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Blood First Edition Paper, prepublished online June 12, 2009; DOI 10.1182/blood-2009-03-209205
Exposure of human megakaryocytes to high shear rates accelerates platelet production
Claire Dunois-Lardé1,2, Claude Capron3-5, Serge Fichelson2,3, Thomas Bauer6, Elisabeth
Cramer-Bordé*3-5, Dominique Baruch*1,2.
* Equal contribution
1
INSERM U765, Paris, France; 2Université Paris Descartes, Paris, France; 3INSERM
U567, Paris, France ; 4Université de Versailles-St Quentin, Versailles, France ;
5
Service d’Hématologie et d’Immunologie, Hôpital Ambroise Paré, Boulogne,
France; 6Service d’Orthopédie, Hôpital Ambroise Paré, Boulogne, France
Corresponding author:
Dominique Baruch, INSERM Unit 765, Faculté des Sciences Pharmaceutiques et Biologiques
de Paris, 4 Avenue de l’Observatoire, 75270 Paris CEDEX 06, France. Phone: 33 (0)1 53 73
99 38 ; Fax: 33 (0)1 44 07 17 72. e-mail : [email protected]
Running heads :
DUNOIS-LARDE et al.
High shear rates promote platelet formation
Scientific category: Thrombosis and Hemostasis
1
Copyright © 2009 American Society of Hematology
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Abstract
Platelets originate from megakaryocytes (MKs) by cytoplasmic elongation into proplatelets.
Direct platelet release is not seen in bone marrow hematopoietic islands. It was suggested that
proplatelet fragmentation into platelets can occur intravascularly, yet evidence of its
dependence on hydrodynamic forces is missing. Therefore, we investigated whether platelet
production from MKs could be upregulated by circulatory forces. Human mature MKs were
perfused at a high shear rate on von Willebrand factor. Cells were observed in real-time by
videomicroscopy, and by confocal and electron microscopy after fixation. Dramatic cellular
modifications followed exposure to high shear rates: 30-45% adherent MKs were converted
into proplatelets and released platelets within 20 minutes, contrary to static conditions that
required several hours, often without platelet release. Tubulin was present in elongated
proplatelets and platelets, thus ruling out membrane tethers. By using inhibitors, we
demonstrated the fundamental roles of microtubule assembly and MK receptor GPIb.
Secretory granules were present along the proplatelet shafts and in shed platelets, as shown by
P-selectin labeling. Platelets generated in vitro were functional since they responded to
thrombin by P-selectin expression and cytoskeletal reorganization. In conclusion, MK
exposure to high shear rates promotes platelet production via GPIb, depending on microtubule
assembly and elongation.
2
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Introduction
Megakaryocyte (MK) differentiation is a continuous process characterized by sequential
steps. MK ploidy increases through endomitosis, with parallel increase in cell size. Synthesis
of storage organelles is enhanced and plasma membrane invaginates, resulting in the
formation of demarcation membranes. Finally, mature MKs undergo complete cytoskeleton
reorganization with microtubule involvement, to induce pseudopodial elongations called
proplatelets1,2. Platelets are released from the tips of proplatelets that contain the organelles3.
In static conditions, platelets are formed from mature MKs cultured in the presence of
thrombopoietin (TPO)3. MK fragmentation into platelets does not occur in the extra-vascular,
hematopoietic space in normal bone marrow. To achieve platelet formation, MKs need to
migrate into the sinusoid capillaries and mature MKs are generally located in the vicinity of
sinusoids, more precisely on their abluminal side4. MK fragmentation only occurs in the
intravascular space and mature MKs have been identified in the blood circulation where they
may interact via their surface receptors with proteins expressed on endothelial cells 4,5,6.
Adhesion of MKs to endothelial cells is essential for robust platelet production in vitro, a
process that is believed to mimic what occurs in vivo when MKs interact with bone marrow
endothelial cells within the vascular niche7. Recently, the process of proplatelet formation was
examined in mouse skull bone marrow in vivo: the authors observed large MK fragments
resembling immature proplatelets protruding into the intravascular luminal side of bone
marrow sinusoids and entering flowing blood8.
Circulating platelets are essential for primary hemostasis by their capacity of adhesion to the
subendothelial matrix and aggregation9. Among the constituents of vascular endothelial cells,
von Willebrand factor (VWF) allows translocation of circulating platelets in conditions of
high shear rates (>1000 s− 1) through binding of their GPIb receptor10. GPIb-mediated
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signaling occurs progressively during tethering of platelets and allows activation of the
αIIbβ3 integrin and platelet aggregation. GPIb is crucial for cytoskeletal reorganization by
linking the actin-binding protein filamin11. The Bernard-Soulier Syndrome (BSS) is a
bleeding disorder characterized by severe thrombocytopenia and giant platelets 12. It is due to
genetic abnormalities of the GPIb-IX-V complex, in particular of the GPIbα subunit that
contains the VWF and thrombin binding sites. Normal numbers of MKs are found in the bone
marrow of BSS patients, suggesting that the macrothrombocytopenia is related to defective
platelet formation from MKs. It is intriguing that in optimal culture conditions, the yield of
shed platelets in vitro is far below what could be expected from the large daily platelet
production in vivo13. Many steps of platelet shedding remain elusive, in particular a role of
shear forces on platelet formation has never been demonstrated. In the present report, we
examined the role of high shear rates on platelet release from mature MKs adherent to a
vascular matrix in flow conditions and found that it required both intact microtubules and the
GPIb receptor.
