impaired megakaryocytopoiesis in type 2b von willebrand disease with severe thrombocytopenia

doi:10.1182/blood-2006-03-009449Prepublished online May 23, 2006;

Corvazier, Robert Combrie, Edith Fressinaud, Dominique Meyer, Alan T Nurden and Jocelyne EnoufPaquita Nurden, Najet Debili, William Vainchenker, Regis Bobe, Raymonde Bredoux, Elisabeth severe thrombocytopeniaImpaired megakaryocytopoiesis in type 2B von Willebrand disease with

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Impaired Megakaryocytopoiesis in Type 2B von Willebrand

Disease with Severe Thrombocytopenia

Paquita Nurden1, Najet Debili2, William Vainchenker2, Régis Bobe3, Raymonde

Bredoux3, Elisabeth Corvazier3, Robert Combrié1, Edith Fressinaud4, Dominique

Meyer4, Alan Nurden1, Jocelyne Enouf3.

From 1Centre de Référence des Pathologies Plaquettaires and Institut Fédératif

de Recherche N°4, Hôpital Cardiologique, Pessac, France; 2Inserm U.790,

Villejuif, France; 3Inserm U.689, Hôpital Lariboisière, Paris, France; 4Inserm

U.143, Le Kremlin-Bicêtre, France.

Short Title: Thrombocytopenia in type 2B VWD

Scientific heading: Hemostasis, Thrombosis and Vascular Biology

Abstract word count: 191

Text word count: 4996

Corresponding address: Paquita Nurden M.D., Ph.D., Centre de Référence des Pathologies

Plaquettaires, Laboratoire d’Hématologie, Hôpital Cardiologique, 33604 Pessac, France. Tel:

33 5 57 65 68 43; Fax: 33 5 57 65 65 31

E-mail. [email protected]

Blood First Edition Paper, prepublished online May 23, 2006; DOI 10.1182/blood-2006-03-009449

Copyright © 2006 American Society of Hematology




2

Abstract

In type 2B von Willebrand disease, there is spontaneous binding of mutated VWF multimers

to platelets. Here we report a family in which severe thrombocytopenia may also be linked to

abnormal megakaryocytopoiesis. A heterozygous mutation in the VWF A1 domain gave a

R1308P substitution in an interactive site for GPIbα. Electron microscopy showed clusters of

platelets in close contact. Binding of antibodies to the GPIbα N-terminal domain was

decreased, whereas GPIX and GPV were normally detected. In Western blotting (WB),

GPIbα, αIIb and β3 were normally present. Proteins involved in Ca2+ homeostasis were

analyzed by quantitating platelet mRNA or by WB. Plasma membrane Ca2+ ATPase (PMCA)-

4b and type III inositol trisphosphate receptor (InsP3-R3) were selectively increased. The

presence of degradation products of polyadenosine diphosphate (ADP)-ribose polymerase

protein (PARP) suggested ongoing caspase-3 activity. These were findings typical of

immature normal megakaryocytes cultured from peripheral blood CD34+ cells with TPO.

Significantly, megakaryocytes from the patients in culture produced self-associated and

interwoven proplatelets. Immunolocalization showed VWF not only associated with platelets,

but already on the megakaryocyte surface and within internal channels. In this family, type 2B

VWD is clearly associated with abnormal platelet production.




3

Introduction

Von Willebrand disease (VWD) is the most common inherited disorder of the platelet-vessel

wall interaction and involves both quantitative and qualitative defects of von Willebrand

factor (VWF), a crucial mediator of platelet function and carrier of the FVIII protein. In type

1 and type 3 VWD, deficiencies or absence of VWF protein are responsible for the bleeding

syndrome; but in Type 2 disease a functionally abnormal protein or the specific lack of large

multimers account for the VWD phenotype (1,2). In hemostasis, GPIbα mediates platelet

attachment to exposed subendothelium by binding through its N-terminus to the A1 domain of

VWF exposed within the subendothelial matrix (3, 4). In normal subjects, soluble VWF

multimers in plasma fail to gain access to their binding site on GPIbα, accessibility being

controlled by a disulfide-linked double loop region just below the leucine-rich repeats of

GPIbα (5). In type 2B VWD, mutations giving rise to a selective number of amino acid

substitutions in the A1 domain provide gain of function to the VWF multimers which then

spontaneously bind to platelets in suspension through a direct interaction with GPIbα (6-8).

This often results in the loss of the largest multimers from plasma although these may be at

least partially preserved in some cases (9). Bleeding results from platelets having blocked

GPIb function despite a heightened ristocetin-induced platelet agglutination in platelet

function testing, and perhaps through the relative hemostatic inefficiency of the remaining

small multimers. The thrombocytopenia that accompanies this disorder in some although not

all patients may also be a factor in defining bleeding severity.

The gain-of-function mutations that give rise to type 2B VWD are clustered within

exon 28 (see ISTH SSC database at http://www.ragtimedesign.com/vwf/mutation) of the

VWF gene. Among the most affected codons is Arginine (R)1308 which is commonly

substituted by a cysteine (C), and in isolated cases by histidine (H), proline (P) or leucine (L)

residues (2, 10-15). Although the basic phenotype of type 2B VWD can be accounted for by




4

the ability of the mutated protein to spontaneously bind to platelets, some reports highlight

platelet abnormalities that are difficult to explain by this interaction alone. For example, the

degree of reported thrombocytopenia is highly variable, with sometimes giant and

morphologically abnormal platelets being observed (16, 17). We ourselves have previously

reported giant platelets in 3 unrelated patients with type 2B VWD (18). Here, we describe a

previously unreported family with an autosomal dominant trait and where severe

thrombocytopenia is associated with the presence of agglutinates of morphologically

abnormal platelets. Analysis of the VWF gene using DNA from both affected members of the

family identified a heterozygous R1308P substitution in VWF. Other studies on platelets of

both patients concentrated on two Ca2+-pump families, the plasma membrane Ca2+ATPases

(PMCAs) and the sarco/endoplasmic reticulum Ca2+ATPases (SERCAs) and in particular

SERCA3 of which several isoforms exist in platelets (19). We noted an increased presence of

PMCA4b and inositol trisphosphate receptor3 (InsP3-R3). As we also showed changes in

Ca2+-ATPase and InsP3-R3 distribution during proplatelet production by normal

megakaryocytes (MKs) in culture, we considered that abnormal megakaryocytopoiesis, might

be part of the phenotype in this family. Significantly, culture of peripheral blood CD34+ cells

of both patients resulted in MKs showing self-associated proplatelets, with VWF already

bound to the MK surface. In this family, type 2B VWD and familial thrombocytopenia due to

an altered megakaryocytopoiesis are clearly related.




5

Methods:

Case reports

The patients are a brother (patient 1) and sister (patient 2), 58 and 57 years old respectively.

