The von Willebrand factor self-association is modulated by a multiple domain interaction

ORIGINAL ARTICLE

The von Willebrand factor self-association is modulated by amultiple domain interaction

H. ULR ICHTS , K . VANHOORELBEKE , J . P . G IRMA,* P . J . L ENT ING,� S . VAUTER IN and H . DECKMYNLaboratory for Thrombosis Research, KU Leuven Campus Kortrijk, Kortrijk, Belgium; *Inserm U143, Le Kremlin-Bicetre, France; �Laboratory for

Thrombosis and Haemostasis, Department of Haematology, University Medical Center Utrecht, Utrecht, the Netherlands

To cite this article: Ulrichts H, Vanhoorelbeke K, Girma JP, Lenting PJ, Vauterin S, Deckmyn H. The von Willebrand factor self-association is

modulated by a multiple domain interaction. J Thromb Haemost 2005; 3: 552–61.

Summary. Background: Platelet adhesion and aggregation at

sites of vascular injury exposed to rapid blood flow require

von Willebrand factor (VWF). VWF becomes immobilized by

binding to subendothelial components or by a self-association

at the interface of soluble and surface-bound VWF.

Objectives: As this self-association has been demonstrated

only under shear conditions, our first goal was to determine

whether the same interaction could be observed under static

conditions. Furthermore, we wanted to identify VWF

domain(s) important for this self-association. Results: Biotin-

ylated VWF (b-VWF) interacted dose-dependently and

specifically with immobilized VWF in an enzyme-linked

immunosorbent assay (ELISA) assay, showing that shear is

not necessary to induce the VWF self-association. Whereas

anti-VWF monoclonal antibodies (mAbs) had no effect on the

self-association, the proteolytic VWF-fragments SpII(1366–

2050) and SpIII(1–1365) inhibited the b-VWF–VWF interac-

tion by 70 and 80%, respectively. Moreover, a specific binding

of b-VWF to immobilized Sp-fragments was demonstrated.

Finally, both biotinylated SpII and SpIII were able to bind

specifically to both immobilized SpII and SpIII. Similar results

were observed under flow conditions, which confirmed the

functional relevance of our ELISA system. Conclusion: We

have developed an ELISA binding assay in which a specific

VWF self-association under static conditions can be demon-

strated. Our results suggest a multiple domain interaction

between immobilized and soluble VWF.

Keywords: von Willebrand factor, platelet adhesion,

A-domains.

Introduction

Vascular damage is followed rapidly by platelet responses

leading to the firm attachment of platelets at the site of injury.

At sites of high blood flow, the vonWillebrand factor (VWF) is

essential for this process [1]. VWF modulates platelet-surface

and platelet–platelet interactions by linking platelet receptors to

the extracellular matrix and to one another [2]. Moreover,

VWF exerts a carrier function for the plasma coagulation

factor VIII [3], protecting it thereby from proteolytic inactiva-

tion. Soluble VWF is not able to interact with non-activated

platelets. However, when immobilized to subendothelial struc-

tures, VWF forms a reactive surface capable of capturing

platelets from flowing blood [4,5]. Earlier studies showed a

shear-dependent conformational change of VWF from a

globular state to an extended chain upon immobilization [6].

However, we recently failed in showing such a large overall

conformational change but did not exclude small-scale effects

of shear on one or more VWF domains [7]. Surface properties

itself have an effect on VWF shape [8]. Therefore, the

discrepancy between these studies can reside in the different

strategy of immobilizing VWF.

Transient interactions of the platelet GPIb-IX-V receptor

complex with the A1-domain of immobilized VWF [9] result in

an initial attachment, characterized by a continuous surface

translocation of the platelets [10]. This leads to stable platelet

adhesion through the platelet collagen receptors GPVI and

GPIa/IIa [11], activation of the platelet GPIIb/IIIa receptor

complex [12] and finally to platelet aggregation [13] and

thrombus growth.

In addition to circulating in plasma, VWF is deposited into

the extracellular matrix of the subendothelium [14]. Regardless

of the presence of this subendothelial VWF, several extracel-

lular matrix components bind plasma VWF rapidly upon

exposure to circulating blood due to vascular injury. VWF

binds directly to collagen types I, III and VI, and also interacts

with collagen type IV [15,16]. In addition, VWF contains two

distinct binding sites for heparin [17,18], which could possibly

interact with matrix proteoglycans that contain sulfated

sugars. The major collagen-binding site resides in the VWF

A3-domain [19]. Sites other than the VWF A3-domain could

Correspondence: Hans Deckmyn, Laboratory for Thrombosis

Research, IRC, KU Leuven Campus Kortrijk, E. Sabbelaan 53,

8500 Kortrijk, Belgium.

Tel.: +32 56 24 64 22; fax: +32 56 24 69 97; e-mail: Hans.Deckmyn@

kulak.ac.be

Received 15 March 2004, accepted 30 November 2004

Journal of Thrombosis and Haemostasis, 3: 552–561

� 2005 International Society on Thrombosis and Haemostasis

bind to extracellular matrix components, such as the VWF

A1-domain which is the primary interaction site for collagen

type VI [16]. However, we showed that the monoclonal anti-

VWF antibody 82D6A3, which inhibits the VWF interaction

with fibrillar collagen, inhibits arterial platelet thrombus

formation in vivo in baboons [20], suggesting that VWF sites

other than the A3-domain are less significant for binding to

the extracellular matrix. Moreover, these findings confirm that

the relevant thrombogenic connective tissue ligand is fibrillar

collagen.

Upon immobilization, VWF sustains a functionally relevant

homotypic self-association with soluble VWF multimers [21],

demonstrating that direct immobilization of VWF is not

strictly needed for interaction with the platelet GPIb-IX–V

complex. This function of circulating VWF in the initiation of

platelet adhesion was demonstrated in flow chamber studies:

in spite of the fact that the A1-domain of immobilized VWF is

essential for platelet translocation under shear [5], platelets

were still able to interact with immobilized DA1-VWF when

soluble plasma VWF was present. Moreover, a shear-

dependent self-association of VWF in suspension was demo-

nstrated in a cone-plate viscometer [22]. The goal of this study

was to elucidate, first, if shear is a prerequisite for the

interaction of soluble VWF with immobilized VWF. Sec-

ondly, we wanted to identify regions in VWF important for

this self-association.