4
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Material and Methods
Proteins
VWF was a gift of Laboratoire français du Fractionnement et des Biotechnologies (Lille,
France). Fibrinogen was from Hyphen BioMed (Neuville-sur-Oise, France). Human VWF
and fibrinogen were purified and depleted of contaminant fibronectin and of fibrinogen and
VWF, respectively14. Equine tendon collagen was from Nycomed (Munich, Germany) and
human fibronectin was from VWR (Fontenay-sous-Bois, France). Wild-type and mutated
(V1316M type 2B) recombinant VWF (rVWF) were obtained as described elsewhere15.
Antibodies
Monoclonal antibody (MoAb) 713, which blocks VWF binding to GPIbα was a kind gift of
Dr Girma (INSERM Unit 770, Le Kremlin-Bicêtre, France)15. Polyclonal anti-glycocalicin
(the extracellular domain of GPIbα containing the VWF binding site) and anti-CD62P
antibodies were a kind gift of Dr Berndt (Monash University, Melbourne, Australia)16.
Phycoerythrin (PE) conjugated anti-CD62P (P-selectin), fluorescein isothiocyanate (FITC)conjugated anti-CD41a (αIIb), anti-CD42b (GPIbα) and non-immune antibodies were from
Beckman Coulter (Villepinte, France). Anti α-tubulin MoAb was from Sigma (Saint Quentin
Fallavier, France). The anti-β3 integrin P37 MoAb was a gift of Dr Gonzalez-Rodriguez
(CSIC, Madrid, Spain)17. The anti-αIIbβ3 Fab (C7E3) Abciximab (Reopro®) was from Lilly
(Suresnes, France).
Cell culture
Human MKs used in this study were cultured from precursor cells isolated either from
umbilical cord blood or from bone marrow. Human bone marrow samples (harvested
during hip surgery), and cord blood samples were obtained after informed consent and
approval from our Institute Ethic Committee (Assistance Publique des Hôpitaux de
Paris) and in accordance with the Declaration of Helsinki. Human umbilical cord blood and
bone marrow CD34+ cells were separated by an immunomagnetic technique (StemCell
Technologies, Grenoble, France) as previously described18. The cells were grown in Iscove's
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Modified Dulbecco's Medium (IMDM) (Gibco-Invitrogen, Cergy-Pontoise, France)
supplemented with 15% BIT 9500 serum substitute (StemCell Technologies) and 20 nmol/L
TPO peptide agonist AF13948 (Sigma). Ten ng/mL Stem Cell Factor (SCF) (Amgen, Neuillysur-Seine, France) were added on day 1 of culture. MKs were cultured for 10 to 16 days in the
presence of TPO. Cells were shown by flow cytometry to express GPIb and αIIbβ3 (60-70%
positive cells). For shear experiments, cells were used between day 10 and 16. Human
endothelial cells were isolated from umbilical veins and grown to confluence on coverslips as
reported19. Cells from a first to third passage were used. Stimulation with 10 µM forskolin
and 100 µM IBMX was performed for 20 minutes before perfusion with MK.
Human platelets
Blood was obtained from healthy individuals who had not ingested any medication during the
previous two weeks. Blood was drawn into 15% (v/v) acid citrate dextrose (ACD) pH 5.8.
Washed platelets were prepared from isolated platelet-rich plasma (PRP) in the presence of
apyrase (1 U/mL) (Sigma) and ACD (1 mL for 40 mL)15. Briefly, after washing, platelets
were resuspended in Hepes buffer (10 mmol/L Hepes (N-[2-hydroxyethyl]piperazine-N'[ethanesulfonic acid]), 136 mmol/L NaCl, 2.7 mmol/L KCl and 2 mmol/L MgCl2), pH 7.5
containing 0.15% bovine serum albumin (BSA). Platelets were counted with an electronic
particle counter (Model Z1, Coulter Electronics, Margency, France), and concentration
8
adjusted to 1.5x10 platelets/mL.
Perfusion studies
Perfusion studies were performed using a previously published flow chamber obtained from
Maastricht Instrumentation14. The flow chamber consisted of a rectangular cavity 0.05 mm
high, 29 mm long, and 5 mm wide, carved in a Plexiglas block. The bottom of the chamber
was lined with a glass coverslip coated overnight at 4°C with VWF (20 µg/mL) diluted in Tris
(tris(hydroxymethyl)aminomethane) buffered saline (TBS, 25 mmol/L Tris-HCl, pH 7.4, 150
mmol/L NaCl). In some experiments, another purified protein (fibronectin, fibrinogen,
collagen or type 2B-rVWF) was tested. MKs in suspension in IMDM (2 mL) at a
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concentration of 0.8-1.5 x 106 cells/mL were drawn in a 5-mL glass gas-tight microsyringe
(Exmine, Ito Corporation, Fuji, Japan) connected to the chamber with an extension set
(Steritex, Codan, Lensahn, Germany). A flow rate of 225 µL/min was applied with an electric
pump, generating a shear rate of 1800 s-1. The cells were perfused for 10 min, followed by
IMDM perfusion at the same shear rate for 10 min. All experiments were performed at 37°C
maintained with a Minitüb heating system (Minitüb Abfüll und Labortechnik, Tiefenbach,
Germany). In some experiments, cells or coverslips were preincubated with antibodies for 10
min prior to perfusion. After the end of the perfusion, the coverslip was fixed with ice-cold
methanol for 5 min, washed with distilled water, dried, and stained with Romanovsky
staining. In some experiments, cells were fixed with a perfusion of 2% paraformaldehyde
(Carlo Erba, Val de Reuil, France) in PBS directly in the flow chamber and processed for
immunofluorescence as described below. Effluents were collected at the exit of the chamber
for further studies, including thrombin activation.
