J Biol Chem, Vol. 274, Issue 51, 36241-36251, December 17, 1999
The von Willebrand Factor-Glycoprotein Ib/V/IX Interaction
Induces Actin Polymerization and Cytoskeletal Reorganization in Rolling
Platelets and Glycoprotein Ib/V/IX-transfected Cells*
Yuping
Yuan
,
Suhasini
Kulkarni,
Philippe
Ulsemer§,
Susan L.
Cranmer,
Cindy L.
Yap,
Warwick S.
Nesbitt,
Ian
Harper,
Nayna
Mistry,
Sacha M.
Dopheide,
Sascha C.
Hughan,
David
Williamson,
Corinne
de la Salle§,
Hatem H.
Salem,
Francois
Lanza§, and
Shaun P.
Jackson¶
From the Australian Centre for Blood Diseases,
Department of Medicine, Monash Medical School, the ¶ Department of
Pathology, Box Hill Hospital, Victoria, Australia, and the
§ Etablissement de Transfusion Sanguine, INSERM,
Unite 311, 67065 Strasbourg Cedex, France
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ABSTRACT |
Platelet adhesion to sites of vascular injury is
initiated by the binding of the platelet glycoprotein (GP) Ib-V-IX
complex to matrix-bound von Willebrand factor (vWf). This
receptor-ligand interaction is characterized by a rapid on-off rate
that enables efficient platelet tethering and rolling under conditions
of rapid blood flow. We demonstrate here that platelets adhering to
immobilized vWf under flow conditions undergo rapid morphological
conversion from flat discs to spiny spheres during surface
translocation. Studies of Glanzmann thrombasthenic platelets (lacking
integrin 
IIb
3) and Chinese hamster
ovary (CHO) cells transfected with GPIb/IX (CHO-Ib/IX) confirmed that
vWf binding to GPIb/IX was sufficient to induce actin polymerization
and cytoskeletal reorganization independent of integrin
IIb3. vWf-induced cytoskeletal
reorganization occurred independently of several well characterized
signaling processes linked to platelet activation, including calcium
influx, prostaglandin metabolism, protein tyrosine phosphorylation,
activation of protein kinase C or phosphatidylinositol 3-kinase but was
critically dependent on the mobilization of intracellular calcium.
Studies of Oregon Green 488 1,2-bis(o-amino-5-fluorophenoxy)ethane-N,N,N',N-tetraacetic acid tetraacetoxymethyl ester-loaded platelets and CHO-Ib/IX cells demonstrated that these cells mobilize intracellular calcium in a
shear-dependent manner during surface translocation on vWf. Taken together, these studies suggest that the vWf-GPIb interaction stimulates actin polymerization and cytoskeletal reorganization in
rolling platelets via a shear-sensitive signaling pathway linked to
intracellular calcium mobilization.
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INTRODUCTION |
The ability of platelets to adhere to subendothelial matrix
proteins and to other activated platelets at sites of vascular injury
is essential for the arrest of bleeding and for subsequent vascular
repair. The first step in the hemostatic process involves the binding
of the platelet adhesion receptor, GPIb/V/IX, to the vascular adhesive
protein, vWf.1 Under
conditions of rapid blood flow this receptor-ligand interaction is
indispensable for tethering platelets to the injured vessel wall as a
prerequisite step for integrin-mediated cell arrest (1, 2). This
multi-step adhesion mechanism is remarkably similar to that utilized by
leukocytes to adhere to post-capillary venules in vivo.
Rolling of leukocytes is mediated by one or more selectin family
members, whereas irreversible cell adhesion requires activation of
2 integrins (3, 4).
vWf is a unique adhesive ligand in that it has the ability to support
both the initial transient phase of platelet adhesion as well as
integrin IIb3-mediated cell arrest. The
A1 domain of vWf contains the binding site for GPIb, whereas the C1
domain peptide sequence Arg-Gly-Asp (RGD) binds integrin
IIb3 (platelet GPIIb/IIIa) (5). Bond
formation between vWf and GPIb is rapid, reversible, and inherently
resistant to detachment by high shear forces. This latter property of
the vWf-GPIb interaction is essential for sustaining platelet tethering
under high shear, presumably as a consequence of the multivalency of
the vWf-GPIb interaction (6) as well as anchorage of the receptor
complex to the membrane skeleton (7). There is growing evidence that
GPIb/V/IX not only mediates platelet adhesion but also transduces
signals required for platelet activation (8). One of these activation
events involves the conversion of integrin
IIb3 from a low affinity to a high
affinity receptor (9, 10). The interaction between activated integrin
IIb3 and vWf is characterized by a slow
dissociation rate that supports irreversible platelet adhesion and
stable platelet aggregation (1, 9, 10). These adhesive interactions are physiologically important as evidenced by the severe bleeding disorders
experienced by individuals with congenital deficiencies of vWf,
GPIb/V/IX, or integrin IIb3.