Methods

Materials

The three fragments SpI (monomer, AA911–1365), SpII

(dimer, AA1366–2050) and SpIII (dimer, AA1–1365) were

produced by digestion of purified VWF with Staphyloccocus

aureus V8 protease [23]. Amino acids were numbered from the

first amino acid of the mature VWF subunit. Different

monoclonal antibodies (mAbs) to VWF have been described

previously: mAb 82D6A3 binds to the VWF A3 domain and

inhibits VWF binding to collagen types I, III and IV [16], mAb

1C1E7 recognizes theN-terminal part of VWF (AA1–272) [24].

mAb 724 and mAb 701 recognize the VWF A1 domain. mAb

724 blocks VWF binding to botrocetin and botrocetin-induced

VWF binding to GPIb [25], while mAb 701 inhibits ristocetin-

and botrocetin-induced platelet agglutination [26]. Recombin-

ant DA3-VWF (D909-1113) and recombinant DA1-VWF

(D478–716) were purified as described previously [19,27].

VWF (Red Cross, Brussels, Belgium), SpII, SpIII, DA3-VWF

and 82D6A3 were biotinylated using EZ-link Sulfo-NHS-

SS-Biotin (Perbio, Helsingberg, Sweden). Botrocetin was

purified from crude Bothrops jararaca venom (Sigma, St Louis,

MO, USA), as described previously [28]. A recombinant

N-terminal, VWF-binding GPIba fragment (AA1-289)

(rGPIba) was expressed in Chinese hamster ovary cells and

purified as described [29,30]. Heparin (Clexane) was obtained

from Aventis (Brussels, Belgium). Fibrinogen was from

Calbiochem (La Jolla, CA, USA).

Binding of VWF and biotinylated VWF to human collagen

type III

Ninety-six-well microtiter plates (Greiner, Frickenhausen,

Germany) were coated overnight (ON) at 4 �C with 25 lgmL)1 human collagen type III (Sigma), in phosphate-buffered

saline (PBS). Wells were blocked with PBS containing 3%

milk powder and incubated with a dilution series of purified

VWF or biotinylated VWF (b-VWF). Bound VWF or

b-VWF was detected for 45 min at RT with a 1/3000 dilution

of anti-VWF polyclonal antibodies labeled with horseradish

peroxidase (anti-VWF-Ig-HRP, Dako, Glostrup, Denmark).

Visualization was obtained with ortho-phenylenediamine

(Sigma) and H2O2 and the coloring reaction was stopped

with 4 mol L)1 H2SO4, after which the absorbance was

determined at 490 nm.

Botrocetin-induced binding of b-VWF and VWF to a

recombinant fragment of GPIba

The botrocetin-induced binding of b-VWF and VWF to

rGPIba was performed as described previously [30,31]. Briefly,

microtiter plates were coated ON at 4 �C with anti-GPIbamAb 2D4 (5 lg mL)1 in PBS) and blocked with Tris-buffered

saline (TBS) containing 3%milk powder.Wells were incubated

for 2 h at 37 �Cwith rGPIba (1 lgmL)1 in TBS, 0.1%Tween-

20) and incubated for 2 h at 37 �C with a dilution series of

VWF or b-VWF in TBS, 0.1% Tween-20 containing 0.01 lgmL)1 botrocetin. Bound VWF or b-VWF was detected with

anti-VWF-Ig-HRP as described above.

Multimeric analysis

The multimeric pattern of VWF and b-VWF was determined

essentially as described [32]. Briefly, VWF and b-VWF were

separated on sodium-dodecyl sulfate (SDS) 0.8–1.5% IEF

agarose gels. Gels were fixed on Gelbond (Cambrex Bio

Science Rockland, Inc., ME, USA) and VWF was immuno-

detected using anti-VWF-Ig-HRP andH2O2 and 3–3¢ diamino-

benzidine as substrate (DAB, Sigma).

Binding of soluble biotinylated VWF to immobilized VWF,

DA3-VWF, SpI, SpII or SpIII

Ninety-six-well microtiter polystyrene plates were directly

coated ON at 4 �C with VWF, DA3-VWF, bovine serum

albumin (BSA, Sigma), human serum albumin (HSA, Sigma),

SpI, SpII or SpIII (10 lg mL)1 in PBS) and blocked for 2 h at

room temperature (RT) with PBS containing 3%milk powder.

Wells were incubated for 2 h at 37 �C with a dilution series of

b-VWF in PBS containing 0.3% milk powder. Bound b-VWF

was detected for 45 min at RT with a 1/10 000 dilution of

streptavidin-HRP (Roche Molecular Biochemicals, Mann-

heim, Germany). Visualization was performed as described

above. The binding of soluble b-BSA to immobilized BSA or

VWF was performed in parallel.

Functional VWF self-association 553


Kinetic binding assays

Ninety-six-well microtiter plates were coated ON at 4 �C with

VWF and blocked as described above. For the association

experiments, wells were incubated at 37 �C for different

intervals with b-VWF (50 lg mL)1 in PBS containing 0.3%

milk powder). For the dissociation experiments, wells were first

equilibrated for 1.5 h with b-VWF (25 lg mL)1 in PBS

containing 0.3% milk powder), after which the supernatant

was removed and exchanged by a 10 · excess of unlabeled

VWF in PBS containing 0.3% milk powder. Bound b-VWF

was detected with streptavidin-HRP and visualization was

performed as described above.

Binding of soluble VWF to immobilized DA3-VWF: detection

of bound VWF with anti-VWF mAb 82D6A3

Ninety-six-well microtiter plates were coated ON at 4 �C with

DA3-VWF and blocked as described above. Wells were

incubated for 2 h at 37 �C with a dilution series of VWF in

PBS containing 0.3%milk powder, followed by the addition of

1 lgmL)1 of biotinylatedmAb 82D6A3 (b-82D6A3) for 1 h at

RT. Bound b-82D6A3 was detected with streptavidin-HRP

and visualization was performed as described above.