Videomicroscopy system
The perfusion chamber was positioned on the stage of an inverted microscope (Axiovert 135,
Zeiss, Germany). A CCD camera (Sony, Tokyo, Japan) was used to visualize cells, using a
20x Hoffman modulation contrast objective. Continuous recording was performed with a
digital image recorder (Replay Software, Microvision Instruments, Evry, France) connected
to a videotimer (VTG, Tokyo, Japan) at 0.25 image/s. For visualization, using “Archimed”
software (Microvision Instruments), frames were read at a velocity of 10 images/s (40-fold
acceleration). Image frames were analyzed with Histolab or Videomet quantification software
(Microvision Instruments).
Cell activation studies and flow cytometry.
Effluent cell suspensions were collected and centrifuged in the presence of 0.5 mmol/L EDTA
at 1800 rpm for 12 min, and the pellets were resuspended in IMDM. Aliquots were incubated
in the absence or presence of thrombin (0.5 U/mL) for 10 min at 37°C. Cells in suspension
(100 µL) were incubated for 20 min at room temperature with FITC-anti-CD62P and PE-anti-
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CD41a (5 µL of each) or non immune conjugated control antibody, before adding 300 µL
PBS. The cells were then analyzed with a FACSort flow cytometer (Becton-Dickinson, Le
Pont-de-Claix, France); 10 000 events were acquired with settings on the platelet region
identified by their characteristic profile on a right-angle scatter (SSC) and forward-angle
scatter (FSC) plot determined in separate experiments with unstimulated washed platelets.
Static adhesion
Aliquots (150 µL) of unactivated and thrombin-activated cells were added to fibrinogencoated glass Lab-Tek chamber slides (Nunc, Rochester, NY) incubated for 30 min at 37°C in
static conditions. Non adherent cells were removed and the slides were washed three times
with PBS, before fixation with 2% paraformaldehyde in PBS for 30 minutes and storage at
4°C for confocal immunofluorescence the next day.
Confocal immunofluorescence
Paraformaldehyde-fixed cells were permeabilized for 5 min with 0.1% Triton X-100 in PBS,
non-specific binding was blocked in PBS-BSA 2%, cells were incubated 90 min at 37°C with
the anti-β3 P37 MoAb (10 µg/mL), anti-α-tubulin MoAb (2 µg/mL) or polyclonal antiCD62P (1/100) in PBS-BSA, washed and incubated with 1/200 dilution of a secondary
antibody conjugated to AlexaFluor488 or AlexaFluor546 (Molecular Probes, Eugene, OR).
The negative control used purified IgG at the same concentration. For actin labeling,
permeabilized cells were incubated at room temperature for 30 min with 50 μL of 30 nmol/L
AlexaFluor546-phalloidin (Molecular Probes) in PBS containing 2% BSA. The coverslips
were then washed twice and incubated with TOPRO3 before mounting in Vectashield
mounting solution (Vector Laboratories, Peterborough, UK). AlexaFluor488/AlexaFluor546
staining was analyzed with a Leica TCS SP2 AOBS confocal microscope.
Electron microscopy
Cells present on the coverslips, or alternatively effluent cell suspensions after passage over
the various matrices, were fixed in glutaraldehyde fixative (1.5% final concentration in
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phosphate buffer 0.1M, pH 7.4) and were then processed for electron microscopy as
previously described20. Cell monolayers were detached from the coverslips with liquid
nitrogen before thin sectioning. Some cells in suspension were activated by thrombin.
Examination was performed on a Jeol 10-11 electron microscope (Jeol Ltd, Tokyo, Japan).
Statistics
We used Student’s unpaired t-test for statistical analysis; P values<0.05 were considered
significant. Error bars represent the standard error of the mean (SEM).
9
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Results
Deformations of MKs under shear
Cord blood mature MKs were perfused at varying shear rates on a surface coated with
purified VWF. At 1800 s-1, MKs established transient interactions with VWF, slowing down
the velocity of MKs rolling on VWF and progressively leading to profound morphological
changes that finally led to platelet shedding from MKs. The different steps of platelet
formation are shown in Supplementary Video 1 and 2 (online) and summarized in Figure 1.