In addition to supporting irreversible platelet adhesion, vWf binding
to platelets induces dramatic cytoskeletal reorganization transforming
resting disc-shaped platelets to fully spread forms (11, 12). The
physiological importance of vWf in promoting cytoskeletal
reorganization has been highlighted by in vivo experiments on pigs with von Willebrand disease (vWD) (13). In these studies, platelet adhesion to injured coronary arteries of normal pigs was
associated with platelet filopodial extension and cell spreading. In
contrast, platelets from vWD pigs extended few filopodia and failed to
spread following adhesion to the subendothelium, indicating that
cytoskeletal reorganization of adherent platelets in vivo is
primarily a vWf-dependent process. Despite the
potential importance of vWf-induced cytoskeletal reorganization, the
molecular basis for this phenomenon has been poorly defined. Studies of
human platelets congenitally deficient in integrin
IIb3 (Glanzmann thrombasthenia) have
demonstrated an important role for this receptor in inducing platelet
spreading at sites of vessel wall injury (14, 15). The role of the
vWf-GPIb/V/IX interaction in inducing cytoskeletal reorganization is
less clear. A previous study by Cunningham et al. (16) has
demonstrated that the vWf-GPIb/IX interaction can induce pseudopodial
extension and cell spreading in GPIb/IX-transfected CHO cells, although
a follow-up study by the same group has suggested that these
cytoskeletal changes require activation of endogenous CHO cell
integrins (17). The ability of the vWf-GPIb interaction to induce
cytoskeletal reorganization and shape change is a potentially important
issue. Other rolling receptors, such as the selectins, do not induce
cytoskeletal reorganization during the rolling process (3). This is not
surprising given that the spherical shape of resting leukocytes is
ideally suited for a rolling-type adhesion and that morphological
change in these cells reduces their ability to adhere under flow (3).
Circulating platelets, however, exhibit a flat discoid morphology, well
suited for efficient cell transport through the microvasculature but less ideal for cell rolling.
In this study we have examined the possibility that the vWf-GPIb/V/IX
interaction not only mediates platelet rolling but also transduces
signals leading to cytoskeletal reorganization during the rolling
process. We demonstrate that platelets tethering to a vWf matrix under
flow undergo dramatic cytoskeletal reorganization, leading to platelet
sphering and filopodial extension. These morphological changes were
also observed in Glanzmann thrombasthenic platelets and
GPIb/IX-transfected cells demonstrating that these cytoskeletal changes
occur independently of integrin IIb3. The
ability of the vWf-GPIb interaction to induce these cytoskeletal
changes did not require calcium influx, activation of PKC, PI 3-kinase, protein tyrosine phosphorylation, or prostaglandin metabolism but was
critically dependent on the mobilization of calcium from intracellular stores.
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EXPERIMENTAL PROCEDURES |
Materials and Antibodies--
DNA, DNase I, FITC-conjugated
phalloidin, Arg-Gly-Asp-Ser (RGDS) peptide, wortmannin, and
cytochalasin D were purchased from Sigma. Latrunculin B, methotrexate,
and EGTA-AM were obtained from Calbiochem. Oregon Green 488 BAPTA-AM-1
was from Molecular Probes Inc. (Eugene, OR). G418 and polymerase chain
reaction reagents were from Roche Molecular Biochemicals. GmbH and
Zeocin was from Invitrogen (San Diego, CA). Pfu polymerase
was from Stratagene (GmbH, Germany). Genistein, tyrphostin, erbstatin,
calphostin, and bisindolylmaleimide were from Biomol Research
Laboratories (Plymouth Meeting, PA). LY294002 was from ICN (Coats Mesa,
CA). Human (HvWf) and bovine vWf (BvWf) were purified from plasma
cryoprecipitate according to the method of Montgomery and Zimmerman
(18). Ristocetin was supplied by Paesel and Lorei Inc. (Germany).
Botrocetin and the anti-GP Ib monoclonal antibodies (mAb) AK2 and
WM23 were generous donations from Prof. Michael Berndt (Baker Medical
Research Institute, Melbourne, Australia).
Anti-IIb3 chimeric Fab fragment of mAb
7E3 (c7E3 Fab-abciximab) was from Eli Lilly (Centocor, Leiden,
Netherlands). All other materials were from sources we have described
previously (12, 19).
Platelet Aggregation Studies--
Washed platelets were prepared
and finally resuspended in Tyrode's buffer (10 mM Hepes,
12 mM NaHCO3, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, and 5 mM glucose) as described previously (12). Washed platelets
(3 × 108/ml) were aggregated with the indicated
concentration of HvWf in the presence of 1 mg/ml ristocetin or with
BvWf alone. Aggregation was initiated by stirring the reaction mixture
at 920 rpm, 37 °C, in a four-channel automated platelet analyzer
(Kyoto Daiichi, Japan). In some studies, platelets were preincubated
for 10 min with either EDTA (2 mM), RGDS peptide (1 mM), or the anti-IIb3 c7E3
Fab (20 µg/ml) to block ligand binding to
IIb3. vWf binding to GPIb was blocked by
pretreating platelets with the anti-GPIb mAb, AK2 (5 µg/ml).
Calcium influx was prevented by resuspending platelets in Tyrode's
buffer containing EDTA (2 mM) or EGTA (2 mM).
In other studies, platelets were preincubated for 30 min at 37 °C
with inhibitors of PI 3-kinase (wortmannin (100 nM) or LY294002 (10 µM)), PKC (calphostin (1 µM)
or bisindolylmaleimide (200 nM)), or protein tyrosine
kinases (genistein (100 µM), tyrphostin (200 µM), or erbstatin (25 µg/ml)). The prostaglandin
pathway was inhibited by pretreating platelets with aspirin (1 mM) for 90 min at 37 °C or by donor ingestion of 300 mg
of aspirin per day for 3 consecutive days before blood collection. In
each case the pharmacological activity of the inhibitors was confirmed
as follows. Wortmannin and LY294002 caused dose-dependent
inhibition of thrombin-stimulated production of phosphatidylinositol
(3,4)-bisphosphate; genistein, tyrphostin, or erbstatin completely
inhibited vWf-induced protein tyrosine phosphorylation; calphostin or
bisindolylmaleimide abolished PAC-1 binding to washed platelets
adherent to a vWf matrix; aspirin abolished arachidonic acid-induced
platelet aggregation; RGDS and c7E3 Fab blocked ADP-induced platelet
aggregation. Resuspending platelets in EDTA-containing buffers
abolished calpain activation in thrombin-stimulated platelets.