Relevant hemostatic parameters are summarized in Table I. Both have a lifelong bleeding

diathesis and a history of severe thrombocytopenia, with circulating platelet agglutinates a

constant feature (see Fig. 1). Single platelet counts for each patient were always < 25G/L

when performed with an Advia 120 counter (Bayer, Puteaux, France), values that were

confirmed by manual counting using a light microscope. For the brother, bleeding was

frequent during childhood with epistaxis several times a week. The sister additionally had

excessive bleeding at the time of menarche, and she experienced severe and prolonged

bleeding during surgery (appendectomy). The hemorrhagic tendency has decreased with age

for both patients and is now less frequently spontaneous. Arterial hypertension was first noted

for both patients at around 20 years old and they are treated with β-blockers. Although both

have received platelet transfusions, tests for anti-platelet antibodies are negative. Their now

deceased father had the same bleeding profile, with severe thrombocytopenia and agglutinated

platelets. There is no evidence of a bleeding syndrome in other family members although the

paternal grandfather died when very young. Patient 1 has one son in apparent good health but

who was unavailable for study. Controls were from hospital staff. All donors gave informed

consent according to the Declaration of Helsinki and studies were performed in line with local

ethical protocols.

Hemostatic tests and molecular diagnosis of type 2B VWD

Hemostatic tests were normal in routine testing unless stated otherwise. Prothrombin

consumption evaluated by measuring residual prothrombin with the Neoplastine CI plus kit

(Diagnostica Stago, Asnières, France) was abnormal, typical results being 22% for patient 1




6

and 38% for patient 2 (normal range 2-10%). Ristocetin cofactor activity (VWF:RCo) and

VWF:Ag were evaluated as described (18). Typical values are given in Table 1. FVIII:C was

measured using a one stage assay with Factor VIII:C-deficient plasma as the substrate

(Behring Diagnostica, Rueil Malmaison, France). It should be noted that results for the two

patients varied with blood group. Ristocetin-induced platelet agglutination (RIPA) was

hampered for the patient by the spontaneous agglutination; plasma from both patients caused

a weak agglutination of washed control platelets and this response was increased by low doses

of ristocetin (data not shown).

The Inserm network on molecular abnormalities in VWD (see 13) performed

specialized diagnosis. An absence of high molecular weight VWF multimers was shown by

SDS-polyacrylamide gel electrophoresis. ADAMTS 13 activity in the plasma was normal.

Genomic DNA was extracted from EDTA-anticoagulated whole blood using standard

methods and a heterozygous 3923 G->C transition was identified following PCR

amplification of the 5' portion of exon 28 of the VWF gene and direct sequencing. This gave

rise to a heterozygous R1308P substitution in the VWF protein for both patients. Sequencing

of the GPIbα gene according to ref 20 revealed no mutations (data not shown).

Table 1: Hemostatic parameters characterizing the patients Patient 1 Patient 2 Controls Platelet count < 20 < 20 150-400 G/L Platelet volume 8.5 8.5 6-8 µm3 Platelet agglutinates + + No VWF Ag 75-100 40-46 50-150% VWF RCo 100-115 46-55 50-150% VWF: HMW multimers Absent Absent Yes Blood group A+ 0+ RIPA 0.25 0.25 1-1.5 mg/ml Prothrombin consumption

32 32 <10%

ADAMTS 13 100 100 80-150% HMW = high molecular weight multimers




7

The VWF:RCo/VWF:Ag ratios were always > 0.7 for both patients, near normal values seen

for about 20% of type 2B patients in the French VWD network (13). The A blood group of

patient 1 with higher VWF:Ag and VWF:RCo was not associated with a more severe

thrombocytopenia or a clinically more severe form of the disease.

Platelet glycoprotein analysis

Unless stated otherwise, blood was withdrawn on 3.8 % sodium citrate (9:1 v/v). Platelet-rich

plasma (PRP) was prepared by centrifugation at 120 g for 10 min.

(a) Flow cytometry. PRP was incubated with MoAbs specific for αIIbβ3 (AP-2,

provided by Dr. T. Kunicki, Scripps Research Institute, La Jolla, USA), GPIbα (Bx-1,

prepared by the Bordeaux Laboratory; AP-1, from Dr. Kunicki; Alma-12, from Dr. F. Lanza,

EFS-Alsace, Strasbourg, France; SZ1, from Beckman Coulter, Marseille, France; WM23,

from Dr. M Berndt, Melbourne, Australia), GPIX (FMC25, Chemicon, Paris, France) and

α2β1 (Gi9, from Beckman Coulter) according to our standard procedures (21). In some

experiments, PRP prepared from ACD-A anticoagulated blood was supplemented with

apyrase (Sigma, St. Louis, MO, USA) and ACD-A as described (21), and the platelets

sedimented prior to resuspension in phosphate-buffered saline (PBS) buffer, pH7.2. These

platelets were incubated with the following antibodies: rabbit anti-VWF (Dako, Trappes,

France), or murine anti-LIBS MoAb, AP-6 (IgM, from T Kunicki); anti-RIBS, F26 (IgG,

originally given by Dr. H. Gralnick, NIH, Bethesda, USA), VH10 (IgG, anti-P-selectin,

prepared by the Bordeaux Laboratory). Platelets were sequentially incubated with primary

antibody followed by fluorescein isothiocyanate (FITC)-labeled F(ab')2 fragments of a goat

anti-mouse or by goat anti-rabbit immunoglobulins (Dako-France) (21). For the detection of

AP-6, dichlorotriazinylaminofluorescein (DTAF)–conjugated affinity-purified F(ab')2

fragments of donkey antimouse IgM (Jackson ImmunoResearch, West Grove, PA) were used.




8

All antibodies were at pre-determined primary concentrations. Platelets were analyzed in a

Cytomics F 500 (Beckman Coulter,Villepinte, France) or a FACSCalibur (Becton Dickinson)

flow cytometer. Gating to select platelets was based on preliminary determinations of forward

and wide-angle light scatter, agglutinates were excluded. Mean fluorescence intensity (MFI)

was measured after passage through a 530 nm long-pass interference filter. Histograms were

generated from measurements of 10 000 cells, and data were analyzed using RXP software

(Beckman Coulter) or the Cell Quest software (Becton Dickinson).