The process starts with roughly spherical undeformed MKs (stage 1), then the cell periphery
deforms and pseudopods become visible on adherent MKs (stage 2). Elongation of the
cytoplasm then takes place, organized along the flow, at the rear and/or the front of the cell
body (stage 3). Intermediate swellings with a characteristic beads-on-a-thread aspect start to
appear (Supplementary Video 1). Elongation occurred rapidly, at a velocity of 21 µm/min,
thus 25-fold higher than reported in static conditions3. Interestingly, fragmentation occurred
not only at the tip of these extensions, but also between the swellings, in particular in thinner
constrictions, releasing fragments of varying sizes, and most often several beads still attached
together, but devoid of the main cell body (Figure 1 and Supplementary Video 2). Finally,
these fragments were further broken down into smaller particles, the size of a platelet, clearly
visible during late time points (Figure 1 and Supplementary Video 2). Cord blood and bone
marrow derived MKs produced a similar proportion of fragmented proplatelets and platelets,
reaching 27.6 ± 4.9 % of adherent cord blood MKs and 42.8 ± 3,7 % of adherent bone
marrow MKs at 20 min (Figure 1). This effect was dependent on the extent of shear rate and
on cell concentration (not shown). Shear exposure of immature MKs, cultured less than 9
days, did not allow translocation and fragmentation.
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Effect of high shear rate and VWF on proplatelet and platelet formation
The morphology of MKs incubated in static conditions for 20 min on VWF remained
unchanged with respect to that of the cells before contact with VWF (Figure 2a). As
proplatelet formation was recently shown to occur in 16 hours after MK plating on VWF20,
we reproduced these findings in static conditions, demonstrating that MKs were fully mature
and equipped for platelet biogenesis (Figure 2c). When exposed to a shear rate of 1800 s-1,
these MKs formed long extensions and rapidly released proplatelets and platelets within 1020 min (Figure 2b, 2d). To study shear-induced proplatelet formation on a physiologically
relevant surface, we used VWF released from stimulated endothelial cells in inflammatory
conditions21. We obtained evidence that high molecular weight multimers of VWF supported
MK rolling, adhesion and proplatelet formation within 20 min (Figure 2e, 2f).
Alpha-granule proteins and microtubule network in shear-induced proplatelets and platelets
Proplatelets transport individual α-granules on their microtubule tracks2. To identify the
presence of α-granule protein, coverslips were fixed at the end of perfusion on VWF at high
shear rate, and punctuate positive staining with anti human P-selectin antibody was visualized
along the microtubule tracks (Figure 3), as previously reported22. Proplatelet elongation
requires the sliding of overlapping microtubules that stain for α and β1 tubulin3. We found
that MK extensions were stained by an anti-tubulin antibody in different territories from those
staining for actin, confirming that these extensions displayed a similar structure as the one
identified in proplatelet-forming MKs, by the work of Italiano et al.1,3,23,24. Actin labeling
showed diffuse staining in cytoplasmic areas, as previously shown3, whereas tubulin labeling
followed the proplatelet shaft in elongated MKs and displayed the specific ring pattern on
platelets (Figure 3). The importance of microtubules in shear-induced proplatelet formation
was confirmed by using Nocodazole, an inhibitor of microtubule assembly, in two ways. As
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expected, preincubation with the inhibitor completely prevented MK elongation and
proplatelet formation in flow conditions (data not shown). To demonstrate that Nocodazole
was not acting on preformed platelets in cell suspensions, the microtubule inhibitor (10 µM)
was added after the elongation process had started: the effect visualized in real-time showed
that Nocodazole could revert the shear-induced elongation when added after it had occurred
(Figure 3 and Video 3). The specificity of reversal was confirmed by tubulin staining.
Interestingly, following Nocodazole action at 25 min, tubulin was no longer organized in
proplatelet-forming MK, thus indicating that the elongated processes were different from
tethers (Figure 3). Indeed, membrane tethers are dynamic structures extending from small,
localized adhesion contacts under the influence of flow, are induced by VWF-GPIb
interaction, but they do not display any tubulin staining25. Taken together, these experiments
demonstrate that platelet-sized elements were indeed generated by MK exposure to shear and
did not simply arise in suspensions of mature MKs before their passage through the flow
chamber.
Structure of shear-exposed MK in the process of proplatelet and platelet release
Having shown that proplatelets and platelets were truly formed in real-time by MK exposure
to a high shear rate, effluents were compared before and after perfusion in the flow chamber.
Non adherent flow-through of cells exposed to shear contained naked nuclei, whereas non
adherent cells in static conditions were devoid of them (data not shown). Electron microscopy
showed long cytoplasmic shafts extending from the cell core, containing parallel bundles of
microtubules and were sometimes swollen with cytoplasmic organelles (Figure 5, panel a).
Nuclear lobes with dense heterochromatin were located at one pole of the cell and elongated
naked nuclei were occasionally seen, in contrast to static cultures where they are virtually
never observed (Figure 5, panel b). Large cytoplasmic fragments, devoid of nuclei, were
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roughly spherical, dumbbell-shaped or elongated with slender extremities (Figure 5, panels c
and d). Platelet-like fragments were also visible (Figure 5, panel e). In addition, ultrastructural
data were obtained by sectioning the monolayer of the cells present on the VWF-coated
coverslips removed from the chamber after they had been exposed to shear. It shows platelet
size fragments located close to adhering MKs, displaying ultrastructural organization
characteristic of platelets, and whose distribution pattern resembles that of the putative mother
cell (Figure 5, panel f).
