Laboratory for Thrombosis ResearchLaboratory for Thrombosis Research
Interdisciplinary Research CenterInterdisciplinary Research Center
KU Leuven Campus Kortrijk, Belgium KU Leuven Campus Kortrijk, Belgium
Antibodies that interfere with the collagen-vWF-GPIb axisAntibodies that interfere with the collagen-vWF-GPIb axis
anti-GPIb antibody anti-GPIb antibody 6B46B4anti-vWF A3 domain antibody anti-vWF A3 domain antibody 82D6A382D6A3
- epitope mappingepitope mapping- antithrombotic effectantithrombotic effect
collagen
GPIb/IX/VGPIb/IX/V
vWFvWF
Platelet adhesionPlatelet adhesion
von Willebrand Factorvon Willebrand Factorvon Willebrand Factorvon Willebrand Factor
NN CC
D1D1 D2D2 D’D’ D3D3 A1A1 A2A2 A3A3 D4D4B1B1 B2B2 C1C1 C2C2
PropeptidePropeptide Mature vWFMature vWF
CollagenCollagenGPIbGPIbCollagenCollagenHeparinHeparin
HeparinHeparinFVIIIFVIII
GPIIb/IIIaGPIIb/IIIa
GPIb/IX- vWF-interactionGPIb/IX- vWF-interaction
Shear applied Shear applied by AFM probeby AFM probe
35 Dyn/cm35 Dyn/cm22 applied by applied by rotating diskrotating disk
No shearNo shear
Siedlecki et al, Siedlecki et al, Blood 88, 2939, 1996Blood 88, 2939, 1996
Deckmyn, 1997Deckmyn, 1997
Platelet GPIb/V/IX complexPlatelet GPIb/V/IX complex
GP IbGP Ib
GP VGP V
GP IXGP IXGP IXGP IX
GP IbGP Ib GP IbGP Ib
GP IbGP Ib
plasma membraneplasma membrane
vWFvWF
S-SS-S
S-S S-S
collagencollagen
A1A1
A3A3
vWFvWF
6B4 6B4
82D6A382D6A3
Dynamic : Perfusion chamberShear-induced platelet adhesion
collagen coverslip
pump
Blood
perfusion chamber
water bath 37°C
anti-GPIb MoAb 6B4
+ anti-GPIb MoAb 6B4
control - PBS
imaging
00 11 22 33 44 55 202000
2020
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6060
8080
100100
maxim
al p
late
let
ad
hesio
n (%
)m
axim
al p
late
let
ad
hesio
n (%
)
µg/mLµg/mL
Mid (1300 s-Mid (1300 s-11 ) I ) ICC5050 : 1.0 : 1.0 µg/mlµg/ml
Low (650 s-Low (650 s-11 ) I ) ICC5050 : 3.3 : 3.3 µg/mlµg/ml
High (2600 sHigh (2600 s-1-1) I) ICC5050 : 0.5 : 0.5 µg/mlµg/ml
Shear dependent effect of anti-GPIb Fab 6B4 Shear dependent effect of anti-GPIb Fab 6B4 on platelet adhesion to collagenon platelet adhesion to collagen
650650 13001300 26002600
00
1010
2020
3030
4040
% s
urf
ace
cov
erag
e%
su
rfac
e co
ver
age
shear sshear s-1-1
00
1010
2020
3030
% s
urf
ace
cov
erag
e%
su
rfac
e co
ver
age
00 2.52.5 55 1010 2020
82D6A3 Fab (µg/ ml)82D6A3 Fab (µg/ ml)
82D6A3 inhibits platelet adhesion to collagen under flow82D6A3 inhibits platelet adhesion to collagen under flow
2600 s2600 s-1-1
5 µg/ml5 µg/ml
I. Inhibitory anti-GPIb antibodies I. Inhibitory anti-GPIb antibodies
I. Inhibitory anti-GPIb antibodies I. Inhibitory anti-GPIb antibodies
