R
February 19, 2004
K
EVER
A
AAAA
February 19, 2004
Variants Mutated residues(1) KA (1/M) (2)
MA-55F4C12 MA-33H1F7
PAI-1-wt none 2.7 ± 1.6 109 5.4 ± 1.7 109
(1) residues mutated into an alanine(2) mean SD (n = 4 to 11)
Affinity constants for PAI-1 mutants
PAI-1-EVER E128V129E130R131 9.9 ± 6.0 106 4.2 ± 4.4 108
PAI-1-K K154 4.0 ± 3.1 107 3.0 ± 2.0 107
PAI-1-EVER/K E128V129E130R131 / K154 no binding no binding
February 19, 2004
Molecular
explanation
neutralizing
properties
mobility of the hinge is restricted
t-PA not locked in covalent complex
Epitope hinge region between
-helix F and main part of PAI-1
February 19, 2004
February 19, 2004
neutralizing monoclonal antibody
specific binding to the active conformation
no reactivity with rat PAI-1
MA-56A7C10
February 19, 2004
+Human PAI-1
-Rat PAI-1
Rat26hum-PAI-126
-
Human/rat PAI-1 chimeras
February 19, 2004
26
187
1 379
81
81
277
327
187
327
Human PAI-1
Rat PAI-1
Rat26hum-PAI-1
Rat81hum-PAI-1
Rat187hum-PAI-1
Rat327hum-PAI-1
Hum81rat-PAI-1
Hum187rat-PAI-1
Hum277rat-PAI-1
Hum327rat-PAI-1
Human/rat PAI-1 chimeras
February 19, 2004
Mean SD (n = 2 to 6)
PAI-1 variant MA-33H1F7 MA-56A7C10
Human PAI-1 4.9 0.06
Rat PAI-1 2.7 0.05
Affinity constants KA (109 1/M)
Rat 26 Hum 2.9 0.05
Rat 81 Hum 8.5 0.02
Rat 187 Hum 6.2 0.84
Rat 327 Hum 2.0 0.03
Hum 81 Rat 2.9 0.06
Hum 187 Rat 5.2 0.70
Hum 277 Rat 4.6 0.12
Hum 327 Rat 6.7 0.44
1.2 0.05
no binding
0.61 0.02
1.0 0.07
1.3 0.16
0.61 0.004
no binding
no binding
no binding
no binding
February 19, 2004
MA-56A7C10
1.2 0.05
no binding
3791
Affinity of MA-56A7C10 (109 1/M)
260.61 0.02
1.0 0.07
1.3 0.16
0.61 0.004
187
81
327
81
277
187
327
no binding
no binding
no binding
no binding
February 19, 2004
Species cross-reactivity1 379
Human PAI-1327
Human +Porcine +Rat -Murine -
EETT
350
February 19, 2004
Alanine-scanning
E350
February 19, 2004
Alanine-scanning
E350
EKE242-4RR185-7
HK190-1
D193
DR355-6
February 19, 2004
Variants Mutated residues(1) KA (1/M) MA-56A7C10 (2)
PAI-1-wt none 5.4 ± 1.7 109
(1) residues mutated into an alanine(2) mean SD (n = 2 to 6)
EKE242-4A E242K243E244 0.11 ± 0.01 109
R356A R356 0.067 ± 0.003 109
E350A E350 0.18 ± 0.01 109
Affinity constants for PAI-1 mutants
February 19, 2004
Molecular
explanation
• neutralizing
properties
MA-56A7C10
Epitope in the vicinity and comprising
the distal hinge of the reactive site
loop
• higher affinity
for active PAI-1
February 19, 2004
Active PAI-1 Latent PAI-1
Higher affinity for PAI-1 in the active form
February 19, 2004
Monoclonal Antibodies
• Detailed characterization
• Epitope localizaton
• Cloning of smaller fragments ( scFv)
February 19, 2004
VH VL
Neutralization of PAI-1 by MA-8H9D4 andscFv-8H9D4
molar excess
% n
eutr
aliz
atio
n o
f P
AI-
1 ac
tivi
ty
0 1 2 3 4 50
50
100
MA-8H9D4
scFv-8H9D4
February 19, 2004
Paratope of scFv-8H9D4
February 19, 2004
General conclusions
• PAI-1 is a putative therapeutic target
• PAI-1 can be targeted at various positions
February 19, 2004
February 19, 2004
General conclusions
• PAI-1 is a putative therapeutic target
• PAI-1 can be targeted at various positions
• Molecular and structural information on epitopes and paratopes allows further rational design of PAI-1 neutralizing compounds
February 19, 2004