(b) Western blotting. Washed platelets were prepared as described (21) and lysed in

buffer containing 10 mM Tris-HCl (pH 7.0), 3 mM EDTA (ethylenediaminetetraacetic acid),

5 mM N-ethylmaleimide, and 2% sodium dodecyl sulfate (SDS) (20). When performed,

disulfide reduction was with dithiothreitol. Then, 10 µg or 20 µg protein was separated by

SDS–polyacrylamide gel electrophoresis (SDS-PAGE) on minigels and transferred

electrophoretically onto nitrocellulose membrane using Trans-Blot Transfer Medium

(Amersham Life Science, Bucks, United Kingdom). Nonspecific binding of protein was

blocked by incubating the membrane for 1 h in a solution of 5% fat-free milk in 20 mM Tris-

HCl, pH 8.2, containing 0.05% (vol/vol) Tween 20 (TB-T). Individual membrane strips were

then incubated with MoAbs to GPIbα (Bx-1; 1 µg/mL), αIIb (SZ22, Beckman Coulter; 0.5

µg/mL), or β3 (Y2/51, Dakopatts, Glostrup, Denmark; 0.5 µg/mL) for 12 h at room

temperature. After washing, the membrane was further incubated with a 1:10,000 dilution of

horseradish peroxidase–conjugated antimouse or anti-rabbit IgG (Jackson ImmunoResearch)

and bound antibody was detected using a chemiluminescence procedure (Amersham Life

Science) (20).

Studies on calcium-binding proteins and caspase activities in platelets




9

Total RNA and protein lysates were simultaneously prepared from EDTA-anticoagulated

blood using TRIZOL solution as recommended by the manufacturer (Invitrogen, Cergy-

Pointoise, France).

(a) Western blotting. Electrophoresis of proteins and WB analysis was performed as

previously described (22,23). Individual nitrocellulose membrane strips were incubated with

MoAbs to total PMCA (5F10, Affinity BioReagents, NJ, USA), PMCA4b (JA3, Labvision,

Fremont, USA), total SERCA3 (PL/IM430, originally obtained from Dr. N Crawford,

London, UK), total SERCA2 (IID8, Affinity BioReagents; platelets only express SERCA2b),

caspase-9 (Upstate, Lake Placid, NY, USA), caspase-12 (Sigma), InsP3-R3 (Transduction

Laboratories, Lexington, KY, USA), µ-calpain (Calbiochem, San Diego, CA, USA) and

PARP (Calbiochem). Also used were rabbit antibodies to SERCA3a and SERCA3b (see ref

24), β3 (a gift from Dr. D. Pidard, Paris, France), calreticulin (Novus Biological Inc, Littleton,

CO, USA), InsP3-R1 (ABR) and InsP3-R2 (Santa Cruz Biotechnology). Anti-mouse and anti-

rabbit peroxidase-conjugated antibodies were from Jackson Immunoresearch.

(b) RT-PCR based analyses. These were essentially performed according to published

procedures (22, 23). The primers used to amplify mRNA for PMCA1b, PMCA4b, SERCA2b,

SERCA3a-c and InsP3-R1-R3 were detailed previously by us (22,25,26). PCR was performed

acording to Martin et al (22) where a touchdown-PCR was performed using 10 cycles with

annealing temperature decrements from 65°C to 55°C. PCR was conducted for different

cycles, each of them consisting of successive periods of denaturation at 95°C for 1 min,

annealing at 58°C for 1 min, and extension at 72°C for 1 min. GAPDH amplifications were

used as controls of the amounts of RNA. Other controls included amplifying SERCA2b in the

absence of RT. PCR products were visualized on ethidium bromide-stained gels. The gels

were scanned using ADOBE Photoshop and quantified by Molecular Analyst, version NIH

Image 1.62b7.




10

In vitro liquid cultures of megakaryocytes from CD34+ cells

CD34+ cells were obtained from umbilical cord blood, from normal adult donors undergoing

leukapheresis, or from the peripheral blood of the patients. Precursor cells were separated

over a Ficoll-metrizoate gradient (Lymphoprep; Nycomed Pharma, Oslo, Norway) to obtain

an enriched fraction of mononuclear cells. CD34+ cells were purified using an

immunomagnetic cell sorting system according to the manufacturer's protocol (AutoMacs;

Miltenyi Biotec, Bergisch Gladbach, Germany) and cultivated in serum-free medium prepared

as previously reported and containing TPO (10 ng/ml; Kirin Brewery, Tokyo, Japan) (27).

Samples were taken between days 8 and 17 (see Results). Total RNA and protein lysate were

simultaneously prepared and treated for RT-PCR or WB analysis for Ca2+-ATPases or

calcium-binding proteins as described for platelets.

Analysis of proplatelet formation and immunofluorescence studies

Cultured megakaryocytes derived from CD34+ cells from the patients were plated on poly-L-

lysine-coated slides (Menzel-Glaser, Braunschweig, Germany) at day 12 of culture for 1 hour

in a cell incubator (37°C, 5% CO2 in air, 100 % humidity). Cells were then fixed with 2%

paraformaldehyde for 5 min, permeabilized with 0.1 % Triton X-100 for 5 min and incubated

with a mouse anti-P-selectin (VH10, see section on platelet glycoprotein analysis) and rabbit

anti-VWF (Dako) antibodies at predetermined saturating amounts for 1 hour at room

temperature. After 3 subsequent washes, cells were incubated with the appropriate secondary

antibodies conjugated with FITC or tetramethylrhodamine isothiocyanate (TRITC) (27).

When performed, counter staining for nuclei was with 4'-6-Diamidino-2-phenylindole

dihydrochloride (DAPI). The images were acquired using a Zeiss laser scanning microscope




11

(LSM 510 Zeiss, Jena, Germany) by using a 63X1/NA oil objective or an inverted Leica DM

IFBE microscope (Nikon Eclipse 600, Tokyo, Japan) again with a 63X1/NA oil objective.

Electron microscopy

PRP was prepared from citrated blood by centrifugation for 10 min at 80 g, incubated for 20

min at 37° C, and fixed in 1.25% glutaraldehyde (Fluka) for 1 h at room temperature. Samples

were processed for standard electron microscopy (EM) as previously described (18). Cultured

megakaryocytes from the patients or control donors were fixed under the same conditions for

1 h, washed, and transported to Bordeaux overnight in 0.5% glutaraldehyde. For immunogold

labeling, sucrose-loaded megakaryocytes were frozen in propane and then in liquid nitrogen

using a Reichert KF 80 freezing system (Leica, Vienna, Austria) (28),Ultrathin sections were

cut at –120° C with an Ultracut E ultramicrotome (Reichert, Villepinte, France) equipped with

an FC 4E cryokit attachment and placed on collodion-coated nickel grids (28). After washing,

sections were floated on buffer containing anti-VWF (10 µg/ml) (Dako). Bound antibody was

detected using goat anti-rabbit IgG conjugated to 10 nm gold particles (Amersham, Orsay,

France). The sections were stained with uranyl acetate and osmium according to our standard

procedures (28). Sections were embedded in a thin film of methylcellulose and observed with

a Jeol JEM-1010 transmission electron microscope (Jeol, Croissy-sur-Seine, France) at 80 kV.

Controls included the absence of primary antibody or its substitution with an irrelevant IgG of

the same species and at the same concentration.