Analysis of Platelet Adhesion Under Flow--
Platelet adhesion
under flow was performed using a modified method of Cranmer et
al. (7). Microslides (flat, rectangular microcapillary tubes) were
coated with HvWf (100 µg/ml) in phosphate-buffered saline (PBS) for
2 h at room temperature or overnight at 4 °C. For flow studies
using citrated whole blood, the microcapillary tubes were left
unblocked; however, for washed platelet studies the vWf-coated tubes
were blocked with either 25% heat-inactivated human serum (containing
50 µg/ml phenylmethylsulfonyl fluoride) or 1% bovine serum albumin
for 60 min. In general, the results were similar with either blocking
method although there was less nonspecific adhesion on the
serum-blocked vWf matrices. For whole blood studies, platelets were
fluorescently labeled by incubating citrate anti-coagulated blood with
DiOC6 (1 µM) for 10 min. The labeled whole
blood was perfused through the vWf-coated microcapillary tubes at a
shear rate of 150 or 750 s
1, and the non-adherent blood
cells were removed by perfusing Tyrode's buffer. c7E3 Fab (20 µg/ml)
was present throughout the experiments to prevent irreversible platelet
adhesion. For washed platelet flow studies, cells were washed twice in
platelet washing buffer, pH 6.5, containing the platelet activation
inhibitors, PGE1 (200 ng/ml), and theophylline (10 mM). Platelets were finally resuspended in the same buffer
(3 × 108/ml) and perfused over a vWf matrix at a
shear rate of 150 s1. In the majority of experiments with
washed platelets, flow was ceased for 5 min to maximize platelet
adhesion onto the matrix. Flow was reinitiated at a shear rate of 750 s1 and platelets washed with platelet washing buffer
(containing platelet activation inhibitors) or Tyrode's buffer. In
some studies, platelets were preincubated with cytochalasin D (10 µM) or latrunculin B (500 ng/ml) for 10 min, or with
inhibitors of tyrosine kinases, PKC, PI 3-kinase, the prostaglandin
pathway, or with EGTA-AM or BAPTA-AM for 30 min, as described under
"Platelet Aggregation Studies." Rolling platelets were visualized
using fluorescence or differential interference contrast (DIC)
microscopy (100× objective) (Leica DMIRB) and videotaped for
subsequent image analysis.
DNase I Inhibition Assays--
Actin polymerization in
vWf-aggregated platelets, transfected K562, and CHO cells was analyzed
using a quantitative DNase I inhibition assay as described by Blikstad
et al. (20) and Fox et al. (21). Washed platelets
(3 × 108/ml) and transfected K562 or CHO cells
(3 × 106/ml) were aggregated with either HvWf (20 µg/ml, unless otherwise specified) in the presence of ristocetin (1 mg/ml) or BvWf (20 µg/ml) alone with stirring for the indicated time
points. Cells were then lysed in 1% Triton X-100-containing buffer (20 mM Tris, pH 7.4, 1 mM phenylmethylsulfonyl
fluoride, 2 mM EDTA, 1 mM benzamidine, and 0.1 µM phallacidin) and immediately subjected to DNase I
inhibition assay. An alternative method (22), involving sedimentation
of filamentous actin by ultracentrifugation (100,000 × g for 3 h) of whole platelet lysates, yielded similar
results to the DNase I inhibition assay.
Expression of the GPIb-IX Complex on the Surface of CHO and K562
Cells--
Full-length cDNAs for GPIb, Ib and IX cloned into
the pDX vector were generous donations from Dr. J. Lopez (Houston, TX). GPV was cloned into the pZeoSV vector. K562 cells were cultured in RPMI
media supplemented with 10% fetal calf serum. CHO cells expressing
GPIb and IX (CHOIX, obtained from Dr. J. Lopez, Houston) were
cultured in Dulbecco's modified Eagle's/F-12 media supplemented with
10% fetal calf serum, 400 µg/ml G418, and 100 µM
methotrexate. K562 cells were transfected with the cDNAs for
GPIb, GPIb, GPIX, and GPV, whereas CHOIX cells were
transfected with the cDNA for full-length wild type GPIb cloned
into pDX, using FuGene TP6 Transfection Reagent (Roche Molecular
Biochemicals). Briefly, FuGene TP6 (3 µl) and each plasmid (0.9 µg)
were mixed with pZeoSV (0.1 µg) and incubated with K562 or CHOIX
cells for 3-7 h. Selection was initiated 3 days after transfection
with 200 µg/ml Zeocin or G418. The individual Zeocin or
G418-resistant CHO cell clones were isolated with glass cloning rings,
and K562 clones were isolated by limiting dilution 2 weeks following
selection. CHO cell clones expressing GPIbIX (CHOIX) and
K562VIX cells were cultured for a further 2 weeks and subjected
to immunofluorescence and fluorescence-activated cell sorter analysis
to examine protein expression on the cell surface.