Epitope mapping of inhibitory antibodies against Epitope mapping of inhibitory antibodies against platelet GPIb platelet GPIb reveals interaction between the reveals interaction between the leucine-rich repeat N-terminal and C-terminal leucine-rich repeat N-terminal and C-terminal
domains of GPIb domains of GPIb
N. Cauwenberghs, K. Vanhoorelbeke, S. Vauterin, G. Romo, N. Cauwenberghs, K. Vanhoorelbeke, S. Vauterin, G. Romo, DF. Westra, E. Huizinga, J. Lopez, MC. Berndt, H.Deckmyn DF. Westra, E. Huizinga, J. Lopez, MC. Berndt, H.Deckmyn
Blood Blood 98:652-660, 2001 98:652-660, 2001
Platelet GPIb/V/IX complexPlatelet GPIb/V/IX complex
GP IbGP Ib
GP VGP V
GP IXGP IXGP IXGP IX
GP IbGP Ib GP IbGP Ib
GP IbGP Ib
plasma membraneplasma membrane
vWFvWF
S-SS-S
S-S S-S
Structure of the N-terminal domain of GPIbStructure of the N-terminal domain of GPIb Structure of the N-terminal domain of GPIbStructure of the N-terminal domain of GPIb
Uff Uff et al, et al, J Biol Chem. 2002;277:35657J Biol Chem. 2002;277:35657
Huizinga et al, Huizinga et al, Science 2002Science 2002;; 297: 1176 297: 1176
Static: Inhibition of vWF - GPIb interaction Static: Inhibition of vWF - GPIb interaction in ELISA by anti-GPIb MoAbsin ELISA by anti-GPIb MoAbs
vWF
bin
din
g (
OD
492
nm
)vW
F b
ind
ing
(O
D 4
92n
m)
00 22 44 66 88 1010 12120,00,0
0,10,1
0,20,2
0,30,3
0,40,4 26D126D1
27A1027A10 12G112G1 12E412E4 6B46B4
24G1024G10
µg/mLµg/mL
ristocetinristocetin
00 55 1010 1515 2020 25250,00,0
0,10,1
0,20,2
0,30,3
0,40,4
0,50,5 26D126D1
27A1027A10 12G112G1 12E412E4
6B46B4 24G10 24G10
vWF
bin
din
g (
OD
492
nm
)vW
F b
ind
ing
(O
D 4
92 n
m)
µg/mLµg/mL
botrocetinbotrocetin
Cross-blocking ELISA’s between MoAbs Cross-blocking ELISA’s between MoAbs for binding to plateletsfor binding to platelets
++++
++++
++++
++++
++++
++++
++++ ++++ ++++ ++++ ++++
++++
++++
++++
++++
-- -- --
--
--
--
--
--
--
TM60TM60
LJIb1LJIb1
AK2AK2
--
--
--
--
--
--
--
--
--
++++
++++
++++
++++
++++
++++
TM60TM60 LJIb1LJIb1 AK2AK2
bind to two regions on GPIbbind to two regions on GPIbTwo groups of intercompeting MoAbsTwo groups of intercompeting MoAbs Where ?Where ?
++++
++++
++++
++++
++++
++++
++++
++++
++++
--
--
--
--
--
--
++++
++++
++++
++++
--
--
--
--
--
--
12G112G1
12E412E4
27 A1027 A10
24G1024G10
6B46B4
24G1024G10 6B46B412G112G1 12E412E4 27A1027A10biotinylatedbiotinylated
Human - canine chimeras of GPIbHuman - canine chimeras of GPIb((Shen et al.,Shen et al., Blood 2000) Blood 2000)
LRR 1 LRR 2 LRR 3 LRR 4 LRR 5 LRR 6 LRR 7LRR 1 LRR 2 LRR 3 LRR 4 LRR 5 LRR 6 LRR 7
11 3535 5959 8181 200200104104 128128 152152 176176 268268 282282aaaaN-flankN-flank
Human Human Dog Dog C 35C 35
C 59C 59
C C 81 ...81 ...