Inhibition of association of soluble VWF with immobilized

VWF, SpI, SpII or SpIII

Ninety-six-well microtiter plates were coated ON with VWF,

SpI, SpII or SpIII and blocked as described above. Wells were

incubated at RT with a dilution series of VWF, SpI, SpII,

SpIII, BSA, heparin, fibrinogen or the anti-VWF mAbs

1C1E7, 82D6A3, 701 or 724 in PBS containing 0.3% milk

powder to which a constant amount of b-VWFwas next added

after 30 min (final concentration of 25 lg mL)1 in PBS

containing 0.3% milk powder). After 2 h incubation at 37 �C,residual bound b-VWF was detected with streptavidin-HRP

and visualization was performed as described above.

Binding of soluble biotinylated SpII- and SpIII-fragment to

immobilized VWF, SpII- and SpIII-fragment

Ninety-six-well microtiter plates were coated ON with SpII- or

SpIII-fragment and blocked as described above. Wells were

incubated for 2 h at 37 �C with a dilution series of biotinylated

SpII- and SpIII-fragment (b-SpII and b-SpIII) in PBS

containing 0.3% milk powder. Bound b-SpII and b-SpIII

was detected with streptavidin-HRP and visualization was

performed as described above.

Inhibition of binding of soluble biotinylated SpII- and

SpIII-fragment to immobilized SpII- and SpIII-fragment by

soluble VWF

Ninety-six-well microtiter plates were coated ON with SpII-

and SpIII-fragment and blocked as described above. Wells

were incubated at RT with a dilution series of VWF in PBS

containing 0.3% milk powder to which a constant amount of

b-SpII or b-SpIII was next added after 30 min (final concen-

tration of 50 lg mL)1 in PBS containing 0.3% milk powder).

Residual bound b-SpII and b-SpIII was detected with

streptavidin-HRP and visualization was performed as des-

cribed above.

Flow studies

Glass coverslips were coated overnight at 4 �CwithDA1-VWF

or SpII fragment (40 lg mL)1 in PBS). Reconstituted perfu-

sates were prepared as described [27]. Briefly, platelet rich

plasma was obtained from whole blood by centrifugation and

platelets were washed in Krebs–Ringer buffer (4 mmol L)1

KCL, 107 mmol L)1 NaCl, 20 mmol L)1 NaHCO3, 2 mmol

L)1 Na2SO4) containing 19 mmol L)1 sodium citrate, pH 5.0.

Platelets were resuspended in Krebs–Ringer buffer containing

4% albumin to a platelet count of 190 000 lL)1. Autologous

red blood cells were washed with PBS containing 5 mM

glucose. At 5 min before perfusion, washed red blood cells

were added in a volume fraction of 40% of total volume.

Platelet adhesion was studied at 1600 s)1 at 37 �C for 5 min.

Perfusions were performed in the absence or presence of 20 lgmL)1 VWF, SpIII fragment orDA1-VWF.After perfusion, the

coverslips were rinsed in Hepes buffered saline (10 mmol L)1

Hepes, 145 mmol L)1 NaCl, pH 7.35), fixated by 0.5%

glutaraldehyde and stained with May–Grunwald/Giemsa.

Platelet adhesion was evaluated with a light microscope at

400 · magnification, connected to an Image Analyzer (Lucia,

Laboratory Imaging Ltd, Analis, Namur, Belgium). Platelet

adhesion was expressed as percentage of surface covered with

platelets. For each experiment, the mean ± SE of 10 images

was determined.

Results

Functional self-association of soluble and immobilized

VWF under static conditions

Our first goal was to determine whether soluble VWFwas able

to interact with immobilized VWF under static conditions. An

enzyme-linked immunosorbent assay (ELISA) assay was set up

in which the binding of soluble biotinylated VWF (b-VWF) to

immobilized VWF was determined. Soluble b-VWF bound

dose-dependently to immobilized VWF in this assay, demon-

strating that shear is not essential for the VWF self-association

(Fig. 1). The data were fitted using the specific binding model

characterized by the equation y ¼ Bmax x/(Kd,app + x), where

y represents the absorbance at 490 nm · the soluble b-VWF

concentration and Kd,app the apparent dissociation constant

[33]. In order to compare individual experiments, OD values

were recalculated as percentage binding using the extrapolated

parameter Bmax as 100% binding. Soluble b-VWF interacted

with a Kd,app of 93 ± 3 nmol L)1 using aMr of 270 kDa for a

VWF monomer [34]. Biotinylation had no major influence on

554 H. Ulrichts et al


VWF function as neither the binding to human collagen type

III nor the botrocetin-induced binding to rGPIba was altered

significantly (Fig. 2A). Ristocetin could not be used in the assay

as amodulator as this resulted in a too high background signal.

Moreover, themultimericVWFstructurewas not influenced by

the biotinylation process (Fig. 2B).

Specificity of the VWF self-association

In order to be sure that the binding of soluble b-VWF to

immobilized VWF was not caused by putative conformational

changes induced by the biotinylation process, the binding of

soluble b-VWF and soluble VWF to immobilized DA3-VWF

was compared: bound b-VWF was detected with streptavidin-

HRP, whereas bound VWF was measured with b-mAb

82D6A3 followed by streptavidin-HRP. As mAb 82D6A3

recognizes an epitope in the A3-domain of VWF, it does not

bind to immobilized DA3-VWF. Both VWF as well as b-VWF

interacted comparably with immobilized DA3-VWF (Fig. 3A),

showing that the measured self-association of VWF in the

assay is not caused by the biotinylation.

The interaction of soluble b-VWF with immobilized VWF

under static conditions was shown to be specific as soluble

b-VWF did not interact with immobilized BSA or immobilized

HSA, nor could soluble biotinylated BSA (b-BSA) interact

with immobilized VWF (Fig. 3B). Also, no self-association of

soluble b-BSA with immobilized BSA could be demonstrated.

Moreover, the binding of soluble b-VWF to immobilized VWF

could already be inhibited by at least 80% at a 12 molar excess

of unlabeled soluble VWF, whereas soluble BSA or fibrinogen

had no effect (Fig. 3C), suggesting that the measured self-

association is specific.

Reversibility of the VWF self-association

In order to verify if the relatively rapid reversibility of the VWF

self-interaction under flow can also be observed in our ELISA

system, we determined the association rate and the dissociation

rate of the binding of b-VWF to immobilized VWF. Compar-

able experiments were performed for the binding of FVIII to

immobilized VWF [35].