K562 and CHO Cell Adhesion Studies--
CHO and K562 cells
(1 × 106/ml) in Tyrode's buffer were applied to a
HvWf (10 µg/ml) matrix in the presence of botrocetin (1 µg/ml) and
allowed to adhere for 60 min at 37 °C, unless otherwise specified.
In some studies, the cells were pretreated for 10 min with anti-GPIb
mAb (AK2), RGDS peptide (1 mM), EDTA (2 mM),
cytochalasin D (1 µM), or 30 min with 80 µM
EGTA-AM prior to application to vWf matrices. Where indicated, cells
were also fixed in suspension with 3.7% formaldehyde for 10 min and
then allowed to adhere onto poly-L-lysine (100 µg/ml)-coated coverslips. Non-adherent cells were removed by three
gentle washes with PBS. Adherent cells were fixed with 3.7%
formaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 30 min, and stained with FITC-conjugated phalloidin for 30 min. The cells
were then washed three times with PBS and subjected to confocal
fluorescence microscopy (Leica TCSNT, Germany) (16 and 100×
objective). The images were reconstructed using VoxBlast software. The
presence of membrane projections on a given cell was arbitrarily
defined as membrane extensions (>2 µm in length) covering the entire
surface of the cell.
When CHO cell adhesion studies were performed under flow conditions,
CHOIX cells (1 × 106/ml) were resuspended in
Ca2+-free Tyrode's buffer containing 1 mM
EDTA. Cells were perfused through a BvWf (100 µg/ml)-coated
microcapillary tube at a shear rate of 150 or 1500 s1,
and flow was allowed for 10 min by perfusing Tyrode's buffer (containing 1 mM EDTA). The adherent cells were then fixed,
permeabilized, stained with FITC-conjugated phalloidin and subjected to
fluorescence microscopy.
Analysis of Intracellular Calcium Mobilization in Platelets and
GPIb/IX-transfected CHO Cells--
Washed platelets (1 × 109/ml) or CHOIX cells (1 × 106/ml)
were resuspended in Ca2+-free Tyrode's buffer containing
EGTA (1 mM). The cells were then incubated with the
Ca2+-indicator dye, Oregon Green 488 BAPTA-AM-1 (1 µM), for 45 min at 37 °C. The platelets were then
pelleted by centrifugation (2,000 × g for 5 min) and
either resuspended in calcium-free Tyrode's buffer or washed
autologous red blood cells to a final concentration of 1 × 107/ml. Oregon Green 488 BAPTA-AM-1-loaded CHOIX
cells were resuspended in calcium-free Tyrode's buffer. In all
experiments, EGTA (1 mM) was included in the cell
suspensions to chelate trace quantities of extracellular calcium.
Platelets were maintained at 37 °C for 30 min in the presence of Fab
c7E3 (20 µg/ml), prior to perfusion through HvWf (10 µg/ml)-coated
microcapillary tube at a shear rate of 600, 1800, or 3000 s1. In the indicated studies PGE1 (1 µg/ml)
was added to the platelet suspensions to inhibit platelet activation.
Changes in the cytosolic concentration of calcium were monitored with
an upright confocal microscope (63× water lens) at a frame scan rate
of 1 frame/s. For CHOIX studies, cells were perfused through BvWf
(20 µg/ml)-coated microcapillary tubes at 150 s1 and
allowed to preadhere for 5 min. The non-adherent cells were removed and
the adherent cells subjected to the indicated shear rates.
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RESULTS |
Distinct Roles for GPIb/V/IX and Integrin
IIb3 in Mediating vWf-induced
Cytoskeletal Reorganization in Human Platelets--
A striking feature
of the platelet adhesion process is the dramatic change in cell
morphology induced by vWf binding to GPIb/V/IX and integrin
IIb3. Two actin-based structures are
required for platelet spreading, filopodial bundles, and lamellipodial
networks. The filopodial bundles are formed from long actin filaments
radiating from the center of the platelet, whereas lamellipodial
networks are formed from a circumferential zone of orthogonally arrayed short actin filaments (23). The mechanism(s) by which vWf induces platelet spreading remains poorly understood, although previous studies
with Glanzmann thrombasthenic platelets (congenitally deficient in
integrin IIb3) indicate that integrin
IIb3 plays an important role in this
process (14, 15). In our initial studies we compared the ability of
normal and Glanzmann thrombasthenic platelets to extend filopodia and
lamellipodia following adhesion onto a purified vWf matrix. As
demonstrated in Fig. 1A,
normal platelets adherent to a vWf matrix initially adopted a spherical morphology and extended multiple filopodial projections (Fig. 1A,
10'). This was followed by the extension of broad lamellipodial sheets between adjacent filopodia, converting the platelet from a
dendritic to a fully spread form (20' and 60').