Platelets generated by MK shear exposure are functional
Platelet adhesion to fibrinogen mediated by αIIbβ3 integrin occurs in the absence of
activation and is reinforced upon thrombin activation of this receptor. To confirm that
platelets generated by MK shear exposure were functionally similar to blood platelets, cells
collected in the flow-through were compared to isolated blood platelets, in a static adhesion
assay to fibrinogen26. Washed blood platelets prepared in a separate assay without exposure to
shear were firmly adherent to fibrinogen in the absence of thrombin, demonstrating that
αIIbβ3 was functional (Figure 6, panel a). Similar findings were seen on shear-induced
platelets and proplatelets in the absence of thrombin, as the cell elements positive for αIIbβ3
displayed diffuse actin staining (Figure 6, panel b). Following thrombin activation, actin
filaments were reorganized in nucleated (not shown) and anucleated elements, showing
lamellipod and filopod formation, and integrin membrane staining (Figure 6, panel b). These
MK-derived platelets generated by shear exposure displayed a similar cytoskeletal
organization to the one of washed blood platelets (Figure 6, panel a). Flow cytometry analysis
of shear-exposed cells showed three populations of CD41 positive cells, according to their
size. The smaller sized population overlapped with that of blood platelets and responded to
thrombin stimulation, as seen by upregulation of P-selectin (CD62P) on the surface of shear-
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derived platelets (11.8±1.8% compared to 6.2±0.9% for non activated platelets, p=0.0005).
Similarity with isolated blood platelets was confirmed by ultrastructural data: following
thrombin activation, these cells displayed morphological changes typical of activated
platelets, a spherical shape, surface pseudopods, no cytoplasmic granulation but dense
material within centralized and dilated cisternae of surface connected canalicular system
(SCCS) and a central bundle of microfilaments (Figure 6, panel d).
Involvement of MK receptors and of matrix proteins in platelet formation
MK translocation and subsequent steps including proplatelet formation and platelet release
were completely abolished in the presence of an antibody directed against glycocalicin or the
GPIb-binding domain of VWF (Figure 7), demonstrating the crucial importance of the VWFGPIb interaction. In the presence of the αIIbβ3 inhibitor Abciximab, the proportion of cells
without proplatelet (undeforming and early deforming MKs) remained constant in time,
whereas proplatelet elongation was strongly reduced and platelet formation was almost
completely abolished (Figure 7). As shown on Supplementary Video 4 online, this inefficient
proplatelet formation was due to loose contact with the VWF surface. Finally, to further
establish the specificity of adhesive surface in the regulation of proplatelet formation, we
examined the extent of proplatelet formation by MKs perfused over fibrinogen, collagen or
fibronectin at a high shear rate. No proplatelet was formed on these surfaces; the only change
was a mild early MK deformation on collagen (Figure 7). In addition, we studied proplatelet
formation from MKs perfused on a surface coated with a mutated type 2B-rVWF. We
selected the V1316M substitution, a well-defined mutation, characterized in patients, by
enhanced VWF binding to platelet GPIb with mild thrombocytopenia and giant platelets15,27.
We found that the mean velocity of MK translocation on type 2B-rVWF (4.6±0.3 µm/s) was
much lower than on wild type (wt)-rVWF (33.6±2.5 µm/s, p<0.0001). All steps of proplatelet
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formation by MKs exposed to high shear rates were slower on 2B-rVWF than wt-rVWF,
leading to decreased platelet production and increased proplatelet accumulation
(Supplementary Video 5 online).
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Discussion
Mechanical forces play an important role in cell remodeling and cytoskeletal reorganization.
Here, we studied platelet formation from human MKs perfused in a flow chamber
reproducing high shear rate conditions of the microcirculation. A major finding was that
during exposure to high shear rates (>1000 s-1), cytoplasmic MK deformation produced
proplatelet extensions organized along the flow and rapidly leading to platelet formation. The
involvement of shear forces on platelet production by mature MKs is supported by several
observations. First, circulating differentiated MKs are frequently encountered5,28. Second,
direct images of MK fragmentation, platelet release and naked nuclei are not seen in normal
bone marrow hematopoietic islands29. Third, although in vitro MK maturation has been
greatly improved by the use of TPO, the yield of platelet shedding in culture has remained
minimal13. MKs respond to chemotactic stimuli and are able to cross the sinusoid barrier and
enter the blood stream4. Recently, intravital microscopy of mouse skull microvessels
indicated that MKs can extend protrusions or even penetrate inside the lumen of bone marrow
capillaries, giving rise to large proplatelets connected to the MK cell body8. In that in vivo
model, the authors observed the detachment of large cytoplasmic fragments into circulating
blood and suggested that high shear rates appeared to free them from the parent cells and
might contribute to platelet formation. Our results are in agreement with this model and
suggest an effect of MK exposure to high shear rates encountered in small vessels that
contribute to the generation of single, mature platelets. We used a shear rate of 1800 s-1 which
is encountered in small arterioles and capillaries of human organs30 and is higher than the
ones existing in murine bone marrow sinusoids (50 to 165 s-1) .8,31
The latter work was performed with murine species, and noteworthy many differences have
been emphasized in megakaryocyte localization and ultrastructure of proplatelet formation,
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between mice and men32. The present study performed on human MKs sheds additional light
on how these large protrusions can be induced to fragment into small platelets by shear forces
in flowing blood. This is also in keeping with evidence that the pulmonary circulation could
be an important site of platelet production, as the lung capillaries would be the first to be
encountered by cells leaving the bone marrow28. Indeed, the large cytoplasmic fragments and
the isolated platelet-sized fragments that we observed in real-time to form on the coverslip
during the flow assay, resembled those seen downstream of the pulmonary circulation in
vivo33. In our conditions, high shear rates were essential to proplatelet and platelet formation
during an exposure time of 20 minutes. In contrast, no proplatelet or platelet were generated
in static conditions during this short period. The two-dimensional distribution of proplatelets
generated in static conditions during several hours was multipolar24, whereas it was bipolar or
polar in our flow conditions. We demonstrated that this process was driven by microtubule
polymerization to form proplatelets, as platelet formation was completely blocked when
microtubule assembly was inhibited by Nocodazole. This also indicated that the process we
observed was indeed platelet production and not an artifact of cell death3. Elongation
following MK deformation shows similarities with the recently reported platelet cytoskeleton
remodeling and shear-specific morphological changes during surface translocation on VWF34.
