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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3796-3803
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the INSERM U143, Paris, France.
The aim was to better understand the function of von Willebrand
factor (vWF) A1 domain in shear-induced platelet aggregation (SIPA), at
low (200) and high shear rate (4000 seconds-1) generated by
a Couette viscometer. We report on 9 fully multimerized recombinant
vWFs (rvWFs) expressing type 2M or type 2B von Willebrand disease (vWD)
mutations, characterized respectively by a decreased or increased
binding of vWF to GPIb in the presence of ristocetin. We expressed 4 type 2M (-G561A, -E596K, -R611H, and -I662F) and 5 type 2B
(rvWF-M540MM, -V551F, -V553M, -R578Q, and -L697V). SIPA was strongly
impaired in all type 2M rvWFs at 200 and 4000 seconds-1.
Decreased aggregation was correlated with ristocetin binding to
platelets. In contrast, a distinct effect of botrocetin was observed,
since type 2M rvWFs (-G561A, -E596K, and -I662F) were able to bind to
platelets to the same extent as wild type rvWF (rvWF-WT).
Interestingly, SIPA at 200 and 4000 seconds-1 confirmed the
gain-of-function phenotype of the 5 type 2B rvWFs. Our data indicated a
consistent increase of SIPA at both low and high shear rates, reaching
95% of total platelets, whereas SIPA did not exceed 40% in the
presence of rvWF-WT. Aggregation was completely inhibited by monoclonal
antibody 6D1 directed to GPIb, underlining the importance of vWF-GPIb
interaction in type 2B rvWF. Impaired SIPA of type 2M rvWF could
account for the hemorrhagic syndrome observed in type 2M vWD. Increased
SIPA of type 2B rvWF could be responsible for unstable aggregates and
explain the fluctuant thrombocytopenia of type 2B vWD.
(Blood. 2000;95:3796-3803)
Under static conditions, platelet activation induced by
agents such as adenosine 5'-diphosphate or thrombin
involves an interaction between fibrinogen and the activated
vWF binding to GPIb involves a complex mechanism, which is reproduced
in the presence of nonphysiological agents, such as ristocetin or
botrocetin, and involves different sequences of vWF A1 domain,
extending from amino acids 497 to 716.6,7 Ristocetin binds
to 2 proline-rich, negatively charged regions (amino acids 474-488 and
695-708) flanking the disulfide bridge between Cys 509 and Cys 695. Botrocetin binds to predominantly positively charged sequences in the
A1 loop (amino acids 514-542, 539-553, 569-583, and
629-643).7-9 Interestingly, both inducers may act through
different mechanisms.10 Binding of ristocetin to vWF may
relieve the effect of inhibitory sites responsible for maintaining vWF
in an inactive conformation, thus indirectly inducing vWF binding to
GPIb.10,11 In contrast, botrocetin favors direct binding of
vWF to GPIb vWD is a heterogeneous, hereditary bleeding disorder that results from
the quantitative or qualitative deficiency in vWF. Type 2 vWD consists
of qualitative variants; type 1 refers to a partial quantitative
defect; while type 3 refers to the absence of detectable vWF in
plasma. Type 2 vWD comprises 3 types of variants with
impaired binding to GPIb: 2A, 2B, and 2M.12 Type 2A and type 2M (multimer) vWD patients have decreased platelet-dependent functions but differ by the multimeric composition of vWF: Type 2A is
associated with the absence of the HMWM, whereas type 2M has all
multimers. Until now, several type 2M variants have been described and
confirmed by expression of mutant recombinant vWF (rvWF). Mutations
F606I, I662F, and G561S are responsible for an impaired
ristocetin-mediated but normal botrocetin-mediated binding to
platelets.13-15 In contrast, type
2MMilwaukee-1 (deletion between amino acids 629 and
639) and the mutation of R611H are characterized by a decreased
ristocetin-mediated and botrocetin-mediated binding to
platelets.16,17 However, in some cases, the classification
has been debated and the rvWF-R611H reported as
unclassified.17 (For the sake of simplicity, we have
included the rvWF-R611H into type 2M.)