C-flankC-flank
DogDogHuman Human H 35H 35
H 59H 59
H H 81 ...81 ...
Expression on CHO-cells Expression on CHO-cells Binding of Abs: FACS analysisBinding of Abs: FACS analysis
6B4, 6B4, TM60TM60
AK2AK2
24G1024G10
LJIb1LJIb1
27A10, 12E4, 12G127A10, 12E4, 12G1
Chimeras : resultsChimeras : results
Selected 6B4-binding sequencesSelected 6B4-binding sequences
C7 and L15 library:C7 and L15 library: a consensus sequence a consensus sequence C N C N K P G EK P G E R T C R T C
C D T L C D T L K P G EK P G E C C
C A D Q C A D Q K P G EK P G E C C
C C K P G EK P G E V Q Q C V Q Q C
C Y C Y K P G EK P G E W A C W A C
C C K P K P VV E E N R A C N R A CC C K P G EK P G E V Q Q C V Q Q C K P G EK P G E M R M R K P G EK P G E M R G A A M R G A A K P G K P G D P S A L H V V R C W I CD P S A L H V V R C W I C
D G R R D V V V R S A T F Y LE G L Y S P W W P R S L P V L
S S K T R S F G V H L V G P YL P P G L H V F P L A S N R S
S D K F P V Y K YS D K F P V Y K Y P GP G K G C PK G C PGPIbGPIb 251-265 251-265
W K Q G V D V K A M T S N N V A S
GPIbGPIb 230-245230-245
Vincente JBC 1990, 265, 274
L15 libraryL15 library
6B46B4phage peptidesphage peptides
‘‘gain-of-function’gain-of-function’Platelet-type vWDPlatelet-type vWDaaaa G G233233V, MV, M239239VV
6B4 epitope : Phage display peptide libraries6B4 epitope : Phage display peptide libraries
Biacore binding of 6B4 and 24G10 to Biacore binding of 6B4 and 24G10 to wild-type and mutant rec GPIbwild-type and mutant rec GPIb (1-280)(1-280)
6B46B4
5050
100100
150150
200200
250250
300300
450450 550550 650650 750750 850850
RURU
Time (sec)Time (sec)
Wild-typeWild-type
M239VM239V
G233VG233VMutantsMutants
6B4: reduced binding to PT-vWD mutants6B4: reduced binding to PT-vWD mutants
00
5050
100100
150150
200200
250250
350350 450450 550550 650650 750750 850850
RURU
Time (sec)Time (sec)
24G1024G10
Wild-typeWild-type
M239VM239V
G233VG233VMutantsMutants
24G10: enhanced binding to PT-vWD mutants24G10: enhanced binding to PT-vWD mutants
6B46B4‘‘gain-of-function’gain-of-function’Platelet-type vWDPlatelet-type vWDaaaa G G233233V, MV, M239239VV
24G1024G10
Mutants: resultsMutants: results
‘‘platelet-type von Willebrand’s disease mutationsplatelet-type von Willebrand’s disease mutations close proximity close proximity
• Two groups of anti-GPIb MoAbs with distinct inhibitory Two groups of anti-GPIb MoAbs with distinct inhibitory characteristicscharacteristics
ConclusionsConclusions
• Two regions on GPIbTwo regions on GPIb important for binding of vWF : important for binding of vWF :
I.I. Leucine-rich repeat N-terminal flanking region and first adjacent LRRs Leucine-rich repeat N-terminal flanking region and first adjacent LRRs
(aa 1-81)(aa 1-81)
II.II. Leucine-rich repeat C-terminal flanking region (aa 200-268) Leucine-rich repeat C-terminal flanking region (aa 200-268)
Regulates binding properties of aa 1-81 of GPIbRegulates binding properties of aa 1-81 of GPIb
Uff et al, J Biol Chem. 2002 Jun 26
Structure of the N-terminal domain of GPIbStructure of the N-terminal domain of GPIb and the vWF A1-domain- GPIband the vWF A1-domain- GPIbcomplexcomplexStructure of the N-terminal domain of GPIbStructure of the N-terminal domain of GPIb and the vWF A1-domain- GPIband the vWF A1-domain- GPIbcomplexcomplex
Huizinga et al, Science 2002; 297: 1176-1179
I. Inhibitory anti-GPIb antibodies I. Inhibitory anti-GPIb antibodies Antithrombotic effectAntithrombotic effect
Cauwenberghs N, Cauwenberghs N, Meiring M, Meiring M, Lamprecht S, Lamprecht S, Roodt JP, Roodt JP, Vauterin S, Vauterin S, Deckmyn H, Deckmyn H, Kotzé HFKotzé HF
Antithrombotic effect of platelet glycoprotein Ib blocking monoclonal antibody Antithrombotic effect of platelet glycoprotein Ib blocking monoclonal antibody Fab-fragments in a baboon model. Fab-fragments in a baboon model.