100

80

60

40

% b

indi

ng

20

00 20 40 60 80

350nM

280210140

b-VWF

700

100µg/mL

Fig. 1. Binding of soluble b-VWF to immobilized VWF under static

conditions. Microtiter plates were coated with VWF and incubated

subsequently with a dilution series of soluble b-VWF after which

bound b-VWF was detected with streptavidin-HRP (n ¼ 3).

1.6A

B

1.4

1.2

1.0

0.8

OD

490

nm

0.6

0.4

0.2

0.00 2

VWF b-VWF

4VWF or b-VWF (µg/ml)

6 8 10

Fig. 2. Effect of biotinylation on VWF function and multimeric structure.

(A) Binding of VWF and b-VWF to human collagen type III and to

rGPIba. Microtiter plates were coated with (i) human collagen type III

and were incubated with a dilution series of VWF (d) or b-VWF (s) or

(ii) were coated with anti-GPIba mAb 2D4, followed by incubation with

rGPIba and incubated with a dilution series of VWF (j) or b-VWF (h) in

the presence of 0.01 lg mL)1 botrocetin. Bound VWF and b-VWF were

detected with anti-VWF-Ig-HRP. Representative of three independent

experiments. (B) Purified VWF and b-VWF were separated on SDS-IEF

agarose gels. Gels were fixed on Gelbond and VWF or b-VWF multimers

were immunostained with anti-VWF-Ig-HRP and DAB as substrate.



For the association experiments, we observed the time-

dependent binding of b-VWF to immobilized VWF (Fig. 4A).

In order to compare the results of the different repeats of this

experiment, we recalculated the OD values as percentage

binding, using the OD value for the binding of b-VWF after

1.5 h incubation as 100% binding. These results demonstrated

that binding of b-VWF increased with time and that maximal

binding was reached after 45 min incubation. After 10 min

incubation, more than 50% of the maximal binding was

reached.

For the dissociation experiments, the removal of bound

b-VWF by a 10 · excess of unlabeled VWF was measured

as a function of time (Fig. 4B). Unlabeled VWF could

completely remove bound b-VWF from the surface, clearly

demonstrating the reversibility and specificity of the VWF

self-interaction measured in the ELISA assay. Comparable

with the association data, bound b-VWF was almost

completely removed after 45 min incubation with unlabeled

VWF, and after 10 min incubation more than 50% bound

b-VWF was removed.

80A

B

C

60

40

% b

indi

ngO

D 4

90nm

% in

hibi

tion

20

00

0.8

0.6

0.4

0.2

0.00

100

80

60

40

20

0VWF BSA Fibrinogen

20b-VWF or b-BSA (µg/mL)

40 60 80

20 40VWF or b-VWF (µg/mL)

60 80 100

Fig. 3. Specificity of the VWF self-association. (A) Interaction of VWF

and b-VWF with immobilized DA3-VWF. Microtiter plates were coated

with DA3-VWF and were incubated with a dilution series of VWF (j) or

b-VWF (d). Bound VWF was detected with b-82D6A3 followed by

incubation with streptavidin-HRP. Bound b-VWF was detected with

streptavidin-HRP; representative of three independent experiments. (B)

Interaction of soluble b-VWF or b-BSA with immobilized VWF or BSA.

Microtiter plates were coated with VWF (squares), BSA (circles) or HSA

(triangles) and incubated with a dilution series of soluble b-BSA (open

symbols) or b-VWF (filled symbols). Bound b-BSA or b-VWF was

detected with streptavidin-HRP; representative of three independent

experiments. (C) Inhibition of VWF self-association by soluble VWF,

BSA or fibrinogen. Microtiter plates were coated with VWF and prein-

cubated with soluble unlabeled VWF (300 lg mL)1), BSA (300 lg mL)1)

or fibrinogen (300 lgmL)1). Soluble b-VWFwas added (25 lgmL)1 final

concentration) for 1.5 h after which residual bound b-VWF was detected

with streptavidin-HRP. The percentage inhibition is shown. As 0% inhi-

bition reference, the binding of 25 lg mL)1 soluble b-VWF in the absence

of inhibitor was chosen (n ¼ 3).

125

100

100

75

50

25

0

75

% b

indi

ng%

bin

ding

50

25

0

0 30

time (min)

60 90

0 30

time (min)

60 90

Fig. 4. Binding kinetics of VWF self-association. (A) Association studies.

Microtiter plates were coated with VWF and incubated with 50 lg mL)1

soluble b-VWF for different time intervals. Bound b-VWF was detected

with streptavidin-HRP (n ¼ 4). (B) Dissociation studies. Microtiter plates

were coated with VWF and equilibrated with soluble 25 lg mL)1 b-VWF

for 1.5 h after which bound b-VWF was removed by excess unlabeled

VWF (250 lg mL)1) for different time intervals. The residual bound

b-VWF was detected with streptavidin-HRP. The percentage binding is

shown. As 100% binding reference, the binding of 25 lg mL)1 soluble

b-VWF in the absence of unlabeled VWF was chosen (n ¼ 5).



These kinetic binding experiments of b-VWF to immobilized

VWF revealed a comparable association and dissociation rate,

reflecting a rapid reversibility. Moreover, the time-scale of this

VWF self-interaction in the ELISA system is comparable with

the self-association observed under flow conditions [21].

Effect of proteolytic fragments of VWF, anti-VWF mAbs and

heparin on VWF self-association

Proteolytic digestion of VWF by V8 protease results in two

non-overlapping VWF fragments, SpII (1366–2050) and SpIII

(1–1365). The SpIII-fragment can be digested further, resulting

in a smaller VWF fragment SpI (911–1365). These fragments

were used to determine if the VWF self-association is modu-

lated by domains located in either the N-terminal part or the

C-terminal part of VWF or in both parts. Soluble b-VWF is

able to interact with immobilized SpI-, SpII- or SpIII-fragment

(Fig. 5A) with a Kd,app of, respectively, 586 ± 140, 286 ± 37,

196 ± 31 nmol L)1, suggesting that neither of these domains

can solely account for the self-association of VWF. This

interaction could be inhibited by soluble unlabeled VWF

(Fig. 5B), demonstrating a specific interaction. Also, the

binding of soluble b-VWF to immobilized VWF could be

inhibited by the SpI-, SpII- and SpIII-fragments with a

maximum of, respectively, 40%, 70% and 80% (Fig. 5C).