The organization of shear-induced MK elongations resembled that previously reported in
mouse1,3,23,24, as shown by the distribution of P-selectin, an α-granule membrane protein and
by the characteristic tubulin staining. In addition, the reversal of proplatelet elongation and
platelet shedding that we obtained by addition of Nocadazole after the elongation had started,
demonstrates that proplatelet and platelet processes induced by shear are completely different
from the previously reported membrane tethers. These tethers are dynamic structures
extending from small, localized adhesion contacts under the influence of flow, induced by
VWF-GP Ib interaction, but that do not display any tubulin staining25. Thus, not only
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platelets, but also MKs, may show a shear-sensitive functional response. Platelets generated
in vitro by MK exposure to high shear rates were similar to normal human blood platelets as
they responded to thrombin stimulation by upregulating surface P-selectin expression,
confirming a previous report in static conditions35. The functional nature of shear-derived
platelets
was
confirmed
by
their
thrombin-induced
cytoskeletal
reorganization,
indistinguishable from blood platelets, in a fibrinogen adhesion assay and by their
ultrastructural aspect.
At high shear rates, human MKs rolled and attached to a VWF matrix in a fashion close to
that reported with blood platelets. The latter adhere in a two-step process consisting of a
reversible initial contact (GPIb-dependent translocation) followed by an irreversible
attachment mediated by integrin αIIbβ3 activation36. Indeed, MK adhesion and platelet
generation from MKs exposed to high shear rates were completely abrogated by antibodies
inhibiting the VWF-GPIb interaction. In vitro proplatelet formation was previously reported
to be inhibited by an anti-GPIb monoclonal antibody in static conditions20,37. Our results thus
suggest that the macrothrombocytopenia observed in BSS patients, characterized by a genetic
defect of the GPIb-IX-V complex12, may be in part related to impaired shear-induced
thrombopoiesis, the giant platelets thus corresponding to incompletely fragmented
proplatelets. Moreover, in a BSS mouse model, reconstitution of the GPIb-alpha null mouse
with the GPIb intracellular domain fused to the extracellular domain of human IL4-R did not
completely correct the macrothrombocytopenia, confirming the critical role of the GPIb alpha
extracytoplasmic domain in platelet formation38. Intriguingly, the platelet count and size are
normal in severe hereditary VWF deficiency, the type 3 von Willebrand disease (VWD), but
it is conceivable that other matrix proteins such as fibronectin or thrombospondin might
replace VWF for GPIb-binding in shear conditions in vivo39,40. Recently, membrane-bound
VWF was detected on proplatelet forming MKs, suggesting an autocrine stimulation of VWF
18
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bound to GPIb20. Moreover, our findings obtained with a mutated type 2B-rVWF support a
role for VWF interaction with GPIb in proplatelet formation under shear conditions. Indeed,
in type 2B VWD patients, mutated VWF displays increased binding to GPIb, and a low
dissociation rate from GPIb27. This gain-of-function mutation accounts for the transient
thrombocytopenia leading to platelet aggregates that are cleared from the circulation41.
Interestingly, macrothrombocytopenia has also been reported in type 2B VWD42,43. The
decreased rolling velocity of MKs on a 2B-rVWF matrix compared to wt-rVWF that we
describe, suggests a slower and less efficient process of platelet formation at high shear rates,
responsible for an increased proportion of giant platelets in these patients.