Arterioscl. Thromb. Vasc Biol, 20, 1347-1353, 2000Arterioscl. Thromb. Vasc Biol, 20, 1347-1353, 2000
D. Wu, M. Meiring, HF Kotzé, H Deckmyn, N. Cauwenberghs D. Wu, M. Meiring, HF Kotzé, H Deckmyn, N. Cauwenberghs
Inhibition of platelet GPIb, GPIIb/IIIa or both by monoclonal antibodies, prevents Inhibition of platelet GPIb, GPIIb/IIIa or both by monoclonal antibodies, prevents arterial thrombus formation in baboons. arterial thrombus formation in baboons.
Arterioscl. Thromb. Vasc Biol,Arterioscl. Thromb. Vasc Biol, 22, 323-328, 2002 22, 323-328, 2002
Baboon Extracorporeal Shunt Model Baboon Extracorporeal Shunt Model Baboon Extracorporeal Shunt Model Baboon Extracorporeal Shunt Model
DetectorDetector
Computer Computer
Collagenic surfaceCollagenic surfaceSilicone rubber tubingSilicone rubber tubing
Teflon tubingTeflon tubing
3 mm
baboonbaboon
111-In 111-In labeled labeled plateletsplatelets
Baboon Models Baboon Models Baboon Models Baboon Models
i.v. anti-GPIb antibody 6B4i.v. anti-GPIb antibody 6B4
IgGIgG profound thrombocytopenia profound thrombocytopenia(Fab’)(Fab’)22 thrombocytopenia thrombocytopenia
FabFab minor drop in platelet counts minor drop in platelet counts
11 1010 10010000
11
22
33
Mo
lecu
les/
pla
tele
t (x
10
Mo
lecu
les/
pla
tele
t (x
10
44 ))
nmol/Lnmol/L
IgGIgG
(Fab’)(Fab’)22
FabFab
0 10 20 30
1
2
3
Time Time (minutes)(minutes)
controlcontrol
160 µg/kg160 µg/kg
4
320 µg/kg320 µg/kg
Pla
tele
ts d
ep
osit
ed
(x 1
0P
late
lets
dep
osit
ed
(x 1
099))
Baboon Extracorporeal Shunt Model Baboon Extracorporeal Shunt Model Baboon Extracorporeal Shunt Model Baboon Extracorporeal Shunt Model
Inhibition of Platelet Deposition by anti-GPIb Fab 6B4Inhibition of Platelet Deposition by anti-GPIb Fab 6B4
640 µg/kg640 µg/kg
80 µg/kg80 µg/kg
Baboon femoral artery Folts’ ModelBaboon femoral artery Folts’ Model Baboon femoral artery Folts’ ModelBaboon femoral artery Folts’ Model
1.1. High shear modelHigh shear model
2. Comparison 2. Comparison anti-GPIb (6B4) anti-GPIb (6B4) anti-GPIIb/IIIa (16NC72) anti-GPIIb/IIIa (16NC72) anti-GPIb + anti-GPIIb/IIIaanti-GPIb + anti-GPIIb/IIIa
Baboon femoral artery Folts’ ModelBaboon femoral artery Folts’ Model Baboon femoral artery Folts’ ModelBaboon femoral artery Folts’ Model
Inhibition of CFR by anti-GPIb Fab 6B4 Inhibition of CFR by anti-GPIb Fab 6B4
0.6 mg/kg 6B4 0.6 mg/kg 6B4
2.0 mg/kg 6B42.0 mg/kg 6B42.0 mg/kg 6B42.0 mg/kg 6B4
Anti-Ib Anti-Ib ( 0.6)( 0.6) + + Anti-IIb/IIIaAnti-IIb/IIIa ( 0.1)( 0.1)
********
**
******
0.00.0
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120120
140140
CF
R (
% o
f p
re-c
on
tro
l)C
FR
(%
of
pre
-co
ntr
ol)
2.02.00.60.6
Anti-IbAnti-Ib
(mg/kg)(mg/kg)