12

Statistics

Data are presented as mean ± S.D. Statistical significance was determined in multiple

comparisons among independent groups of data in which ANOVA indicated the presence of

significant differences. P < 0.01 was considered significant.




13

Results

Platelet ultrastructure:

For the two patients, platelet agglutinates were observed on smears of whole blood. Results

were similar with blood anticoagulated with EDTA, ACD-A or citrate thus ruling out EDTA-

dependent pseudothrombocytopenia. Electron microscopy showed that platelets in PRP were

often round and somewhat enlarged, but not giant (Fig. 1). Interestingly, platelets were often

joined together by well-defined areas of contact. Sometimes, protein bridges could be seen

between the adjacent platelets (Fig 1B), but in others the contact was tight and fuzzy at high

power, as if fusion had occurred (Fig. 1C).. Although platelet agglutinates were present, there

were no signs of platelet activation; the α-granules were not centralized and pseudopodia

were rare. Platelet aggregation with physiologic agonists could not be tested, not only because

of the very low platelet count in PRP, but also because of continued spontaneous platelet

agglutination under stirring.

Studies on platelet glycoproteins by flow cytometry and western blotting:

As shown in Fig 2A, platelets from both patients normally expressed α2β1 (Gi9) and αIIbβ3

integrins (AP2) by flow cytometry while both the αIIb (not shown) and β3 subunits were

normally seen by Western blotting (Fig 2B). A panel of MoAbs directed against different

epitopes of the GPIb-IX-V complex were tested in flow cytometry. Whereas MoAbs directed

against GPIbα (AP-1, Alma-12, Bx-1 and WM23) showed decreased binding, that of MoAbs

directed against GPIX (FMC25) or an epitope depending on the presence of the complex

(SZ1) were normal or increased. By testing platelets sedimented and resuspended in buffer,

we found an increased presence of VWF for both patients but more for patient 2. The use of

anti-RIBS and anti-LIBS MoAbs showed that αIIbβ3 showed little signs of activation (Fig.

2C). Expression of P-selectin at the platelet surface was also minimal thereby confirming the




14

absence of spontaneous secretion. Western blotting showed no evidence of platelet GPIbα

deficiency (Fig 2B) suggesting that the lower expression of GPIbα in flow cytometry was a

question of MoAb accessibility perhaps linked to the presence of VWF.

Studies on proplatelet production and VWF expression by megakaryocytes cultured in vitro

from CD34+ cells isolated from peripheral blood of the patients:

CD34+ cells were isolated from the blood of both patients and cultured in the presence of

TPO. As seen by phase contrast microscopy, control cultures contained megakaryocytes with

the typical long processes of mature cells (Fig. 3A). Megakaryocyte cultures were obtained

for the patients were clearly capable of producing proplatelets. Both short extensions and long

processes were seen. But to our surprise, and unlike those seen for control cultures, the latter

were interwoven with touching membranes (Fig. 3B). The distribution of VWF in the

megakaryocytes was next examined by fluorescence microscopy and compared to that of P-

selectin. Normally, VWF, which is synthesized in the megakaryocytes, is trafficked from the

trans-Golgi apparatus to the α-granules. This leads to a punctated and largely intracellular

distribution in confocal microscopy (illustrated in ref 29). For the megakaryocytes of both

patients, we unusually detected VWF on the cell surface including on the proplatelets (Figure

3C-K)). In most cells an accompanying intracellular distribution was also observed. In

contrast, P-selectin had a normal intracellular localization (Fig. 3G,J). After double labeling,

VWF was clearly not always co-localized with P-selectin as an α-granule marker, the VWF

surface pool being clearly distinguishable (Fig 3H,K). Immunogold labeling and electron

microscopy confirmed the presence of VWF on the surface of megakaryocytes (including on

protrusions), within the channels of the surface-connected system as well as in α-granules

(Fig. 4).




15

Studies of proteins involved in Ca2+ signaling in the patients' platelets:

A common feature of the binding of various ligands to their receptors is a modulation of Ca2+

homeostasis. Ca2+ signaling is controlled by many Ca2+ ATPases and InsP3-receptors, some

of them being involved in cell differentiation and apoptosis (30,31). This led us to compare

the expression of SERCA- and PMCA- type Ca2+-ATPases as well as InsP3-receptors in

platelet samples isolated from both patients and normal donors. Figure 5A compares the

expression of SERCA-type Ca2+-ATPases (2b, total 3 [lane 6], 3a [lane 7], 3b), total PMCA

(1b + 4b), PMCA4b and InsP3-R1-3 proteins by WB. Calreticulin and β3 were chosen as

control proteins. Quantitative comparisons of the expressions of the different proteins are

shown in Fig 5B. Expression of InsP3-R3 and PMCA4b was 149 ± 8 % and 308 ± 25 %

greater in platelets isolated from patient 1, and 192 ± 18 % and 368 ± 14 % greater in platelets

isolated from patient 2. RT-PCR was used to study mRNAs for these proteins in the platelets

from the controls and the patients. SERCA-type Ca2+-ATPases, InsP3-R1, InsP3-R2 and

PMCA1b showed only slight variations between the controls and the patients. In line with the

altered protein expression, platelets from both patients showed an increased content of InsP3-

R3 and PMCA4b mRNA (Fig 5C and D). Here increases amounted to 175 ± 36 % and 211 ±

23 % in platelets isolated from patient 1 and 221 ± 28 % and 228 ± 29 % in platelets

isolated from patient 2. Specific increases were also seen for mRNAs of some SERCAs with

SERCA 3a increased by 177 ± 22 % for patient 1 and 186 ± 26 % for patient 2. In the context

of our earlier work, these results suggested an abnormal megakaryocytopoiesis (26,31).

Studies of proteins involved in Ca2+-homeostasis in normal megakaryocytes:

Next, CD34+ cells isolated from cord blood or after leukapheresis were cultured in vivo.

Samples taken between days 8 and 17 were analyzed by WB or by RT-PCR for mRNA

content. Typical Western blots for protein expression in the megakaryocytes are shown in Fig.




16

6A and compared to the expression of β3 and calreticulin. A quantitative evaluation of the

data is given in online Fig. 1. Whereas calreticulin levels remained stable, levels of β3

increased with megakaryocyte maturation. A specific increase in SERCA3a protein

expression was accompanied by a net decrease in PMCA4b expression during proplatelet

formation (days 11-17). The kinetics of this are shown in panel 6B and compared to β3

expression. The source of the CD34+CD41+ cells had little influence on the results. InsP3-

receptors were not studied here. Results for platelets are shown for comparison. In terms of

mRNA levels, while those of PMCA1b and InsP3-R1-2 showed modest regulations in the

megakaryocytes, significantly SERCA3-type Ca2+-ATPases are transitionally up-regulated

upon proplatelet formation and both PMCA4b and InsP3-R3 showed a net decrease from day

11 onwards (online Fig. 2).