Type 2B vWD is characterized by a gain-of-function phenotype
(aggregation induced by lower ristocetin concentrations than normal
plasma) because of increased affinity for platelet GPIb. Loss of HMWM
is attributed to their enhanced binding to platelets and their removal
from the circulation.18 Type 2B vWD mutations are localized
in the A1 loop of vWF between amino acids 540 and 578, on a small
fragment that overlaps botrocetin and heparin binding sites. Studies of
crystal structure have demonstrated that most of the type 2B mutations
are located at the interface between the N- and C-terminal parts of the
A1 domain and are responsible for disruption of salt bridges or
hydrophobic packing.19 Thus, type 2B mutations may change
the conformation of the molecule, resulting in a gain of
function.10
Expression of type 2B rvWFs has shown that they display the whole range
of multimers. Several groups20,21 have reported on the
ability of type 2B plasma or rvWF to induce platelet aggregation in the
absence of shear. Interestingly, several type 2B rvWFs (R543Q, V553M,
L697V, and A698V) were shown to induce different extents of activation
and aggregation.21 Mutations located inside the C509-C695
loop were more efficient than those located outside of the
loop.21
Despite an increased affinity of vWF for platelet GPIb, type 2B vWD is
characterized by a hemorrhagic disease and a fluctuating thrombocytopenia. Platelet adhesion studies at high shear rates have
contributed to a better understanding of the physiopathology of this
bleeding disorder. A defect of adhesion and thrombus formation at 2600 seconds-1 has been observed, compared with normal blood,
using a parallel-plate perfusion chamber exposing type III collagen to
type 2B vWD blood.22 The discrepancy between the
gain-of-function phenotype of type 2B mutation and the impaired
adhesion has been confirmed, using type 2B R543Q and R543W rvWFs
exhibiting a normal multimeric pattern.23 It was
hypothesized that in type 2B vWD, the mutated vWF inhibits normal vWF
function. Occupancy of GPIb by soluble type 2B rvWF because of its
increased affinity for this receptor resulted in a decreased platelet
adhesion to collagen type III, compared with wild type (WT). However,
the adhesive capacity of immobilized type 2B vWF was not impaired,
demonstrating the importance of the conformation of vWF in
shear-dependent functions.23
In our study, we have attempted to better understand the involvement of
vWF mutated amino acids on GPIb interaction in shear conditions at 200 and 4000 seconds-1 using rvWF reproducing type 2M or type
2B mutations. We report for the first time on the effect on SIPA of 5 type 2B and 4 type 2M mutations located in the A1 domain.
Purification of protein
Characterization of antibodies
Radiolabeling of IgG IgG was labeled with Na125I (Amersham, Les Ulis, France) and Iodogen (Pierce Chemical Co, Rockford, IL) as described.27 Specific radioactivity varied from 3 to 10 µCi/µg. Labeled antibodies were stored at 4°C and used within 2 months.Plasmid constructs Plasmids with full-length complementary DNA (cDNA) coding for rvWF-WT (pSVL-WT, pSVvWFA, pCDNA3--WT)10,28 or mutated rvWF with either duplication of methionine in position 540, or substitution of phenylalanine in position 551 for valine (pSVL-M540MM, -V551F), were constructed as previously described.29,30 Plasmids coding for substitution of histidine in position 611 for arginine, of methionine in position 553 for valine, of alanine in position 561 for glycine, of glutamine in position 578 for arginine, or valine in position 697 for leucine (pSVvWF-R611H, -V553M, -G561A, -R578Q, -L697V) were a kind gift from Drs C. Mazurier and L. Hilbert (LFB, France).15,17,31,32 The new plasmid with full-length cDNA mutated in position 662 of phenylalanine for isoleucine (pSVvWF-I662F) was obtained by site-directed mutagenesis using QuickChange kit (Stratagene, CA) and the other one in position 596 substituted of lysine for glutamic acid (pCDNA3--E596K) was obtained by Transformer Site-Directed Mutagenesis Kit (Clontech Laboratories, CA).Cell culture and transfection COS-7 cells were cultured with Dulbecco modified essential medium containing L-glutamine (GIBCO-BRL, Cergy Pontoise, France), penicillin (100 U/mL), streptomycin (100 mg/mL), and 10% (v:v) fetal calf serum (Boehringer, Mannheim, Germany). Cells were transfected, using the electroporation method33 or the diethylaminoethyl-dextran method as previously described.31