**
******
Baboon femoral artery Folts’ Model Baboon femoral artery Folts’ Model
****
0.30.30.10.1
Anti-IIb/IIIaAnti-IIb/IIIa
0.00.0
**
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2020
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6060
8080
100100
00 3030 6060 150150 300300 24 h24 h
TimeTime ( (min)min)
% i
nh
ibit
ion
of
ris
toc
eti
n-i
nd
uc
ed
%
in
hib
itio
n o
f ri
sto
ce
tin
-in
du
ce
d
pla
tele
t a
gg
reg
ati
on
p
late
let
ag
gre
ga
tio
n
2.0 mg/kg 6B42.0 mg/kg 6B4
Pla
sm
a c
on
ce
ntr
ati
on
of
6B
4 F
ab
P
las
ma
co
nc
en
tra
tio
n o
f 6
B4
Fa
b
(µg
/ml)
(µg
/ml)
00
1010
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4040
5050
% o
f G
PIb
re
cep
tor
occ
up
anc
y%
of
GP
Ib r
ece
pto
r o
ccu
pan
cy
Plasma levels, receptor occupancy and RiPAPlasma levels, receptor occupancy and RiPAPlasma levels, receptor occupancy and RiPAPlasma levels, receptor occupancy and RiPA
GPIb-occupancy needed for Ripa and CFRGPIb-occupancy needed for Ripa and CFRGPIb-occupancy needed for Ripa and CFRGPIb-occupancy needed for Ripa and CFR
00 2020 4040 8080% r
isto
ceti
n-i
nd
uce
d a
gg
luti
nat
ion
(
)%
ris
toce
tin
-in
du
ced
ag
glu
tin
atio
n (
)
% GPIb Receptor occupancy% GPIb Receptor occupancy
6060
00
2020
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6060
8080
100100
00
2020
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6060
8080
100100
% C
FR
(
)%
CF
R (
)
20% 20% 32% 32%
Bl e
edi n
g t
ime
(min
)B
leed
ing
ti m
e (m
in)
00
55
1010
1515
2020
2525
00 3030 6060
Time after iv (min)Time after iv (min)
**
** •Anti-IIb/IIIa Anti-IIb/IIIa 0.3 mg/kg0.3 mg/kg
•Anti-IIb/IIIa Anti-IIb/IIIa 0.1 mg/kg0.1 mg/kg•Anti-IbAnti-Ib 0.6 mg/kg0.6 mg/kg•Anti-Ib Anti-Ib 2.0 mg/kg2.0 mg/kg•0.6 Anti-Ib + 0.1 Anti-IIb/IIIa0.6 Anti-Ib + 0.1 Anti-IIb/IIIa
Template bleeding time in baboonsTemplate bleeding time in baboonsTemplate bleeding time in baboonsTemplate bleeding time in baboons
ConclusionsConclusionsConclusionsConclusions
1. Inhibition of GPIb is antithrombotic in two baboon models1. Inhibition of GPIb is antithrombotic in two baboon models
2. Administration of anti-GPIb Fab-fragments does not provoke 2. Administration of anti-GPIb Fab-fragments does not provoke thrombocytopeniathrombocytopenia
3. Inhibition of GPIb has minor effects on the bleeding time3. Inhibition of GPIb has minor effects on the bleeding time
4.4. Combination of low dose anti-GPIb and anti-GPIIb/IIIa Combination of low dose anti-GPIb and anti-GPIIb/IIIa antibodies is antithrombotic with moderate effects on antibodies is antithrombotic with moderate effects on the bleeding timethe bleeding time
II. Inhibitory II. Inhibitory anti-vWF A3 domain antibodyanti-vWF A3 domain antibody