Caspase activity in platelets of the patients and in normal megakaryocytes in culture:

Since platelet production is associated with caspase-3 activation and since PMCA4b is a

caspase-3 substrate (29, 30), we looked at possible variations in caspase-3 activation during

proplatelet formation and platelet release. We first looked for evidence of caspase-3 activity

in normal megakaryocytes by testing for cleavage of the caspase-3 substrate PARP. As

shown, in Fig. 6A, the native form of PARP (116 kDa) was cleaved from day 11 onwards and

in parallel with the loss of PMCA4b including its caspase-3 mediated proteolysis (typical 120-

kDa fragment) and the formation of proplatelets.

As sufficient cultured megakaryocytes were unavailable for WB, we re-examined

platelets from the patients and looked for an abnormal caspase-3 activity. Using the same

protein samples as those in Fig 5, we detected full-length PARP in platelets of normal donors

and in the two patients (Fig. 7). In addition, a cleaved 85-kDa PARP degradation product was

specifically observed in the platelets of the two patients (Fig. 7). In contrast, no trace of




17

cleaved PARP was detected in normal platelets. Because caspase-9 and caspase-12 are

activated in parallel to caspase-3 during megakaryocytopoiesis (32,33), we looked for their

presence in the platelets of patient 2. However, only procaspase-9 and pseudoprocaspase-12

were detected and without any degradation (Fig. 7). Finally, because µ-calpain may promote

apoptosis-like events during platelet activation and may degrade PMCA4b (although

proplatelet formation is not a calpain-dependent process) (31,32), we studied calpain

expression in the platelets of the same donors and patients. Again, no trace of autoproteolysis

could be detected under our experimental conditions (Fig 7).

Thus, the two patients studied here had an abnormal caspase-3 activity in their

platelets, and higher PMCA4b and InsP3-R3 expression. Comparison with the relative

expression and/or activity of the same proteins during megakaryocytopoiesis suggests that the

platelets may be produced from immature cells in an apoptotic phase of proplatelet formation.




18

Discussion

Our family was characterized by the presence of a heterozygous R1308P substitution in the

VWF A1 domain, spontaneous binding of VWF to platelets, and autosomal dominant

inheritance that typifies type 2B VWD. Less typical was the constant presence of circulating

platelet agglutinates and a severe thrombocytopenia. Strikingly, the agglutinates were seen in

the presence of all anticoagulants and continued to be seen when blood was taken directly into

anticoagulant containing saturating amounts of blocking antibodies for αIIbβ3 (abciximab)

and GPIbα (alma-12) (data not shown). When characterized by electron microscopy, the

agglutinates were mostly composed of heterogeneously sized and often rounded platelets with

distinct zones of contact. It is probable that spontaneous binding of the mutated VWF is

causing the agglutination. Previous results from our laboratory have visualized VWF bridges

cross-linking platelets following RIPA or shear-induced platelet aggregation (34,35). The

finding that MoAbs to GPIbα bound less well to the patients' platelets than MoAbs to GPIX

or to a complex-dependent determinant on GPIb-IX, are also compatible with a masking

effect of surface-bound VWF.

Despite the above results, such a severe thrombocytopenia and the presence of platelet

agglutinates is rare in patients with type 2B VWD (1,2,13). One possibility is that the nature

of the mutation in this disease can influence phenotype, and studies with recombinant proteins

mutated at different residues within the A1 domain (aa1260-1479) have confirmed that they

bind with different affinities to GPIbα (36,37). While the presence of agglutinates and severe

thrombocytopenia is one variable in type 2B VWD, the presence of giant platelets is another

(16-18). Yet, 3 patients with thrombocytopenia and giant platelets possessed different

mutations in the VWF gene (1304insMet; V1316M, P1337L) showing that such morphologic

changes are not linked to a recurrent mutation (18). Although thrombocytopenia can be

greater during pregnancy or after DDAVP infusion in type 2B VWD, when plasma levels of




19

VWF are increased (1,38), in our family the severe thrombocytopenia was constant. Important

questions therefore arise as to why such platelet variability is seen in type 2B VWD, and what

factors control the 'platelet' phenotype.

It has previously been demonstrated that spontaneous binding of mutated type 2B

VWF induces a cytoplasmic Ca2+ rise in platelets (39). Thus we tested platelets of both

patients for their expression of a series of Ca2+-ATPases or Ca2+-binding proteins.

Significantly, PMCA4b and InsP3-R3 were increased for both protein and mRNA expression

while for SERCA-3a only mRNA was increased. PMCA4b is a substrate for caspase-3, an

enzyme involved in platelet production by megakaryocytes (31,32,40). So we studied its

expression compared to SERCAs during normal megakaryocytopoiesis. To do this,

CD34+CD41+ cells from umbilical cord blood or following leukapheresis were cultured with

SCF and TPO and examined between days 8 and 17. A specific increase in SERCA3 protein

expression in mature megakaryocytes paralleled a striking down-regulation of PMCA4b and

this in cells from each source. Thus a greater presence of PMCA4b in the patient's platelets

suggested that they were being produced from more immature megakaryocytes. This was

supported when the patients' platelets were unusually shown to contain a 85 kDa degradation

product of another caspase-3 substrate, PARP. Procaspase-3 but not caspase-3 has been

previously seen in control platelets (41). Comparison of the kinetics of PMCA4b and PARP

degradation in normal megakaryocytes suggested that both were linked with proplatelet

formation. As degradation of PMCA4b is associated with apoptosis in other cells in vitro (40),

and as InsP3-R3 has recently been shown to preferentially transmit apoptotic signals into

mitochondria (42), our results comfort the hypothesis that the defects in our family extend to

the apoptotic phase of megakaryocyte maturation. This were specific changes for no evidence

of caspase-9 or caspase-12 activities were seen in platelets of patient 2. Both caspase-9 (32)

and caspase-12 (33) are active during normal megakaryocytopoiesis.




20

CD34+CD41+ cells from the peripheral blood of both patients normally gave rise to

megakaryocyte colonies. As cells matured they normally formed stunted processes possibly

resembling immature proplatelets (43), but a major surprise was that the long proplatelet-like

structures were intertwined and incompletely separated. Whereas we had previously shown

that cultured human and murine megakaryocytes gave a punctate pattern on

immunofluorescence analysis for VWF (29), for the patients there was an additional surface

staining for VWF. Confocal microscopy and dual staining with anti-P-selectin antibody

confirmed the normal formation of α-granules within the central megakaryocyte body, the

presence of an intracellular VWF pool and confirmed that P-selectin did not follow the VWF

to the surface. Immunogold staining for VWF confirmed that VWF was on the surface of the

megakaryocytes. One possibility is that it is being excluded from the megakaryocytes and

then rebinds. An intriguing alternative is that the mutated protein is spontaneously binding to

GPIbα during its passage through the Golgi apparatus and is carried to the surface by the

GPIb-IX-V complex.