The SpII-fragment, the SpIII-fragment or a combination of

SpII- and SpIII-fragment inhibited the VWF self-interaction

comparably as unlabeled VWF (Fig. 5C vs. Fig. 3C). These

results together suggest that the VWF self-association is

modulated by multiple domains in both soluble VWF and

immobilized VWF. Comparable results were obtained with

VWF deletion mutants [36]: soluble b-VWF interacted dose-

dependently with D¢-A3 VWF-fragment, with D¢D3 VWF-

fragment and with DD¢-D3 VWF(data not shown).

In order to try to identify specific regions within the Sp

fragments of VWF responsible for the binding of soluble

b-VWF to immobilized VWF, the influence of the anti-VWF

mAbs 1C1E7, 701, 724 and 82D6A3 and of heparin on the

VWF self-association was investigated. mAb 1C1E7 recognizes

an epitope in the N-terminal region of VWF (AA1-272), the

mAbs 701 and 724 bind to the VWF A1-domain and mAb

82D6A3 interacts with the VWFA3 domain. Heparin interacts

with the VWF A1-domain and the N-terminal region of VWF

(AA1-272). Neither one of the mAbs, nor the mixture of the 4

mAbs, nor heparin was able to inhibit the binding of soluble

VWF to immobilized VWF (data not shown, n ¼ 3).

(Self-)association of SpII- and SpIII-fragments

To analyze whether the VWF self-association is modulated by

interaction between identical or different domains, the binding

of soluble b-SpII and b-SpIII to both immobilized SpII and

SpIII was verified. Both soluble b-SpII and b-SpIII are able to

interact with both immobilized SpII and SpIII (Fig. 6A, 6B).

Using a Mr of 110 kDa for the SpII-fragment and a Mr of

170 kDa for the SpIII-fragment [18], soluble b-SpII interacted

80A

B

C

100

80

60

40

20

0

100

80

60

40

20

0

Spl Spll Splll

Spl Spll Splll Spll + Splll

60

40

% b

indi

ng%

inhi

bitio

n%

inhi

bitio

n

20

00 20 40

b/VWF (µg/mL)

60 80 100

Fig. 5. Effect of Sp-fragments on VWF self-association. (A) Microtiter

plates were coated with SpI- (j), SpII- (d) or SpIII. (A) fragment and

incubated with a dilution series of soluble b-VWF, after which bound

b-VWFwas detected with streptavidin-HRP (n ¼ 3). (B)Microtiter plates

were coated with SpI-, SpII- or SpIII-fragment and preincubated with

soluble unlabeled VWF (600 lg mL)1, 300 lg mL)1 and 300 lg mL)1,

respectively). Soluble b-VWF (25 lg mL)1 final concentration) was added

and incubated for 1.5 h after which residual bound b-VWF was detected.

The percentage inhibition is shown. As 0% inhibition reference, the

binding of soluble b-VWF (25 lg mL)1) in the absence of soluble VWF

was chosen (n ¼ 2 or 3). (C) Microtiter plates were coated with VWF and

preincubated with soluble SpI- (100 lgmL)1), SpII- (100 lgmL)1), SpIII-

fragment (100 lg mL)1) or a mixture of SpII- (100 lg mL)1) and SpIII-

fragment (100 lg mL)1). Soluble b-VWF was added (25 lg mL)1 final

concentration) for 1.5 h after which residual bound b-VWF was detected

with streptavidin-HRP. The percentage inhibition is shown. As 0% inhi-

bition reference, the binding of 25 lg mL)1 soluble b-VWF in the absence

of inhibitor was chosen (n ¼ 3).



with immobilized SpII and SpIII with a Kd,app of

62 ± 10 nmol L)1 and 96 ± 19 nmol L)1, respectively, while

soluble b-SpIII interacted with immobilized SpII and SpIII

with a Kd,app of 136 ± 35 nmol L)1 and 121 ± 24 nmol L)1,

respectively. VWF is able to inhibit these interactions, showing

that the measured binding is specific (Fig. 6C).

Functional self-association of soluble and immobilized VWF

and VWF-fragments under flow conditions

In order to verify whether the interaction of Sp-fragments can

also be observed under flow conditions, perfusion experiments

were performed. Coverslips were coated with DA1-VWF

(Fig. 7A) or SpII (Fig. 7B), which both lack the A1-domain

essential for platelet interaction. Plasma-free blood was

perfused over these surfaces at 1600 s)1 for 5 min in the

presence of VWF, DA1-VWF or SpIII fragment after which

adhered platelets were fixed, stained and analyzed postper-

fusion.

Similar to previous experiments [21], platelets failed to

interact with immobilized DA1-VWF unless soluble VWF was

provided (P < 0.01). Moreover, soluble SpIII fragment was

able to sustain platelet adhesion to immobilized DA1-VWF

(P < 0.01). When SpII was immobilized, platelets did not

interact with this surface unless soluble VWFor SpIII fragment

was provided (P < 0.05). In the presence of soluble DA1-VWF, no platelet adhesion to immobilized SpII was observed,

as expected.

Discussion

Platelet adhesion is initiated at regions of high shear stress by

interaction of the GPIb-IX–V platelet receptor complex of

flowing platelets with immobilized VWF. It is believed that

collagen-bound VWF is able to modulate platelet transloca-

tion. However, recent flow chamber studies demonstrated that

immobilized VWF is able to support a homotypic self-

association [21]. Soluble VWF is not only able to become

14

12

10

8

6

4

2

0

Sur

face

cov

erag

e (%

)

12B

A

10

8

6

4

2

0

Sur

face

cov

erag

e (%

)

/ VWF Splll

/ VWF Splll ∆A1-VWF

Fig. 7. Flow studies. Cover slips were coated with DA1-VWF (A) or SpII

(B) and perfused for 5 min at 1600 s)1 with washed blood cells in the

absence or presence of soluble VWF, SpIII or DA1-VWF (20 lg mL)1).

Cover slips were fixated with glutaraldehyde and stained with May–

Grunwald/Giemsa (n ¼ 3 or 4).