II. Inhibitory II. Inhibitory anti-vWF A3 domain antibodyanti-vWF A3 domain antibody
Epitope mappingEpitope mapping
Vanhoorelbeke K, Depraetere H, Romijn RAP, Huizinga E, De Maeyer M, Deckmyn HVanhoorelbeke K, Depraetere H, Romijn RAP, Huizinga E, De Maeyer M, Deckmyn H J. Biol. Chem. 278, 37815–37821, 2003
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00 1010 2020 3030
% i
nh
ibit
ion
% i
nh
ibit
ion
vWF (vWF (g/ml)g/ml)
00
0.20.2
0.40.4
0.60.6
0.80.8
1.01.0
1.21.2
00 55 1010
Phages (10Phages (101010/ml)/ml)
OD
(49
0-63
0)O
D (
490-
630)
82D6A3 binding phages82D6A3 binding phages
Phage binding to Phage binding to 82D6A382D6A3
Inhibition of phage Inhibition of phage binding to 82D6A3 binding to 82D6A3 by vWFby vWF
00
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% i
nh
ibit
ion
% i
nh
ibit
ion
vWF (vWF (g/ml)g/ml)
00
0.20.2
0.40.4
0.60.6
0.80.8
1.01.0
1.21.2
00 55 1010
Phages (10Phages (101010/ml)/ml)
OD
(49
0-63
0)O
D (
490-
630)
82D6A3 binding phages82D6A3 binding phages
Phage binding to Phage binding to 82D6A382D6A3
Inhibition of phage Inhibition of phage binding to 82D6A3 binding to 82D6A3 by vWFby vWF
C6-libraryC6-library CMTCMTSPWRSPWRC C 8/13 clones8/13 clones CRTCRTSPWRSPWRC C 4/13 4/13 CYRCYRSPWRSPWRC C 1/131/13
L15-libraryL15-library GDCFFGFLN GDCFFGFLNSPWRSPWRVVC C RRSSSYWVYSYWVYSPWRSPWRFISR FISR
Peptides displayed on 82D6A3 binding phagesPeptides displayed on 82D6A3 binding phages
vWF(974-989) vWF(974-989) SSITTIDVITTIDVPWPWNNVVVPEKVPEK
Trp 982
Localisation of Localisation of PWPW on vWF-A3-domain on vWF-A3-domain
Pro 981
wtwt A3A3
mock
mock
0
25
50
75
100
125
Per
cen
tag
e 82
D6A
3 a
nti
bo
dy
bo
un
d82D6A3 binding to 28 82D6A3 binding to 28 vWF vWF point mutantspoint mutants
Pro98
1His
Pro98
1His
Pro98
1Ala
Pro98
1Ala
Arg96
3Ala
Arg96
3Ala
Pro96
2His
Pro96
2His
Gln96
6Ala
Gln96
6Ala
Thr977
Ala
Thr977
Ala
Ile97
5Ala
Ile97
5Ala
Ser97
4Ala
Ser97
4Ala
Asp97
9Ala
Asp97
9Ala
Val98
0Ala
Val98
0Ala
Asn98
3Ala
Asn98
3Ala
Val98
4Ala
Val98
4Ala
Val98
5Ala
Val98
5Ala
Glu98
7Ala
Glu98
7Ala
His99
0Ala
His99
0Ala
Ser99
3Ala
Ser99
3Ala
Val99
7Ala
Val99
7Ala
Gln99
9Ala
Gln99
9Ala
Glu10
01Ala
Glu10
01Ala
Gln10
06Ala
Gln10
06Ala
Asp10
09Ala
Asp10
09Ala
Arg10
16Ala
Arg10
16Ala
Ser10
20Ala
Ser10
20Ala
Glu10
21Ala
Glu10
21Ala
Met
1022
Ala
Met
1022
Ala
His10
23Ala
His10
23Ala
Pro10
27His
Pro10
27His
Pro 981Pro 981Trp 982Trp 982
Met 1022Met 1022
His 1023His 1023
Ser 1020Ser 1020
Asp 1009Asp 1009
Arg 1016Arg 1016
Epitope of 82D6A3 on vWF-A3 domain
Epitope of 82D6A3 on vWF-A3 domain
modeled cyclic CMTSPWRC modeled cyclic CMTSPWRC mapped onmapped on VWF-A3 domainVWF-A3 domain