The R1308P mutation (R545P in terms of an older nomenclature) has been located

previously in a family with type 2B VWD, the principal reported propositus had a platelet

count of 100 G/L (14) with plasma lacking high molecular weight multimers. In this same

study, recombinant mutated VWF with P1308 and C1308 substitutions bound spontaneously

to platelets and had greater reactivity with GPIb compared to substitutions at positions 1306

or 1341 (14). In fact, evidence obtained from the crystal structure of the A1 domain suggests

that R1308 is involved in salt bridge formation or intramolecular packing and the P1308

substitution changes A1 domain conformation allowing facilitated access of a closely

localized sequence directly involved in binding to GPIbα (44). As much evidence suggests

that GPIbα (or the GPIb-IX-V complex) is a signaling molecule through the association of

GPIbα or GPIbβ cytoplasmic domains with proteins such as 14-3-3_ζcalmodulin (a regulator




21

protein of PMCA4b) or FcRγ, one possibility is that GPIbα-regulated signaling is involved in

abnormal platelet production (45-47). Yet, in our study, platelets from both patients showed

few signs of activation in that secretion had not occurred and αIIbβ3 inetgrin was not in an

active conformation. Notwithstanding, αIIbβ3-independent shear induced platelet aggregation

has been observed with recombinant VWF mutated for another type 2B VWD mutation,

V1316M which led to pp125FAK phosphorylation (48).

The degree of thrombocytopenia in our family is severe, and while this is undoubtedly

in part due to the clearance of circulating agglutinates, we have presented evidence for

changes in the production of proplatelets during megakaryocytopoiesis. Perhaps the nearest

report in the literature to our family is the description of type 2B Tampa (49,50). Here,

chronic thrombocytopenia (9-46,000/µL for 3 patients) associated with in vivo platelet

aggregate formation and spontaneous platelet aggregation in vitro. Also of interest is type 2B

Hiroshima, where chronic thrombocytopenia was associated with normal plasma levels of

multimers (51). A platelet imbalance between PMCA and SERCA-type ATPases as shown

by us is a new marker of an abnormal megakaryocytopoiesis. This could in our family result

in the release of morphologically abnormal platelets, or even platelets that are incompletely

separated and which circulate as clusters. Abnormal processing of the mutated VWF in the

megakaryocytes could be a cause of this abnormality, although the fact that closely-related

mutations do not result in such a severe thrombocytopenia suggests that other contributory

factors are involved. Perhaps nonidentified modifier genes linked or not to the Ca2+ -binding

protein imbalance contribute to the phenotypic variability in type 2B VWD. Whatever the

cause, our results unambiguously show that this family with type 2B VWD may also be

considered as having familial thrombocytopenia.




22

Acknowledgement: This work was performed in the context of the French network on

'Rare Diseases of Platelet Function and Production' financed by the GIS-Institut des Maladies

Rares and Inserm. We thank Jalil Abdelali, IFR54, Institut Gustave Roussy, for confocal

analysis.




23

References

1. Sadler JE. New concepts in von Willebrand disease. Annu Rev Med. 2005;56:173-

191.

2. Mazurier C, Ribba AS, Gaucher C, Meyer D. Molecular genetics of von Willebrand

disease. Ann Genet. 1998;41:34-43.

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

receptor interactions in platelet thrombus formation under flow. Cell. 1998;94:657-666.

4. Dopheide SM, Maxwell MJ, Jackson SP. Shear-dependent tether formation during

platelet translocation on von Willebrand factor. Blood. 2002;99:159-167.

5. Uff S, Clemetson JM, Harrison T, Clemetson KJ, Emsley J. Crystal structure of the

platelet glycoprotein Ibα N-terminal domain reveals an unmasking mechanism for receptor

activation. J Biol Chem. 2002;277:35657-35663.

6. Ruggeri ZM, Pareti FI, Mannucci PM, Ciavarella N, Zimmerman TS. Heightened

interaction between platelets and factor VIII/von Willebrand factor in a new subtype of von

Willebrand's disease. N Engl J Med. 1980;302:1047-1051.

7. Huizinga EG, Tsuji S, Romijn RA, et al. Structures of glycoprotein Ibα and its

complex with von Willebrand factor A1 domain. Science. 2002;297:1176-1179.

8. Dumas JJ, Kumar R, McDonagh T, et al. Crystal structure of the wild-type von

Willebrand factor A1-glycoprotein Ibα complex reveals conformation differences with a

complex bearing von Willebrand disease mutations. J Biol Chem. 2004;279:23327-23334.

9. Federici AB, Mannucci PM, Stabile F, et al. A type 2b von Willebrand disease

mutation (Ile546-->Val) associated with an unusual phenotype. Thromb Haemost.

1997;78:1132-1137.




24

10. Sadler JE. von Willebrand factor: two sides of a coin. J Thromb Haemost.

2005;3:1702-1709.

11. Cooney KA, Nichols WC, Bruck ME, et al. The molecular defect in type IIB von

Willebrand disease. Identification of four potential missense mutations within the putative

GpIb binding domain. J Clin Invest. 1991;87:1227-1233.

12. Randi AM, Rabinowitz I, Mancuso DJ, Mannucci PM, Sadler JE. Molecular basis of

von Willebrand disease type IIB. Candidate mutations cluster in one disulfide loop between

proposed platelet glycoprotein Ib binding sequences. J Clin Invest. 1991;87:1220-1226.

13. Meyer D, Fressinaud E, Gaucher C, et al. Gene defects in 150 unrelated French cases

with type 2 von Willebrand disease: from the patient to the gene. INSERM Network on

Molecular Abnormalities in von Willebrand Disease. Thromb Haemost. 1997;78:451-456.

14. Hilbert L, Gaucher C, Abgrall JF, Parquet A, Trzeciak C, Mazurier C. Identification of

new type 2B von Willebrand disease mutations: Arg543Gln, Arg545Pro and Arg578Leu. Br J

Haematol. 1998;103:877-884.

15. Baronciani L, Federici AB, Beretta M, Cozzi G, Canciani MT, Mannucci PM.

Expression studies on a novel type 2B variant of the von Willebrand factor gene (R1308L)

characterized by defective collagen binding. J Thromb Haemost. 2005;3:2689-2694.

16. Lopez-Fernandez MF, Lopez-Berges C, Martin-Bernal JA, et al. Type IIB von

Willebrand's disease associated with a complex thrombocytopenic thrombocytopathy. Am J

Hematol. 1988;27:291-298.

17. Moll S, Lazarowski AR, White GC, 2nd. Giant platelet disorder in a patient with type

2B von Willebrand's disease. Am J Hematol. 1998;57:62-67.

18. Nurden P, Chretien F, Poujol C, Winckler J, Borel-Derlon A, Nurden A. Platelet

ultrastructural abnormalities in three patients with type 2B von Willebrand disease. Br J

Haematol. 2000;110:704-714.