We have observed proplatelet formation on HUVEC monolayers stimulated by known
effectors of VWF release. Weibel-Palade bodies (WPB) of endothelial cells release VWF by a
tightly regulated mechanism that allows a rapid response to changes within the vascular
micro-environment21. Both Ca2+-raising agents and cAMP-raising agonists display specific
patterns of cytoskeletal remodeling that lead to opposite effects on endothelial cell barrier
function21. Thus MK fragmentation leading to platelets may occur intravascularly and be
finely regulated depending on the release of VWF from WBP, as well as on the VWF content
of endothelial cells, that is known to be heterogeneous along the vascular bed, and particularly
high in the pulmonary artery44,45. Indeed, there are evidence that platelet counts are higher in
pulmonary venous samples than in pulmonary arterial blood28,33, suggesting that circulating
MK fragments and proplatelets may be processed into platelets when passing through the lung
capillaries, where they are exposed to high shear rates. Shear rates conditions used in this
study appear relevant to the pulmonary microcirculation, which has been suggested to be a
site of platelet production. In humans, the diameter of pulmonary capillaries is 5-8 µm46,47
thus smaller than that of bone marrow sinusoids (22 to 60 µm). 48
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VWF mediates platelet interactions with the vessel wall at the high shear rates encountered in
capillaries and small arteries, through a tightly regulated interaction with GPIb, stabilized by
signaling triggered by GPIb engagement that results in αIIbβ3 integrin activation9. In our
flow model, VWF interaction with αIIbβ3 was necessary for complete MK elongation and
proplatelet formation. This is in agreement with the inhibiting effect of another αIIbβ3
antagonist, lotrafiban, on proplatelet formation in static conditions49. In contrast to our shear
conditions, the latter authors found that in static conditions, fibrinogen yielded a larger
proportion of proplatelets than collagen or VWF. This suggests that shear forces may regulate
the specificity of MK αIIbβ3 interactions with matrix proteins, as previously reported for
platelets50. However, it seems that, in vivo, the involvement of αIIbβ3 in platelet formation is
more dispensable than GPIb, since αIIbβ3 congenital deficiency (Glanzmann thrombasthenia)
does not lead to thrombocytopenia. Interestingly, the cytoplasmic part of the β3 subunit of
αIIbβ3 was recently found to be involved in proplatelet formation, in a familial form of
macrothrombocytopenia51. A possible explanation is that αIIbβ3 is only implicated in MK
anchorage to the surface, whereas GPIb is mainly responsible not only for MK arrest but also
for several other downstream steps such as proplatelet elongation, swellings, and for the
eventual detachment of platelets and proplatelets from the mother cell in a process similar to
cytokinesis, through GPIb interaction with actin via filamin52. In conclusion, our finding that
mature MK interaction with VWF in flow conditions promotes human proplatelet formation
represents a major breakthrough in understanding platelet formation, which may thus occur in
capillaries or small arteries. The exploration of diseases with decreased platelet production
would greatly benefit of a better understanding of platelet shedding from MKs. Finally, our
results may also have important implications for research on platelet production for
therapeutic purposes.
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Acknowledgements
Claire Dunois-Lardé was supported by a grant from Agence Nationale de la Recherche and
LINAT (Leducq International Network Against Thrombosis). We thank Dr Pierre Raynal for
providing umbilical cord blood, Valerie Jalbert and Azzedine Yacia for technical assistance
with MK culture and Laurence Momeux for technical assistance with electron microscopy.
Authorship
Conceived and designed the experiments: CDL, DB. Performed the experiments: CDL, DB,
CC. Analyzed the data: CDL, DB, ECB. Designed the study: DB, ECB. Contributed reagents,
materials, analysis tools: SF, TB. Wrote the manuscript: DB, ECB. Prepared figures and
videos: CDL. Authors report no conflict of interest.
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Figure Legends
Figure 1: MK deformation on a VWF surface at high shear rate. Cells suspended in
IMDM were perfused on VWF at 1800 s-1 for 10 min, then IMDM alone was perfused for 10
min. Adhesion led to major cell shape changes and to proplatelet formation.
Microphotographs during perfusion indicate four different stages starting from undeformed
MKs (stage 1) to early deformation characterized by loss of cell sphericity (stage 2); the stage
3 includes later deformation of MKs with cytoskeletal reorganization at a cell pole or both
ends, also a long thin filament of successive “beads on a thread”; stage 4 comprises
fragmentation of proplatelets and cleavage from the nucleus, as well as platelet formation and
release. Proportion of cells relative to the total number of cells counted in 10 fields, was
plotted as a function of perfusion time classified according to these four stages for cord blood
MKs (bottom left panel) or bone marrow MKs (bottom right panel) perfused on VWF.
Photographs are representative of 15 experiments, curves are mean ± SEM of 8 experiments.
The bar represents 10 µm. Black lines have been inserted to indicate repositioned images.
Figure 2: Comparison of shear or static conditions of MK deformations on a VWFcontaining surface. Coverslips were removed from the flow chamber and rinsed with PBS
prior to fixation in ice-cold methanol and subsequent Romanovsky staining (panel a and b,
magnification x 500): during static 20 min adhesion, cells displayed a spherical shape,
without any proplatelet extension (panel a), but after exposure to high shear rate for 20 min,
MKs extended long filopods at their tip and beaded platelet-like spikes were formed along the
shaft (panel b). In separate experiments, images were recorded on coverslips coated with
VWF (c-d) or on stimulated HUVECs (e-f) during real-time perfusion. Panel c shows
26
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proplatelet formation during 16 hours on VWF-coated slides in static conditions extending in
different directions (panel c). In shear conditions, after 20 minutes, proplatelet formation is
bipolar and organized along the flow (panel d). Similar organization of proplatelet formation
is seen on UL-VWF released by HUVECs stimulated by IBMX and forskolin (panel e and f).
Bar on panels c to e represents 10 µm.
Figure 3: Distribution of α-granule proteins and tubulin in shear-induced proplatelets
and platelets. Confocal microscope analysis of immunofluorescence showing labeling (left
panels) with anti P-selectin, anti-tubulin antibodies and overlay of (from top to bottom) a MK
extending a thick proplatelet, a long and thin proplatelet, a dumbbell shaped proplatelet with
its central narrowing, and two platelets. P-selectin staining is seen in organelles in forming
proplatelets and in platelets in different spots, whereas tubulin stains the entire microtubule
shaft of the proplatelet and the periphery of platelets. Right panels: labeling with phalloidin,
anti-tubulin antibodies and overlay of (from top to bottom) a MK extending proplatelet, a
long and thin proplatelet with its shaft as well as its tip, labeled for tubulin, a dumbbell shaped
proplatelet with its central narrowing, and a platelet exhibiting a circular labeling pattern. No
colocalization between actin and tubulin is visible. The bar represents 10 µm.