In contrast, Glanzmann thrombasthenic platelets adopted a spherical
morphology and extended filopodia; however, lamellipodial formation was
completely absent (Fig. 1A). These results suggest that the
vWf-GPIb/V/IX interaction may be sufficient to induce platelet shape
change and filopodial extension, whereas the binding of vWf to integrin IIb3 is essential for the formation of
lamellipodial networks. Additional evidence in support of this
hypothesis was derived from studies of normal platelets pretreated with
the anti-IIb3 chimeric Fab fragment of
7E3 (c7E3 Fab). Similar to Glanzmann thrombasthenic platelets, blocking
ligand binding to integrin IIb3 abolished
lamellipodial formation but had no effect on the ability of platelets
to change shape and extend filopodia (Fig. 1B). A technical
limitation of using HvWf for these studies was the inability of this
protein to support stable adhesion in the presence of inhibitors of
integrin IIb3. It is possible that
repeated washing may have preferentially removed discoid cells from the
matrix leaving non-representative dendritic platelets. We therefore
performed adhesion assays with purified BvWf, which supports stable
platelet adhesion in the absence of integrin
IIb3. As with HvWf, all platelets
pretreated with c7E3 Fab became spherical and extended filopodia after
adhering to BvWf but were incapable of extending lamellipodia (Fig.
1B).
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Fig. 1.
vWf binding to GPIb/V/IX is sufficient to
induce platelet filopodial formation. A, washed normal
(1 × 107/ml) or Glanzmann thrombasthenic platelets
(1 × 108/ml) were allowed to preadhere onto a vWf
matrix (100 µg/ml) in the presence of platelet inhibitors,
PGE1 (200 ng/ml), and theophylline (10 mM) for
60 min. Non-adherent cells were aspirated, and adherent cells were
allowed to spread for the indicated times following removal of the
platelet inhibitors. Cells were then fixed, permeabilized, stained with
FITC-conjugated phalloidin, and imaged using confocal fluorescence
microscopy. B, washed normal platelets (1 × 107/ml) were pretreated with control buffer or c7E3 Fab (20 µg/ml) (c7E3 Fab) for 10 min and then allowed to adhere for 60 min to
either human vWf (100 µg/ml) or bovine vWf (10 µg/ml). Images were
obtained using confocal fluorescence microscopy (100× objective). The
cells presented are representative of >90% of the total population of
platelets from four independent experiments, and the results for
Glanzmann thrombasthenic platelets were from one experiment.
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Cytoskeletal Reorganization and Filopodial Extension in Rolling
Platelets--
The ability of the vWf-GPIb/V/IX interaction to induce
cytoskeletal reorganization in static adhesion assays raised the
interesting possibility that similar morphological changes may occur
during the process of platelet translocation on a vWf matrix. To
adequately visualize the morphology of platelets under flow conditions,
we performed all adhesion assays in flat rectangular microcapillary tubes, as described under "Experimental Procedures." This assay system enabled high magnification imaging of platelets in real-time with sufficient resolution to distinguish between resting discoid cells
and dendritic forms. We initially performed flow studies on
anticoagulated whole blood using DiOC6-labeled platelets.
DiOC6 stains membrane lipid and enables visualization of
membrane projections during the process of platelet activation (24).
Consistent with previous reports (1), perfusion of whole blood over a
vWf matrix at 150 s1 in the presence of an inhibitor of
integrin IIb3 (c7E3 Fab) resulted in
platelets forming reversible adhesion contacts with the matrix (Fig.
2A). These adhesion contacts
were mediated by the vWf-GPIb interaction as they were abolished by
pretreating platelets with the anti-GPIb antibody, AK2 (Fig.
2A, 1' + AK2). Most platelets translocated in a
stop-start manner, forming stationary adhesion contacts with the matrix
for 1-5 s during surface translocation. As demonstrated in Fig.
2A, the majority of platelets that initially tethered to vWf
did not have filopodial projections (5"); however, during
the process of platelet translocation these cells underwent morphological conversion to dendritic forms (1').
Morphological analysis of translocating platelets by DIC microscopy
after washing out all non-adherent cells demonstrated the majority of
platelets exhibited a spherical morphology and extended multiple
filopodia (Fig. 2A, DIC). In control studies, we
confirmed that the vWf-induced morphological changes were not secondary
to photoactivation of fluorescently labeled platelets, as DIC imaging
of non-labeled platelets in anticoagulated whole blood revealed that
all cells tethering to the matrix underwent shape change during surface translocation. Identical results were obtained using whole blood from
an individual with Glanzmann thrombasthenia, confirming that these
morphological changes occur independently of integrin
IIb3 (data not shown).
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Fig. 2.
Filopodial extension in rolling
platelets. A, whole blood was labeled with
DiOC6 (1 µM) and perfused through HvWf (100 µg/ml)-coated microcapillary tubes at a shear rate of 150 s1 in the presence of c7E3 Fab (20 µg/ml). Where
indicated, whole blood was also incubated with the anti-GP Ib mAb
(AK2) prior to perfusion. Images of rolling platelets were obtained by
fluorescence microscopy at a cell-matrix contact time of 5 s
(5") or 1 min (1'). After removal of non-adherent
blood cells with Tyrode's buffer (containing c7E3 Fab) rolling
platelets were visualized by DIC microscopy (100×). B,
washed platelets (3 × 108/ml) were pretreated with
c7E3 Fab (20 µg/ml) for 10 min. Cells were fixed then applied to
poly-L-lysine (100 µg/ml)-coated coverslips
(Fixed). Alternatively, platelets were perfused through
vWf-coated microcapillary tubes in the presence
(+PGE1/Theo) or absence
( PGE1/Theo) of PGE1 (200 ng/ml)
and theophylline (Theo) (10 mM). The images are
of platelets at the inlet of the microcapillary tube (Inlet) that have
rolled first a short time (less then 1 min) or from the outlet of the
tube (Outlet), in which the majority of cells had rolled for greater
than 5 min. C, c7E3 Fab-treated washed platelets were
perfused through vWf-coated microcapillary tubes in the presence of
cytochalasin D (10 µM) (+ CD). The morphology
of rolling platelets was also examined following removal of
cytochalasin D ( CD). Results presented in this figure are
representative of the total platelet population and were obtained from
one experiment, representative of 10.