100A

80

60

40% b

indi

ng

20

0

100

80

60

40

20

0

0 10 20 30b-Spll (µg/mL)

40 50

B

C

80

60

40

% b

indi

ng

% in

hibi

tion

20

00

Immobilized Spllb-Spll

Splllb-Spll

Splllb-Splll

Spllb-SplllSoluble

10 20 30b-Spll (µg/mL)

40 50

Fig. 6. Binding of soluble b-SpII- and b-SpIII-fragment to immobilized

SpII- and SpIII- fragment. Microtiter plates were coated with SpII (j) or

SpIII (d) and incubated with a dilution series of soluble b-SpII (A) or

b-SpIII (B). Bound b-SpII or b-SpIII was detected with streptavidin-HRP

(n ¼ 3). (C)Microtiter plates were coated with SpII or SpIII fragment and

preincubated with soluble unlabeled VWF (300 lg mL)1). Soluble b-SpII

(50 lg mL)1 final concentration) or soluble b-SpIII (50 lg mL)1 final

concentration) was added and incubated for 1.5 h after which residual

bound b-SpII or b-SpIII was detected. The percentage inhibition is shown.

As 0% inhibition reference, the binding of soluble b-SpII (50 lg mL)1) or

soluble b-SpIII (50 lg mL)1) in the absence of soluble VWF was chosen

(n ¼ 2 or 3).



immobilized on subendothelial collagen, but also on collagen-

bound VWF. This self-interaction was shown to be specific and

was demonstrated only in the presence of shear. Also, VWF in

suspension is able to self-associate in the presence of high shear

[22].

In the present study, we demonstrated that shear is not a

prerequisite for homotypic self-association of VWF as we

could show that this interaction also occurred under static

conditions. This was demonstrated using a solid-phase assay

where soluble b-VWF interacted dose-dependently with immo-

bilized VWF. The observation seen in this ELISA assay was

not due to the biotinylation, as this modification did not alter

the VWF function: b-VWF interacted normally with a

recombinant GPIba fragment or with human collagen type

III and the multimeric structure of VWFwas not influenced by

the biotinylation process. Moreover, a comparable interaction

of soluble VWF and b-VWF with immobilized DA3-VWF

could also be demonstrated. These observations indicate that

biotinylation of VWF does not induce major changes in VWF

structure or function. This is in agreement with earlier results

[37].

The measured VWF self-association in the ELISA setup

was, moreover, specific and reversible as (i) no interaction of

VWF with BSA or HSA could be demonstrated, (ii) unlabeled

VWF could almost completely remove bound b-VWF from

the surface and (iii) kinetic binding experiments showed

comparable association and dissociation rates for the self-

interaction. Taken together, these results show that our

ELISA is a good tool to further investigate the VWF self-

association. To try to elucidate the different domains or

regions necessary for this self-association, mAbs and proteo-

lytic fragments of VWF were used. In agreement with earlier

results [21], our studies consolidate the observation that the

measured VWF self-association does not involve a strictly

homotypic association of the A3-domain, as (i) soluble VWF

could interact with immobilized DA3-VWF and (ii) the

SpI-fragment which contains the A3-domain could inhibit

the VWF self-interaction with a maximum of 40% only. We

extended these results by demonstrating that the VWF self-

interaction is modulated by a multiple domain interaction.

First, none of the mAbs directed against well-defined regions

in VWF were able to inhibit the self-association. Secondly,

neither the N-terminal part (SpIII, AA1-1365) or the

C-terminal part (SpII, AA1366–2050) or a central part (SpI,

AA911–1365) of VWF could account solely for this self-

association, as (i) soluble VWF is able to interact with

immobilized SpI(911–1365), SpII(1366–2050) and SpIII(1–

1365) fragment and (ii) soluble SpI-, SpII- and SpIII-fragment

are able to inhibit the VWF self-association. In agreement

with this hypothesis is the observation that larger VWF-

fragments are able to inhibit the self-interaction more strongly

and soluble b-VWF is able to bind with greater affinity/avidity

to larger VWF-fragments. Indeed, soluble unlabeled VWF

could inhibit the binding of b-VWF to immobilized VWF

with a maximum of 80% while the dimeric SpIII-, dimeric

SpII- and monomeric SpI-fragment could inhibit maximally

by 80%, 70% or 40%, respectively. Soluble b-VWF interacted

with immobilized VWF with a Kd,app of 93 ± 3 nmol L)1,

while the binding of soluble b-VWF with immobilized SpIII-,

SpII- and SpI-fragment was characterized with a Kd,app of,

respectively, 196 ± 31 nmol L)1, 286 ± 37 nmol L)1 and

586 ± 140 nmol L)1.

We next studied whether this multiple domain interaction

between different VWF molecules occurred through a homo-

typic or heterotypic domain association by using N-terminal

parts (SpIII) and C-terminal parts (SpII) of VWF and their

biotinylated counterparts. Both soluble b-SpII and b-SpIII

fragments were able to interact with both immobilized SpII-

and SpIII-fragments. This demonstrates that VWF can

self-associate in different ways combining different regions.

Moreover, this Sp-fragment association was specific as it could

be inhibited by soluble VWF.

A

B

C (i) (ii)

ss

ss ss ss

ssssss

ssss

Spll

SplIl

Fig. 8. Possible model of VWF self-association. (A) In solution, the globular structure of VWF is maintained by multiple interactions between SpII–

SpII, SpIII–SpIII and SpII–SpIII domains. (B) Immobilization of VWF disrupts the multiple domain interactions present in the globular VWF structure.

(C) VWF self-association is mediated by the same interactions which are present in globular VWF. Homotypic (i) and heterotypic (ii) domain interac-

tions from the N- and C-terminal portion of VWF modulate VWF self-interaction.



Finally, we were able to demonstrate that Sp-fragments can

associate under shear conditions as well. These results corro-

borate the data of our ELISA system and give indirect proof

for the functionality and specificity of the results obtained in

the static system. The surface coverage observed when recon-

stituted bloodwas perfused over immobilizedDA1-VWF in the

presence of VWF, was however, significantly higher than what

was observed previously by Lankhof et al. in an analogous

system [27]. In that study, a surface coverage of approximately

3% was observed when whole blood was perfused over

immobilized DA1-VWF. However, in our study a higher

concentration of both immobilized and soluble VWFwas used,

which might explain the higher surface coverage observed. The

analogy between the results under static and under flow

conditions suggests that the ELISA system is a good tool to

investigate further the VWF self-association. On one hand, the

ELISA system is more easy to perform and requires less

materials; on the other hand, several interactions can be studied

in the ELISA system which cannot be studied in the flow

system as this system requires the absence of the A1-domain in

the immobilized VWF fragment or mutant and the presence of

the A1-domain in the soluble VWF.

