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

4040

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

00

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100100

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

**

00

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

00

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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

2020

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6060

8080

100100

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

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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100100

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