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Further evidence confirming that the vWf-GPIb interaction can induce
cytoskeletal reorganization during the process of platelet translocation was obtained from studies using washed platelets. In
these experiments washed platelets were perfused through vWf-coated microcapillary tubes in the presence of the platelet activation inhibitors, PGE1 and theophylline, to avoid platelet
activation prior to cell contact with the vWf matrix. As detailed under
"Experimental Procedures" considerable care was taken to ensure
that platelets were not activated during the washing procedure. This
included maintaining the platelet suspension at pH 6.5 and including
platelet activation inhibitors from the moment of blood collection,
throughout all washing steps and during platelet perfusion experiments.
We confirmed that platelets were maintained in a resting state under these conditions by their lack of surface expression of P-selectin and
by the inability of the activation-dependent antibody,
PAC-1, to recognize integrin IIb3 on the
cell surface (data not shown). Analysis of the morphology of c7E3
Fab-treated washed platelets (in the presence of PGE1 and
theophylline) by DIC microscopy demonstrated that these cells exhibited
a resting discoid morphology (Fig. 2B, Fixed) and rolled in
a stop-start, side-to-side "flipping" manner over the vWf matrix
(Fig. 2B, +PGE1/Theo). Removing
PGE1 and theophylline from the rolling platelets by
perfusing the microcapillary tubes with Tyrode's buffer resulted in
the platelets adopting a spherical morphology and extending filopodia,
in an identical manner to that observed in whole blood. The extent of
filopodial formation reflected the time of platelet contact with the
vWf matrix. Translocating platelets at the inlet of the microcapillary tube (Fig. 2B, PGE1/Theo-Inlet),
which had only a brief contact with the vWf matrix, exhibited a small
number of short filopodia, whereas cells that had translocated to the
outlet of the microcapillary tube (Fig. 2B,
PGE1/Theo-Outlet) extended large prominent
filopodia. The filopodia were dynamic structures that extended and
retracted from the cell surface and, along with the cell body,
participated in the adhesion process by forming transient adhesion
contacts with the matrix. The extension of filopodia from the surface
of rolling platelets was dependent on actin polymerization as it was
completely inhibited by cytochalasin D (Fig. 2C, +CD) or
latrunculin B (data not shown). Cytochalasin D prevents actin filament
elongation in platelets by binding to the fast-growing (barbed) ends of
actin filaments (21, 25), whereas latrunculin B prevents actin
polymerization by binding monomeric actin (26). As with the platelet
activation inhibitors, removing cytochalasin D from the rolling
platelets subsequently resulted in filopodial extension in all cells
(Fig. 2C, CD). These studies, combined with our
experiments in whole blood, provide strong evidence that the
vWf-GPIb/V/IX interaction can induce actin polymerization and
cytoskeletal reorganization during the process of platelet translocation.
vWf-induced Actin Polymerization in Aggregated Platelets--
In
addition to mediating platelet adhesion to the injured vessel wall, vWf
also plays an important role in mediating platelet-platelet adhesion
contacts (aggregation) under conditions of high shear stress (27-30).
We have previously established that a number of signaling processes
occurring in adherent platelets also occur in vWf-aggregated platelets
(12). We therefore investigated the ability of vWf to induce actin
polymerization in suspension-activated platelets by measuring changes
in the cellular level of filamentous (F) actin. As demonstrated in Fig.
3A, aggregation of platelets by HvWf in the presence of ristocetin resulted in a
time-dependent increase in F-actin. The level of F-actin
increased from a basal level of 38 ± 3% (n = 7)
up to a maximum of 72 ± 1% (n = 7) after 10 min
stimulation. Ristocetin alone had no effect on the F-actin content of
platelets, whereas purified BvWf induced an increase in F-actin similar
to that observed with HvWf (Fig. 3B). Actin polymerization
required platelet stirring, and aggregation was inhibited by
pretreating platelets with the anti-GPIb blocking antibody, AK2 (data
not shown). These observations are consistent with previous reports
demonstrating that GPIb signaling in suspension-activated platelets is
an aggregation-dependent phenomenon (19, 31). The
effect of increasing concentrations of vWf on actin polymerization was
also examined. As demonstrated in Fig. 3C, increasing the BvWf concentration from 2 to 20 µg/ml resulted in a
dose-dependent increase in the cellular levels of F-actin
from a resting level of 42% ± 2.6 (n = 4) up to a
maximum of 72.6% ± 3.2 (n = 4).
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Fig. 3.
vWf-induced actin polymerization in
aggregated platelets. A, washed platelets (3 × 108/ml) were aggregated with HvWf (20 µg/ml) in the
presence of ristocetin (1 mg/ml) for the indicated time points while
stirring. In all experiments, the F-actin content in whole cell lysates
was determined using the DNase I inhibition assay, as described under
"Experimental Procedures." Results represent the mean ± S.E.
from seven experiments. B, platelets were stirred in the
presence of buffer (Resting), ristocetin (1 mg/ml) alone
(Ristocetin), HvWf (20 µg/ml) and ristocetin (1 mg/ml)
(HvWf + Ristocetin), or BvWf (BvWf) alone (20 µg/ml) for 10 min. C, platelets were aggregated with the
indicated concentrations of BvWf for 10 min. Results in B
and C represent the mean ± S.E. from four independent
experiments performed in duplicate. Statistical analysis was performed
using the Student's t test comparing resting
versus vWf-aggregated platelets (*** p < 0.0001).