It has to be noted that other groups were not able to

demonstrate VWF self-association using surface plasmon

resonance [21]. These different results are due possibly to the

different coating surfaces that were used. In the present study,

VWF was immobilized on a plastic surface (polystyrene

microtiter plates), while in surface plasmon resonance mainly

carboxymethylated dextran matrices or streptavidin-deriva-

tized surfaces are used [38]. As immobilization of VWF onto a

plastic surface is known to be a prerequisite for platelet

adhesion [39], due probably to a conformational change in

VWF, the same immobilization of VWF (and thus conform-

ational change) might be necessary to observe the VWF self-

association. Moreover, to observe self-interaction of VWF in

solution, a certain threshold of shear stress was needed,

suggesting that also here a conformational change in VWF is

favorable [22,40].

In conclusion, we have demonstrated that soluble VWF is

able to interact with immobilized VWF and that this associ-

ation can be induced in a shear-independent way and

modulated by a multiple domain interaction. A tempting

hypothesis is that the same multiple domain interactions are

responsible for maintaining the globular structure of VWF

under resting conditions (Fig. 8). Once these are disrupted by,

for example, coating onto plastic or shear, homotypic and/or

heterotypic region interaction might result in self-association.

This mechanism might be similar to the process of 3D domain

swapping [41].

Acknowledgements

H.U. and K.V. are supported by a postdoctoral fellowship of

the F.W.O.-Flanders. We thank Hendrik Feys for performing

the multimer analysis. This study is supported by the F.W.O.-

Flanders grant G.0168.02 and GOA-grant 2004/09.

References

1 Sakariassen KS, Bolhuis PA, Sixma JJ. Human blood platelet adhe-

sion to artery subendothelium is mediated by factor VIII-von

Willebrand factor bound to the subendothelium. Nature 1979; 279:

636–8.

2 Ruggeri ZM. Structure of von Willebrand factor and its function in

platelet adhesion and thrombus formation. Best Pract Res Clin

Haematol 2001; 14: 257–79.

3 Foster PA, Fulcher CA, Marti T, Titani K, Zimmerman TS. A major

factor VIII binding domain resides within the amino-terminal 272

amino acid residues of von Willebrand factor. J Biol Chem 1987; 262:

8443–6.

4 SugimotoM,Miyata S. Functional property of vonWillebrand factor

under flowing blood. Int J Hematol 2002; 75: 19–24.

5 Savage B, Almus-Jacobs F, Ruggeri ZM. Specific synergy of multiple

substrate–receptor interactions in platelet thrombus formation under

flow. Cell 1998; 94: 657–66.

6 SiedleckiCA,LestiniBJ,Kottke-MarchantKK,EppellSJ,WilsonDL,

Marchant RE. Shear-dependent changes in the three-dimensional

structure of human von Willebrand factor. Blood 1996; 88: 2939–50.

7 Novak L, Deckmyn H, Damjanovich S, Harsfalvi J. Shear-dependent

morphology of vonWillebrand factor bound to immobilized collagen.

Blood 2002; 99: 2070–6.

8 Raghavachari M, Tsai HM, Kottke-Marchant KK, Marchant RE.

Surface dependent structures of von Willebrand factor observed by

AFM under aqueous conditions. Colloids Surf B Biointerfaces 2003;

19: 315–24.

9 Miura S, Li CQ, Cao Z,WangH,WardellMR, Sadler JE. Interaction

of von Willebrand factor domain A1 with platelet glycoprotein Ibal-

pha-(1-289). Slow intrinsic binding kinetics mediate rapid platelet

adhesion. J Biol Chem 2000; 275: 7539–46.

10 Savage B, Saldivar E, Ruggeri ZM. Initiation of platelet adhesion by

arrest onto fibrinogen or translocation on von Willebrand factor.

Cell 1996; 84: 289–97.

11 Nieswandt B, Watson SP. Platelet–collagen interaction: is GPVI the

central receptor? Blood 2003; 102: 449–61.

12 Savage B, Cattaneo M, Ruggeri ZM. Mechanisms of platelet aggre-

gation. Curr Opin Hematol 2001; 8: 270–6.

13 Yap CL, Hughan SC, Cranmer SL, Nesbitt WS, Rooney MM,

Giuliano S, Kulkarni S, Dopheide SM, Yuan Y, Salem HH, Jackson

SP. Synergistic adhesive interactions and signaling mechanisms

operating between platelet glycoprotein Ib/IX and integrin alpha

IIbbeta 3. Studies in human platelets ans transfected Chinese hamster

ovary cells. J Biol Chem 2000; 275: 41377–88.

14 Rand JH, Glanville RW,WuXX, Ross JM, Zangari M, Gordon RE,

Schwartz E, Potter BJ. The significance of subendothelial von Wille-

brand factor. Thromb Haemost 1997; 78: 445–50.

15 Sixma JJ, van Zanten GH, Huizinga EG, van der Plas RM, Verkley

M, Wu YP, Gros P, de Groot PG. Platelet adhesion to collagen: an

update. Thromb Haemost 1997; 78: 434–8.

16 Hoylaerts MF, Yamamoto H, Nuyts K, Vreys I, Deckmyn H,

Vermylen J. von Willebrand factor binds to native collagen VI pri-

marily via its A1 domain. Biochem J 1997; 324: 185–91.

17 Fujimura Y, Titani K, Holland LZ, Roberts JR, Kostel P, Ruggeri

ZM, Zimmerman TS. A heparin-binding domain of human von

Willebrand factor. Characterization and localization to a tryptic

fragment extending from amino acid residue Val-449 to Lys-728.

J Biol Chem 1987; 262: 1734–9.

18 Fretto LJ, Fowler WE, McCaslin DR, Erickson HP, McKee PA.

Substructure of human von Willebrand factor. Proteolysis by V8 and

characterization of two functional domains. J Biol Chem 1986; 261:

15679–89.

19 Lankhof H, van Hoeij M, Schiphorst ME, Bracke M, Wu YP,

Ijsseldijk MJ, Vink T, de Groot PG, Sixma JJ. A3 domain is essential

for interaction of von Willebrand factor with collagen type III.