25

19. Bobe R, Bredoux R, Corvazier E, et al. How many Ca2+ATPase isoforms are

expressed in a cell type? A growing family of membrane proteins illustrated by studies in

platelets. Platelets. 2005;16:133-150.

20. Hillmann A, Nurden A, Nurden P, et al. A novel hemizygous Bernard-Soulier

Syndrome (BSS) mutation in the amino terminal domain of glycoprotein (GP)Ibβ. Platelet

characterization and transfection studies. Thromb Haemost. 2002;88:1026-1032.

21. Nurden P, Savi P, Heilmann E, et al. An inherited bleeding disorder linked to a

defective interaction between ADP and its receptor on platelets. Its influence on glycoprotein

IIb-IIIa complex function. J Clin Invest. 1995;95:1612-1622.

22. Martin V, Bredoux R, Corvazier E, et al. Three novel sarco/endoplasmic reticulum

Ca2+ATPase (SERCA) 3 isoforms. Expression, regulation, and function of the members of the

SERCA3 family. J Biol Chem. 2002;277:24442-24452.

23. Bobe R, Bredoux R, Corvazier E, et al. Identification, expression, function, and

localization of a novel (sixth) isoform of the human sarco/endoplasmic reticulum Ca2+ATPase

3 gene. J Biol Chem. 2004;279:24297-24306.

24. Kovàcs T, Felfoldi F, Papp B, et al. All three splice variants of the human

sarco/endoplasmic reticulum Ca2+-ATPase 3 gene are translated to proteins: a study of their

co-expression in platelets and lymphoid cells. Biochem J. 2001;358:559-568.

25. Martin V, Bredoux R, Corvazier E, Papp B, Enouf J. Platelet Ca2+ATPases : a plural,

species-specific, and multiple hypertension-regulated expression system. Hypertension.

2000;35:91-102.

26. Lacabaratz-Porret C, Launay S, Corvazier E, et al. Biogenesis of endoplasmic

reticulum proteins involved in Ca2+ signaling during megakaryocytic differentiation: an in

vitro study. Biochem J. 2000;350:723-734.




26

27. Debili N, Wendling F, Katz A, et al. The Mpl-ligand or thrombopoietin or

megakaryocyte growth and differentiation factor has both direct proliferative and

differentiative activities on human megakaryocyte progenitors. Blood. 1995;86:2516-2525.

28. Nurden P, Poujol C, Durrieu-Jais C, et al. Labeling of the internal pool of GP IIb-IIIa

in platelets by c7E3 Fab fragments (abciximab): flow and endocytic mechanisms contribute to

the transport. Blood. 1999;93:1622-1633.

29. Wilcox DA, Shi Q, Nurden P, et al. Induction of megakaryocytes to synthesize and

store a releasable pool of human factor VIII. J Thromb Haemost. 2003;1:2477-2489.

30. Khan AA, Soloski MJ, Sharp AH, et al. Lymphocyte apoptosis: mediation by

increased type 3 inositol 1,4,5-trisphosphate receptor. Science. 1996;273:503-507.

31. Pàszty K, Verma AK, Padanyi R, Filoteo AG, Penniston JT, Enyedi A. Plasma

membrane Ca2+ATPase isoform 4b is cleaved and activated by caspase-3 during the early

phase of apoptosis. J Biol Chem. 2002;277:6822-6829.

32. De Botton S, Sabri S, Daugas E, et al. Platelet formation is the consequence of caspase

activation within megakaryocytes. Blood. 2002;100:1310-1317.

33. Kerrigan SW, Gaur M, Murphy RP, Shattil SJ, Leavitt AD. Caspase-12: a

developmental link between G-protein-coupled receptors and integrin αIIbβ3 activation.

Blood. 2004;104:1327-1334.

34. Hourdillé P, Gralnick HR, Heilmann E, et al. von Willebrand factor bound to

glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin.

Blood. 1992;79:2011-2021.

35. Poujol C, Nurden A, Paponneau A, Heilmann E, Nurden P. Ultrastructural analysis of

the distribution of von Willebrand factor and fibrinogen in platelet aggregates formed in the

PFA-100TM. Platelets. 1998;9:381-389.




27

36. de Romeuf C, Hilbert L, Mazurier C. Platelet activation and aggregation induced by

recombinant von Willebrand factors reproducing four type 2B von Willebrand disease

missense mutations. Thromb Haemost. 1998;79:211-216.

37. Hilbert L, Gaucher C, Mazurier C. Effects of different amino-acid substitutions in the

leucine 694-proline 708 segment of recombinant von Willebrand factor. Br J Haematol.

1995;91:983-990.

38. Pareti FI, Federici AB, Cattaneo M, Mannucci PM. Spontaneous platelet aggregation

during pregnancy in a patient with von Willebrand disease type IIB can be blocked by

monoclonal antibodies to both platelet glycoproteins Ib and IIb/IIIa. Br J Haematol.

1990;75:86-91.

39. Francesconi M, Casonato A, Pontara E, Dalla Via L, Girolami A, Deana R. Type IIB

von Willebrand factor induces phospholipase A2 activation and cytosolic Ca2+ increase in

platelets. Biochem Biophys Res Commun. 1995;214:102-109.

40. Pàszty K, Kovàcs T, Lacabaratz-Porret C, et al. Expression of hPMCA4b, the major

form of the plasma membrane calcium pump in megakaryoblastoid cells is greatly reduced in

mature human platelets. Cell Calcium. 1998;24:129-135.

41. Shcherbina A, Remold-O'Donnell E. Role of caspase in a subset of human platelet

activation responses. Blood. 1999;93:4222-4231.

42. Mendes CC, Gomes DA, Thompson M, et al. The type III inositol 1,4,5-trisphosphate

receptor preferentially transmits apoptotic Ca2+ signals into mitochondria. J Biol Chem.

2005;280:40892-40900.

43. Richardson JL, Shivdasani RA, Boers C, Hartwig JH, Italiano JE, Jr. Mechanisms of

organelle transport and capture along proplatelets during platelet production. Blood.

2005;106:4066-4075.




28

44. Emsley J, Cruz M, Handin R, Liddington R. Crystal structure of the von Willebrand

Factor A1 domain and implications for the binding of platelet glycoprotein Ib. J Biol Chem.

1998;273:10396-10401.

45. Andrews RK, Munday AD, Mitchell CA, Berndt MC. Interaction of calmodulin with

the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX-V complex. Blood.

2001;98:681-687.

46. Dai K, Bodnar R, Berndt MC, Du X. A critical role for 14-3-3zeta protein in

regulating the VWF binding function of platelet glycoprotein Ib-IX and its therapeutic

implications. Blood. 2005;106:1975-1981.