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vWf-GPIb/V/IX Interaction Induces Actin Polymerization
Independently of Integrin
IIb3--
Previous studies have
established an important role for integrin
IIb3 in regulating actin polymerization
and cytoskeletal reorganization in response to platelet stimulation by
soluble agonists (32, 33). To examine the role of integrin
IIb3 in regulating vWf-induced actin
polymerization, we pretreated platelets with inhibitors of integrin
IIb3, including c7E3 Fab, the
tetrapeptide RGDS, or EDTA, and we examined the ability of BvWf to
induce increases in the cellular levels of F-actin. The effects of EDTA
on BvWf-induced platelet aggregation and actin polymerization are
summarized in Fig. 4A. Using
low concentrations of vWf (1-10 µg/ml), blocking ligand binding to
integrin IIb3 resulted in a significant
reduction in the rate and extent of platelet aggregation and a
corresponding decrease in the extent of F-actin accumulation.
Interestingly, when platelets were stimulated with high concentrations
of BvWf (>15 µg/ml), blocking ligand binding to integrin
IIb3 had minimal inhibitory effect on
platelet aggregation and also on the cellular accumulation of F-actin
(Fig. 4A). Similar findings were apparent with
HvWf-aggregated platelets pretreated with EDTA, c7E3 Fab, or RGDS (Fig.
4B). In all experiments, we observed a strong correlation
between the rate and extent of platelet aggregation and the degree of
actin polymerization (Fig. 4A). Studies on vWf-aggregated
Glanzmann thrombasthenic platelets confirmed the ability of the
vWf-GPIb/V/IX interaction to induce actin polymerization independently
of integrin IIb3 (Fig.
4C).
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Fig. 4.
The vWf-GPIb/V/IX interaction induces actin
polymerization independently of
IIb3.
A, effect of EDTA on BvWf-induced platelet aggregation and
F-actin accumulation. Washed platelets (3 × 108/ml)
were aggregated with the indicated concentration of BvWf for 5 min in
the presence (+) or absence () of 2 mM EDTA. The rate of
aggregation was determined by changes in light transmission in the
washed platelet suspension using a platelet aggregometer. F-actin
content in whole cell lysates was determined as described in Fig. 4.
Results are from one experiment, representative of three experiments
performed in duplicate. B, effect of EDTA, c7E3 Fab, and
RGDS on HvWf-induced actin polymerization. Platelets were either
unstimulated (Resting) or stimulated with HvWf (20 µg/ml)
and ristocetin (1 mg/ml), in the presence of control buffer
(Control), EDTA (2 mM), c7E3 Fab (20 µg/ml),
or RGDS (1 mM). Note: in these experiments, using high
concentrations of vWf in the presence of inhibitors of
IIb3 did not reduce the rate of platelet
aggregation. Results are the mean ± S.E. from four individual
experiments performed in duplicate. ***, p < 0.0001. C, vWf-induced actin polymerization in Glanzmann
thrombasthenic platelets. Washed normal or Glanzmann thrombasthenic
platelets were aggregated with HvWf (20 µg/ml) in the presence of
ristocetin (1 mg/ml) for 10 min. Results are derived from one
experiment.
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vWf-induced Cytoskeletal Reorganization in
GPIb/V/IX-transfected K562 Cells--
To investigate
further the relationship between vWf binding to GPIb and subsequent
actin polymerization and cytoskeletal reorganization, we performed
studies on GPIb/V/IX-transfected K562 cells. K562 cells are derived
from a human leukemic cell line and express many of the signaling
components present in platelets but do not express detectable levels of
GP Ib/V/IX or 3 integrin on the cell surface (data not
shown). These cells therefore represent a potentially useful cell model
to express the GPIb-V-IX complex and examine adhesion and signaling
processes linked to this receptor. In initial studies we compared the
ability of non-transfected (K562) or transfected K562 cells
(K562VIX) to adhere to immobilized HvWf in the presence of
botrocetin. As demonstrated in Fig.
5A (upper panel),
only transfected cells formed stationary adhesion contacts with the vWf
matrix. This adhesion was dependent on GPIb as it was effectively
blocked by pretreating K562VIX cells with the anti-GPIb antibody,
AK2. Examination of the morphology of adherent K562VIX cells
demonstrated that ~80% of these cells extended numerous membrane
projections (Fig. 5A, lower panel, and 5B). This
contrasted with K562VIX cells that exhibited a featureless round
morphology (Fig. 5A, lower panel) prior to adhesion to the
vWf matrix, suggesting that the vWf-GPIb interaction induced these
morphological changes. The extension of membrane projections required
actin polymerization as they were completely abolished by pretreating
the cells with cytochalasin D (Fig. 5A, lower panel). Furthermore, EDTA or RGDS had no effect on the ability of K562VIX cells to adhere to the vWf matrix or extend membrane projections (Fig.
5, A and B), excluding a role for integrins in
this process.