““exposed form” of Trp982exposed form” of Trp982 ““buried form” of Trp982 (crystal)buried form” of Trp982 (crystal)
modeled cyclic CMTSPWRC modeled cyclic CMTSPWRC mapped onmapped on VWF-A3 domainVWF-A3 domain
Fluorescence decay measurements yielded two lifetime components of 2,08 ns and of 6,26 ns
Energy minimalisation calculationShows two rotamer clusters
M Hellings, Y Engelborghs, H Deckmyn, ME Schiphorst, JW Akkerman, M De Maeyer submitted
00
2020
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00 1010 2020 3030 4040 5050 6060 7070 8080 9090 100100
Coll III bindingColl III binding
82D
6A3
bin
din
g8
2D6A
3 b
ind
ing
S968TS968T
D1009AD1009A
S974A I975A T977AS974A I975A T977AV997A E1001AV997A E1001A
Correlation between mutant binding to collagen and 82D6A3Correlation between mutant binding to collagen and 82D6A3
H1023AH1023A
S1020AS1020A
P981HP981H
R1016AR1016A
ConclusionsConclusions
- 82D6A3 prevents vWF binding to collagen in vitro 82D6A3 prevents vWF binding to collagen in vitro
- 82D6A3 binds to the vWF-A3 domain82D6A3 binds to the vWF-A3 domain
- by phage display aby phage display a Ser-Pro-Trp-Arg-mimotopeSer-Pro-Trp-Arg-mimotope sequence is identifiedsequence is identified
- by analysis of mutant-vWF the binding area is identified at the ‘by analysis of mutant-vWF the binding area is identified at the ‘front face’ ’ of A3, withof A3, with Pro981, Asp1009, Arg1016, Ser1020, Met1022, His1023 Pro981, Asp1009, Arg1016, Ser1020, Met1022, His1023 as dominant residuesas dominant residues
- His1023,His1023, Ser1020 Ser1020 and lessand less Arg1016,Arg1016, (Pro981)(Pro981) are necessary are necessary for binding to both 82D6A3 and to collagen confirming for binding to both 82D6A3 and to collagen confirming the localisation of the collagen binding site within vWF-A3 the localisation of the collagen binding site within vWF-A3 and explaining the inhibitory activity of 82D6A3and explaining the inhibitory activity of 82D6A3
D. Wu, K. Vanhoorelbeke, N. Cauwenberghs, G. Vandecasteele, S. D. Wu, K. Vanhoorelbeke, N. Cauwenberghs, G. Vandecasteele, S. Vauterin, M. Meiring, S. Lambrecht, JP Rood, H. Depraetere, Vauterin, M. Meiring, S. Lambrecht, JP Rood, H. Depraetere,
H. Kotzé, H. DeckmynH. Kotzé, H. Deckmyn
Blood 99, 3623-3628, 2002Blood 99, 3623-3628, 2002
Laboratory for Thrombosis Research, KU Leuven Campus Kortrijk, Laboratory for Thrombosis Research, KU Leuven Campus Kortrijk, Kortrijk, BelgiumKortrijk, Belgium
Department of Hematology and Cell Biology, U Orange Free State, Department of Hematology and Cell Biology, U Orange Free State, Bloemfontein, South-AfricaBloemfontein, South-Africa
II. Inhibitory II. Inhibitory anti-vWF A3 domain antibodyanti-vWF A3 domain antibody Antithrombotic effectAntithrombotic effect