rvWF:Ag determination The amount of vWF:Ag in conditioned media after transfection was determined by enzyme-linked immunoadsorbent assay (ELISA) using a pool of 12 MoAbs to vWF (5 µg/mL) for coating wells of Maxi-sorp Nunc-Immuno Plates (A/S Nunc, Roskilde, Denmark) for 2 hours at 37°C, and a pool of 35 MoAbs to vWF coupled to horseradish peroxidase as a second antibody.28 In both pools, epitopes were distributed along the vWF subunit with an approximate ratio of 2:1 in the amino-terminal versus carboxy-terminal region. Levels of vWF:Ag present in the media of transfected cells were expressed relative to a control pool plasma of 15 healthy donors that had been calibrated against the Third International Standard for Willebrand factor in plasma (code 91/666, National Institute for Biological Standards and Control). Data were expressed in µg/mL.rvWF multimer analysis Multimeric analysis of rvWFs was performed by 0.1% SDS-1% agarose gel electrophoresis, as previously described, except that a different source of agarose was used (IEF Pharmacia Fine Chemical, Uppsala, Sweden).35Preparation of washed platelets Blood was obtained from healthy individuals who had not ingested any medication for 2 weeks before donation. The blood was drawn into 15% (v/v) acid citrate dextrose pH 5.8. Washed platelets were prepared from isolated platelet-rich plasma in the presence of apyrase (1 U/mL) (Sigma) and acid citrate dextrose (1 mL for 40 mL) as described.4 Briefly, after washing, platelets were resuspended in Hepes buffer,10 mmol/L Hepes (N-[2-hydroxyethyl]piperazine-N'-[ethanesulfonic acid]), 0.136 mol/L NaCl, 2.7 mmol/L KCl, and 2 mmol/L MgCl2 pH 7.5 containing BSA 0.15%, and they were used after 1 hour of incubation at 37°C. CaCl2 (1 mmol/L) was added after the incubation period. Platelets were counted with an electronic particle counter (Model 1, Coulter Electronics, Margency, France), and the concentration was adjusted to 3.5 × 108 platelets/mL.Shear-induced platelet aggregation The rotating device is a Couette type viscometer used as previously described4 with the following minor modifications. Washed platelet suspensions (0.5 × 108/mL) were exposed for 5 minutes at 20°C to a continuous shear rate of 200 or 4000 seconds-1 in the presence of rvWF (1 µg/mL) diluted in culture medium with 0.15% BSA, in a final volume of 210 µL. In some experiments, MoAb 6D1 (20 µg/mL) was pre-incubated with platelets for 5 minutes at 20°C. Following exposure to shear, samples were fixed with 1% paraformaldehyde by addition of a 10-fold concentrated solution and mixed for 30 seconds. An aliquot (10 µL) of the sheared or control platelet sample as defined above was diluted in 1 mL of Facs-flow buffer (Becton Dickinson, Le Pont-de Claix, France). SIPA was measured in a FACScan flow cytometer (Becton Dickinson) as reported.4 Data acquisition was performed by counting the particle number during a constant time (50 seconds) to measure identical volumes in different samples. In each sample, 1000 events were at least counted. Washed platelets were analyzed by forward light scatter and side light scatter. As negative control, platelet suspensions were incubated with mock-transfected cell medium in the cylinder gap for 5 minutes without exposure to shear, followed by fixation as above. This sample was used as the reference for gating the region of single platelets in the absence of shear. SIPA in the sheared samples was calculated by counting the gated population of single platelets, and results were expressed as the percentage of disappearance of single platelets: DSP = [(no n)/no] × 100,
where no represents the single platelet population of the
negative control platelet sample and n represents the sheared sample
containing WT or mutated rvWF. Means ± SEM were
calculated from 3 experiments performed in duplicate.
Binding of 125I-MoAb/vWF complexes to platelets Binding of rvWF to platelets was performed as described with some modifications.36 Platelets were isolated from outdated platelet concentrates by centrifugation at 200g for 15 minutes and fixed with paraformaldehyde (2%) in 0.15 mol/L NaCl, 25 mmol/L Tris-HCl buffer, pH 7.4 (TBS) containing 0.1% BSA. rvWF (0.4 µg/mL) diluted in TBS containing 1% BSA was pre-incubated with 125I-MoAb 505 (10 ng/mL, 900 000 cpm/mL) during 30 minutes at 20°C. The final mixture contained 108 platelets/mL, 125I-MoAb 505/rvWF complexes and varying concentrations of ristocetin (ABP, New York, NY) (0-1 mg/mL) or botrocetin (0-1 µg/mL). After 1 hour of incubation at 20°C, duplicate aliquots (100 µL) were layered onto 200 µL of 25% sucrose in Microfuge tubes and centrifuged for 3 minutes at 10 000g. The tube tip containing bound ligand was separated from the supernatant. Bound and free radioactivities were counted in a counter (LKB Instruments SA,
Bromma, Sweden). The percentage of total bound radioactivity was
calculated as bound/(free + bound) radioactivity. Nonspecific binding
was obtained by incubating platelets with 125I-MoAb 505 and
ristocetin or botrocetin in the absence of rvWF. Specific binding was
obtained by subtracting nonspecific binding from total binding. Means ± SEM were calculated from 3 experiments performed in duplicate. We
verified that the binding of MoAb 505 to mutated and rvWF-WT, which was
used for indirect labeling of rvWF, was similar to that of a pool of 35 MoAbs. Figure 1 shows that MoAb 505 binds
to type 2M or 2B rvWF with a similar affinity as a pool of MoAbs.