Thromb Haemost 1996; 75: 950–8.



20 Wu D, Vanhoorelbeke K, Cauwenberghs N, Meiring M, Depraetere

H, Kotze HF, DeckmynH. Inhibition of the vonWillebrand (VWF)–

collagen interaction by an antihuman VWF monoclonal antibody

results in abolition of in vivo arterial platelet thrombus formation in

baboons. Blood 2002; 99: 3623–8.

21 Savage B, Sixma JJ, Ruggeri ZM. Functional self-association of von

Willebrand factor during platelet adhesion under flow. Proc Natl

Acad Sci USA 2002; 99: 425–30.

22 Shankaran H, Alexandridis P, Neelamegham S. Aspects of hydro-

dynamic shear regulating shear-induced platelet activation and self-

association of von Willebrand factor in suspension. Blood 2003; 101:

2637–45.

23 Girma JP, Chopek MW, Titani K, Davie EW. Limited proteolysis of

human vonWillebrand factor by Staphylococcus aureus V-8 protease:

isolation and partial characterization of a platelet-binding domain.

Biochemistry 1986; 25: 3156–63.

24 Tornai I, Arnout J, Deckmyn H, Peerlinck K, Vermylen J. A

monoclonal antibody recognizes a von Willebrand factor domain

within the amino-terminal portion of the subunit that modulates the

function of the glycoprotein IB- and IIB/IIIA-binding domains. J Clin

Invest 1993; 91: 273–82.

25 Depraetere H, Ajzenberg N, Girma JP, Lacombe C, Meyer D,

DeckmynH, BaruchD. Platelet aggregation induced by amonoclonal

antibody to the A1 domain of von Willebrand factor. Blood 1998; 91:

3792–9.

26 Obert B, Houllier A, Meyer D, Girma JP. Conformational changes in

the A3 domain of von Willebrand factor modulate the interaction of

the A1 domain with platelet glycoprotein Ib. Blood 1999; 93: 1959–68.

27 Lankhof H, Wu YP, Vink T, Schiphorst ME, Zerwes HG, de Groot

PG, Sixma JJ. Role of the glycoprotein Ib-binding A1 repeat and the

RGD sequence in platelet adhesion to human recombinant von

Willebrand factor. Blood 1995; 86: 1035–42.

28 Fujimura Y, Titani K, Usami Y, Suzuki M, Oyama R, Matsui T,

Fukui H, Sugimoto M, Ruggeri ZM. Isolation and chemical char-

acterization of two structurally and functionally distinct forms of

botrocetin, the platelet coagglutinin isolated from the venom of

Bothrops jararaca. Biochemistry 1991; 30: 1957–64.

29 Ulrichts H, Vanhoorelbeke K, Cauwenberghs S, Vauterin S, Kroll H,

Santoso S, Deckmyn H. Von Willebrand factor but not a-thrombin

binding to platelet glycoprotein Iba is influenced by the HPA-2

polymorphism. Arterioscler Thromb Vasc Biol 2003; 23: 1302–7.

30 Vanhoorelbeke K, Cauwenberghs N, Vauterin S, Schlammadinger A,

Mazurier C, DeckmynH. A reliable and reproducible ELISAmethod

to measure ristocetin cofactor activity of von Willebrand factor.

Thromb Haemost 2000; 83: 107–13.

31 Cauwenberghs N, Vanhoorelbeke K, Vauterin S, Westra DF, Romo

G, Huizinga EG, Lopez JA, Berndt MC, Harsfalvi J, Deckmyn H.

Epitope mapping of inhibitory antibodies against platelet glycopro-

tein Ibalpha reveals interaction between the leucine-rich repeat

N-terminal and C-terminal flanking domains of glycoprotein Ibalpha.

Blood 2001; 98: 652–60.

32 Ruggeri ZM, Zimmerman TS. The complex multimeric com-

position of factor VIII/von Willebrand factor. Blood 1981; 57:

1140–3.

33 Li CQYeP, Cao Z, Wang H, Lu L, Nicastro P, Wood E, Robert JJ,

Ouwehand WH, Hill F, Lopez JA, Wardell MR. Expression of the

amino-terminal domain of platelet glycoprotein Ib alpha: exploitation

of a calmodulin tag for determination of its functional activity.Protein

Expr Purif 2001; 22: 200–10.

34 Budde U, Schneppenheim R. Von Willebrand factor and von Wille-

brand disease. Rev Clin Exp Hematol 2001; 5: 335–68.

35 Bendetowicz AV, Wise RJ, Gilbert GE. Collagen-bound von Wille-

brand factor has reduced affinity for factor VIII. J Biol Chem 1999;

274: 12300–7.

36 Lenting PJ, Westein E, Terraube V, Ribba AS, Huizinga EG, Meyer

D, de Groot PG, Denis CV. An experimental model to study the

in vivo survival of Von Willebrand Factor: basic aspects and

application to the Arg1205His mutation. J Biol Chem 2004; 279:

12102–09.

37 Miura S, Nishida S, Makita K, Sakurai Y, Shimoyama T, Sugimoto

M, Yoshioka A, Ishii K, Kito M, Kobayashi T, Fujimura Y. Inhi-

bition assay for the binding of biotinylated von Willebrand factor to

platelet-bound microtiter wells in the presence of ristocetin or botr-

ocetin. Anal Biochem 1996; 236: 215–20.

38 McDonnell JM. Surface plasmon resonance: towards an under-

standing of the mechanisms of biological molecular recognition.

Curr Opin Chem Biol 2001; 5: 572–7.

39 Van Os E, Wu YP, Pouwels JG, Ijsseldijk MJ, Sixma JJ, Akkerman

JW, de Groot PG, VanWilligenG. Thrombopoietin increases platelet

adhesion under flow and decreases rolling. Br J Haematol 2003; 121:

482–90.

40 Loscalzo J, Fisch M, Handin RI. Solution studies of the quaternary

structure and assembly of human vonWillebrand factor.Biochemistry

1985; 24: 4468–75.

41 Bennett MJ, Schlunegger MP, Eisenberg D. 3D domain swapping:

a mechanism for oligomer assembly. Protein Sci 1995; 4: 2455–68.



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