Figure 4: Reversal of proplatelet elongation by Nocodazole addition during proplatelet
elongation. Frames from experiment shown on Video 4 were selected (from top to bottom)
before (13 min), during elongation (21 min), after addition of Nocodazole (at 21’20’’), during
effect of Nocodazole (25 min), start of elongation reversal (28 min), progress (31 min) and
completion of elongation reversal (38 min). Notice the modifications between the cell body
and its anchorage point (depicted by a white arrow) during different steps. Confocal
microscope analysis of indirect immunofluorescence labeling with anti-tubulin antibody of a
27
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MK extending proplatelet before and after Nocodazole treatment. MKs exhibit diffuse
staining after action of Nocodazole, indicating tubulin depolymerization. Bar represents 10
µM.
Figure 5: Ultrastructure of MKs exposed to high shear rates leading to platelet
formation. Effluent cell suspensions (panels a-e) or alternatively cells present on the
coverslips (panel f), were processed for electron microscopy as described in the Methods
section. Mature MKs were elongated, extending an often unique long cytoplasmic filopod
enclosing parallel longitudinal microtubules (inset). These proplatelets exhibited regular
swellings containing cytoplasmic organelles. A large spherical cytoplasmic fragment,
probably a detached proplatelet (pp) was located nearby (panel a). The nuclear lobes (N)
containing dense chromatin were elongated and located at one pole of the cell. Naked nuclei
with an oval shape and compact nuclear lobes containing dense chromatin, which are
normally absent form MK cultures, were retrieved in the effluents (panel b). Proplatelets (PP)
filled with cytoplasmic organelles appeared as large cytoplasmic fragments, devoid of nuclei,
roughly spherical, dumbbell-shaped or elongated with slender extremities (panels c and d).
Several isolated platelet-sized fragments (P) were observed (panel e). Panel f shows the
section of the cell monolayer present on a VWF-coated coverslip removed from the chamber
after exposition to shear. It shows two platelet size fragments (P) located close to an adhering
MK, displaying alpha granules and surface connected canalicular system characteristic of
platelets, and whose distribution pattern resembles that of the putative mother cell (MK). The
bar represents 2 µm.
Figure 6: Platelets obtained from MK exposure to high shear rates can be activated by
thrombin. Cell effluents in the flow-through of MKs exposed to high shear rates were
28
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analyzed in a fibrinogen adhesion assay in static conditions, followed by confocal
microscopy, with cell staining with phalloidin-Alexa546 to visualize actin and Alexa488 to
visualize αIIbβ3. Washed blood platelets (panel a) and MK-derived platelets generated by
shear exposure (panel b) adhered to fibrinogen in the absence (left panels) or in the presence
of thrombin (right panels). Non-activated cells display diffuse actin staining and αIIbβ3
membrane localization. Following thrombin activation, actin filaments are organized as stress
fibers. They display a similar cytoskeletal organization as washed blood platelets. The bar
represents 10 µm. Flow cytometry assay (panel c): Samples were labelled with anti-CD62PFITC (FL1, P-selectin)) and anti-CD41-PE (FL2, αIIb). Settings of FSC-SSC profiles of
washed blood platelets were used to analyze flow-through cells. Histogram plot of activated
platelets in the flow-through, in the absence or presence of thrombin, following labeling with
non-immune IgG (thin line, grey background), anti-CD62P or anti-CD41a (thick line).
Electron microscopy (panel d) in the presence of thrombin, the platelet-sized fragment
displayed morphological changes characteristic of activated platelets, namely a spherical
shape, surface pseudopods (p), dense material within dilated cisternae of SCCS, no
granulation in the cytoplasm and a central bundle of microfilaments, reminiscent of activated
blood platelets. The bar represents 2 µm.
Figure 7: MK deformations at high shear rate on different surfaces coated with matrix
proteins and effect of VWF inhibitors. Cord blood MK suspended in IMDM were perfused
on VWF (in the absence or presence of inhibitors), fibrinogen, collagen or fibronectin at
1800 s-1 for 10 min, then IMDM alone was perfused for 10 min. Cell numbers were counted
in 10 fields at the end of perfusion (20 min) and classified according to the four stages defined
in figure 1. Intense cell deformation and proplatelet formation occurs on VWF. Cell perfusion
on VWF in the presence of GPIb-VWF interaction inhibitors, either a blocking antibody to
29
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glycocalicin, or an anti-VWF MoAb directed against VWF binding site to GPIb, indicated a
major inhibition of MK adhesion to VWF and of subsequent steps, thus abolishing proplatelet
formation. Abciximab, blocking the interaction of αIIbβ3 with VWF, prevented proplatelet
and platelet formation. MK deformations and proplatelet formation on non-VWF surfaces
were minimal in shear conditions as shown for fibrinogen, fibronectin and collagen.
30
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Prepublished online June 12, 2009;
doi:10.1182/blood-2009-03-209205
Exposure of human megakaryocytes to high shear rates accelerates
platelet production
Claire Dunois-Larde, Claude Capron, Serge Fichelson, Thomas Bauer, Elisabeth Cramer-Borde and
Dominique Baruch
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