Statistical analysis Means ± SEM were calculated from 3 experiments performed in duplicate. Two statistical approaches were carried out, depending on the experimental design. The first approach was performed when the WT and all mutated rvWFs were analyzed in the same series of experiments and was applied to binding studies. Comparison of the 6 groups (1 WT and 5 type 2B rvWFs) was performed by using analysis of variance (ANOVA). Each ANOVA was performed at 4 different concentrations of agonist, ristocetin, or botrocetin. When the global F test was significant (P < .05), multiple comparison procedures were performed to define which recombinant differed from the other. To this end, we used the Student-Newman-Keuls multiple-comparison test with a 5% significance level. The analysis was performed using the procedure proc GLM type III of the SAS software (SAS Institute).
Characterization of rvWFs Expression levels of COS-7 cells transfected with rvWF-WT, mutated type 2M rvWF-I662F, -G561A, -E596K, and -R611H, or mutated type 2B rvWF-V551F, -M540MM, -V553M, -R578Q, and -L697V ranged between 1.1 and 4.9 µg/mL, depending on the transfection method. Conditioned medium from mock-transfected cells was used as negative control in each set of experiments. The multimeric structure of the recombinant proteins was analyzed by 0.1% SDS-1% agarose gel electrophoresis as shown in Figure 2. All multimeric forms were present in type 2M mutated rvWF (-E596K, -G561A, and -I662F) compared with rvWF-WT (Figure 2). As previously reported, HMWM of rvWF-R611H were present but displayed a decreased intensity compared with rvWF-WT.17 All type 2B rvWFs exhibited a normal multimeric pattern as depicted in Figure 2 for a representative type 2B rvWF (L697V).
Type 2M rvWFs interaction with GPIb induced by nonphysiological agonists To test the functional characteristics of the mutated rvWF, platelet-binding assays were performed with rvWF-G561A, -E596K, -I662F, and -R611H in the absence or in the presence of varying concentrations of ristocetin or botrocetin. In the absence of agonist, neither rvWF-WT nor mutated type 2M rvWF was able to bind to platelets. Binding of rvWF-WT to platelets reached 24% at 1 mg/mL of ristocetin, whereas rvWF-G561A, -E596K, -I662F, and -R611H were not able to bind to platelets in these conditions (data not shown). In these samples, the percentage of specific binding was similar to that of the mock-transfected cell medium sample, reaching 2%.
SIPA of type 2M rvWFs
Type 2B rvWFs interaction with GPIb induced by
nonphysiological agonists
Effect of type 2B rvWFs on SIPA
Effect on SIPA of MoAb to GPIb interfering with platelet-vWF
interaction
With the use of a rotating device to apply shear rates ranging from
0 to 4000 seconds-1, we report on the effect on SIPA of 9 different mutations localized in the A1 domain of vWF that reproduce
type 2B or type 2M vWD, characterized by an increased or a decreased
affinity of vWF for GPIb, respectively.
We thank Drs L. Hilbert and C. Mazurier from LFB, Lille, France, for
the kind gift of plasmids coding for V553M, G561A, R578Q, R611H, and
L697V. We thank B. Obert and P. Legendre for expert technical
assistance. Dr C. Mazurier is gratefully acknowledged for critical
reading of the manuscript. We would like to thank Dr J. Warszawski for help in statistical analysis.
Submitted July 23, 1999; accepted February 11, 2000.
Supported by an INSERM fellowship (Poste d'accueil) to N.A.
and a fellowship from Ministère de l'Education Nationale, de la
Recherche et de la Technologie to G. R.-L.
Reprints: Dominique Baruch, INSERM U143, 84 rue du
Général Leclerc, 94276 Bicêtre Cedex, France; e-mail:
baruch{at}infobiogen.fr.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Top
Abstract
Introduction
IIb
3 integrin (GPIIb/IIIa). Shear-induced
platelet aggregation (SIPA) occurs in high shear conditions and
involves binding of von Willebrand factor (vWF) to platelet
glycoprotein (GP) Ib. High molecular weight multimers (HMWM) of vWF are
required to induce SIPA.
A1), obtained by stable expression in
baby hamster kidney cells (kind gift from Dr J.J. Sixma,
Utrecht, The Netherlands).
), rvWF-WT (
), or mutated rvWF-I662F (x), -E596K
(
), -G561A (
), and -R611H (
). (B) Mutated rvWF-L697V (+),
-R578Q (
), -M540MM (