47. Ozaki Y, Asazuma N, Suzuki-Inoue K, Berndt MC. Platelet GPIb-IX-V-dependent

signaling. J Thromb Haemost. 2005;3:1745-1751.

48. Mekrache M, Bachelot-Loza C, Ajzenberg N, Saci A, Legendre P, Baruch D.

Activation of pp125FAK by type 2B recombinant von Willebrand factor binding to platelet

GPIb at a high shear rate occurs independently of αIIbβ3 engagement. Blood. 2003;101:4363-

4371.

49. Saba HI, Saba SR, Dent J, Ruggeri ZM, Zimmerman TS. Type IIB Tampa: a variant of

von Willebrand disease with chronic thrombocytopenia, circulating platelet aggregates, and

spontaneous platelet aggregation. Blood. 1985;66:282-286.

50. Takimoto Y, Imanaka F. Type 2B Hiroshima: a variant of von Willebrand disease

characterized by chronic thrombocytopenia and the presence of all von Willebrand factor

multimers in plasma. Int J Hematol. 1999;70:127-131.

51. Saba HI, Fujimura Y, Saba SR, et al. Spontaneous platelet aggregation in type IIB

Tampa von Willebrand disease is inhibited by the 52/48-kDa fragment of normal von

Willebrand factor, which contains the GPIb binding domain. Am J Hematol. 1989;30:150-

153.




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Figure Legends

Figure 1: Ultrastructure of platelets in PRP from patient 1 as seen by standard transmission

electron microscopy. To be noted are the presence of platelet agglutinates and platelets of

different size and shape that include round forms. Granules are randomly distributed and the

platelets show no signs of activation (A). Platelets are often attached through discrete areas of

surface contact, arrows highlight how at some interfaces protein bridges can be clearly seen

(B) while at other interfaces the membrane surfaces appear to be in direct contact (C). Bar in

A = 1 µm, in B and C = 200 nm.

Figure 2: Platelet glycoprotein analysis by flow cytometry (A,C) and Western blotting (B). In

(A), PRP from patients 1 and 2 or a control donor were incubated with a battery of MoAbs

recognizing αIIbβ3 (AP2), α2β1 (Gi9), GPIbα (AP1, Alma12, Bx1, WM23), GPIX (FMC25)

or the GPIb-IX complex (SZ1). In (C), platelets from patients 1 and 2 or a control donor were

sedimented and resuspended in washing buffer prior to incubation with a polyclonal antibody

to VWF, a MoAb to P-selectin (VH10), and anti-LIBS and anti-Ribs MoAbs respectively. In

(B) lysates of platelets from patients 1 (P1) and 2 (P2) and a control donor were analyzed by

SDS-PAGE and immunoblotted with MoAbs to β3 and GPIbα.

Figures 3: Phase contrast and immunofluorescence analysis of megakaryocytes cultured in

vitro from CD34+ peripheral blood samples from patient 1. Phase contrast microscopy shows

that megakaryocytes from a control donor at day 12 of culture have typical long and discrete

proplatelets (arrows) (A). In contrast, megakaryocytes from patient 1 give rise to proplatelets

that are intertwined (arrow heads). Fixed and permeabilized cells from the patient were

subjected to immunofluorescence labeling for VWF (green). Results show that at day 12 for




30

the patient, VWF has both intracellular and surface staining (white arrows) (C-E). Nuclei are

shown in blue by DAPI. Confocal microscopy in F-K shows the distribution of VWF and P-

selectin in cultured megakaryocytes from patient 1. Cells taken at day 12 were fixed and

permeabilized prior to being labeled for VWF (green fluorescence) (F,I) and P-selectin (red

fluorescence) (G, J). When the colors were merged (H,K), much of the VWF has a different

localization to the α-granule marker, P-selectin, with the labeling for VWF clearly extending

to the surface of the cell.

Figure 4: Immunogold labeling of VWF on cultured cells. Megakaryocytes at day 12 from

cultures of CD34+ cells isolated from the peripheral blood of patient 1 were fixed and frozen-

thin sections stained sequentially using a polyclonal anti-VWF antibody and a secondary

antibody to rabbit IgG adsorbed onto 10 nm gold beads. Transmission electron microscopy

revealed that VWF was present on the cell surface (arrow heads, A) as well as in intracellular

canals (arrows, A-C) and in well-developed α-granules (D, E). Multivesicular bodies (MVB)

are seen in (E). Bars = 500 nm

Figure 5: Abnormal expression of proteins involved in Ca2+ signaling in the patients.

Platelets isolated from 3 normal donors (C1-3) and the two patients P1 and P2 were treated

for simultaneous isolation of total lysate proteins and RNA. (A) Proteins were subjected to

SDS-PAGE and Western Blotting using the isoform-specific antibodies indicated to detect

PMCA, InsP3-R and SERCA-type proteins as well as β3 and calreticulin (internal controls).

In this Figure and the following, the size of proteins is given in kDa. (B) The intensity of the

bands was quantified by densitometry and the mean values obtained for the 3 normal donors

were arbitrarily taken as 100%. The mean ± SD values (n = 8) of relative intensity

(normalized to calreticulin) are shown in arbitrary units. *P < 0.01 compared with values




31

from normal donors. (C) Total RNA was subjected to specific RT-PCR to detect PMCA-,

InsP3-R- and SERCA- RNA species, as well as GAPDH ( internal control). Numbers indicate

the sizes of PCR products in base pairs (bp). (D) The intensity of the bands was quantified as

above except that the mean values (n = 6 to 10) of relative intensity were normalized to

GAPDH. *P < 0.01 compared with values from normal donors.

Figure 6: The proteins involved in Ca2+ signaling in the patients exhibit similarities with

immature megakaryocytes. (A) CD34+CD41+ cells isolated from both umbilical cord blood

(Left) and leukapheresis (Right) samples were FACS-purified, at day 8 of culture. Different

sorted cell samples (3) were cultured for either up to 14 or to 16-17 days and cells were

further treated as above. Proteins were treated for Western blotting to detect same SERCA

and PMCA-type Ca2+-ATPases and control proteins as in Fig. 6 and PARP (n = 5). The

intensity of the bands was quantified by densitometry and the mean values obtained at days 8

or 9 were arbitrarily taken as 100% (On-line supplemental Fig 1). (B) The summary of

densitometric measurements was used to draw the schematic representation of the relative

expressions of PMCA4b, SERCA3a and β3 along with megakaryocytopoiesis (Left) and to

position those of the platelets of the patients (Right).

Figure 7: Platelets of the patients exhibit abnormal caspase-3 processing.

Proteins from the same samples as those used in Fig. 5 were subjected to Western blotting

using the antibodies indicated, to detect the expression of proteins involved in the apoptotic

pathways. The Figure is typical of 3 independent experiments.




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impaired megakaryocytopoiesis in type 2b von willebrand disease with severe thrombocytopenia

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