• Further in vitro characterization of 82D6A3
00 22 44 66 88 1010
0.00.0
0.10.1
0.20.2
0.30.3
0.40.4
0.50.5
0.60.6
0.70.7
OD
490
nm
OD
490
nm
µg/ml 82D6A3µg/ml 82D6A3
0.10.1 11 1010 100100 1000100000
2020
4040
6060
8080
100100
120120
%
% v
WF
vWF
bin
din
g b
ind
ing
ng/mng/mll 82D6A3 82D6A3
Inhibition of baboon vWF binding to collagen by
82D6A3
Removal of collagen-bound baboon vWF by 82D6A3 ( )
In vivoIn vivo inhibition of arterial thrombus formation : inhibition of arterial thrombus formation : Folts’ modelFolts’ model
– e.g. administration of 100 µg/kg 82D6A3e.g. administration of 100 µg/kg 82D6A3
0.1 mg/kg 82D6A3
Antithrombotic effect of 82D6A3Antithrombotic effect of 82D6A3
00
2020
4040
6060
8080
100100
120120
0 100 300 6000 100 300 600
µg/kg 82D6A3µg/kg 82D6A3
% r
edu
ctio
n i
n C
FR
sC
FR
s
P<0.05
P<0.01
P<0.01
In vivoIn vivo inhibition of CFRs by inhibition of CFRs by 82D6A382D6A3
Relation between Relation between in vivoin vivo reduction in CFRs and reduction in CFRs and ex ex
vivovivo vWF-collagen vWF-collagen bindingbinding
00 2020 4040 6060 8080 100100
00
2020
4040
6060
8080
100100
% r
edu
ctio
n in
CF
R's
% r
edu
ctio
n in
CF
R's
% % ex vivoex vivo collagen binding collagen binding
82D6A3 in vivo82D6A3 in vivo
• Coagulation tests, platelet count, Coagulation tests, platelet count, vWF-Ag levels: vWF-Ag levels:
no significant changesno significant changes
• Bleeding timeBleeding time: no significant prolongation : no significant prolongation
even not with ‘overdose’ (600 µg/kg)even not with ‘overdose’ (600 µg/kg)
RESULTS
00
2020
4040
6060
8080
100100
120120
00
22
44
0’ 30’ 60’ 150’ 300’ 24h 48h0’ 30’ 60’ 150’ 300’ 24h 48h
Ex vivo analysis after administration of 300 µg/kg 82D6A3
% v
WF
-co
llag
en
bin
din
g (
% v
WF
-co
llag
en
bin
din
g (
)
)
% v
WF
-occ
(
)%
vW
F-o
cc (
)
µg
/ml 8
2D6A
3 (
µ
g/m
l 82D
6A3
( ))
ConclusionsConclusions
– Blocking VWF-A3 domain is anti-thrombotic
– Without major prolongation of the bleeding timereason why so far few patients with defect in vWF-collagen interaction have been
identified?
– The importance of the vWF-collagen interaction in primary hemostasis under high shear was indicated by in vitro experiments
This study provided the first in vivo proof
collagencollagen
General conclusionsGeneral conclusions
The collagen-vWF-GPIb axis is an The collagen-vWF-GPIb axis is an interesting target for the interesting target for the develoment of new antithrombotic develoment of new antithrombotic agentsagentswith little effect on bleeding timewith little effect on bleeding time
A1A1
A3A3
vWFvWF
VCL, 6B4 VCL, 6B4
AJvW2 AJvW2
saratinsaratin
82D6A382D6A3