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Fig. 5.
vWf-induced cytoskeletal reorganization in
GPIb/V/IX-transfected K562 cells. K562 cells transfected with the
full receptor complex (K562VIX) or non-transfected
control cells (K562) (1 × 106/ml) were
allowed to adhere to HvWf (10 µg/ml)-coated coverslips for 60 min in
the presence of botrocetin (1 µg/ml) (HvWf + Botro). Where
indicated, cells were incubated with the anti-GP Ib mAb AK2 (5 µg/ml), RGDS (1 mM), EDTA (2 mM), or
cytochalasin D (CD) (1 µM) prior to adhesion.
A, adherent cells were fixed, permeabilized, stained with
FITC-conjugated phalloidin, and visualized by confocal fluorescence
microscopy (upper panel, 16× objective; lower
panel, 100× objective). K562VIX cells were also fixed in
suspension (K562VIX - fixed*) prior to adhesion onto
poly-L-lysine (100 µg/ml)-coated coverslips (lower
panel). B, the number of cells adherent or extending
membrane projections was determined in 5 random fields (40× objective)
and expressed as the mean number of cells per field. These results are
from one experiment, representative of five performed in
duplicate.
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Cytoskeletal Reorganization in GPIb/IX-transfected CHO
Cells--
Previous studies examining vWf-induced cytoskeletal
reorganization in GPIb/IX-transfected CHO cells have suggested an
important role for endogenous CHO cell integrins in this process (16, 17). To investigate possible differences between CHO and K562 cells, we
performed CHO cell adhesion studies on HvWf using cells transfected
with GPIb, Ib, and IX (CHOIX). As demonstrated in Fig.
6A, CHOIX cells adhered
to HvWf in the presence of botrocetin. In control studies we
demonstrated that this adhesion required the vWf-GPIb interaction as it
was blocked by AK2 and was not observed with CHO cells expressing
GPIb and GPIX alone (CHOIX). Morphological analysis of adherent
CHOIX cells revealed that >80% of the cells extended membrane
projections similar to those observed with transfected K562 cells (Fig.
6A, lower panel, 6B). Furthermore, approximately
a third of the cells exhibited stress fibers and spread on the vWf
matrix. As with K562VIX, these cytoskeletal changes appeared to
be dependent on the vWf-GPIb interaction as cells fixed in suspension
prior to exposure to a vWf-matrix exhibited a round morphology (Fig.
6A, lower panel). The addition of RGDS or EDTA to
the adhesion assays had no effect on the level of adhesion or the
extension of membrane projections; however, stress fibers and spreading
were completely abolished (Fig. 6, A and B).
Furthermore, all cytoskeletal changes were prevented by pretreating
CHOIX cells with cytochalasin D (Fig. 6A, lower
panel). These results support our findings in
GPIb/V/IX-transfected K562 cells suggesting that the vWf-GPIb/IX
interaction is sufficient to induce cytoskeletal reorganization
independently of integrins; however, subsequent spreading and stress
fiber formation appear to be an integrin-dependent
phenomena. They also establish that cytoskeletal reorganization is not
dependent on the presence of GPV. To confirm that the vWf-GPIb
interaction was able to induce cytoskeletal reorganization in the
absence of an artificial modulator, CHO-Ib/IX cells were perfused
through BvWf-coated microcapillary tubes at venous (150 s1) or arteriole (1500 s1) shear rates. As
demonstrated in Fig. 6C, CHOIX cells exhibited a
smooth round appearance prior to the performance of flow experiments. Exposure of these cells to low shear resulted in the extension of
numerous small filopodial projections over the entire cell surface. At
high shear, filopodia were much more prominent and specifically
localized to the upstream edge of the cell. The reason for this
polarity is unclear but may reflect the shear differential experienced
by the leading and trailing edge of the cell. It should also be noted
that in the static adhesion assays filopodia developed slowly, within
5-10 min of adhesion and were maximal by 60 min. In contrast, cells
exposed to shear extended filopodia within 1-2 min with maximal
extension by 5 min.
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Fig. 6.
vWf-induced cytoskeletal reorganization in
GPIb/IX-transfected CHO cells. A, CHO cells transfected
with partial receptor complexes (GPIb, Ib, and IX)
(CHOIX) or GPIb and IX (CHOIX) were
allowed to adhere for 60 min onto HvWf-coated coverslips (10 µg/ml)
in the presence of botrocetin (1 µg/ml) (HvWf + Botro).
Where indicated, cells were incubated with the anti-GPIb mAb AK2 (5 µg/ml), RGDS (1 mM), or cytochalasin D (CD) (1 µM), prior to adhesion. Adherent cells were fixed,
permeabilized, stained with FITC-conjugated phalloidin and visualized
by confocal fluorescence microscopy (upper panel, 16×
objective; lower panel, 100× objective). CHOIX cells
were also fixed (CHOIX -fixed*) in suspension prior to
adhesion onto poly-L-lysine (100 µg/ml)-coated
coverslips. B, the number of cells adherent, spread, and/or
extending membrane projections was determined in 5 random fields (40×
objective) and expressed as the mean ± S.E. (cells per field)
from four experiments performed in duplicate. C, CHOIX
cells were adhered to BvWf (100 µg/ml)-coated microcapillary tubes in
the presence of 1 mM EDTA. The adherent cells were either
fixed (0 s1) or subjected to shear (150 s1
or 1500 s1) for 10 min prior to fixation. Cells were then
permeabilized, stained with FITC-phalloidin, and subjected to
fluorescence microscopy.
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vWf-induced Increase in Filamentous Actin in GPIb/IX-transfected
CHO and K562 Cells--
To investigate the extent of actin
polymerization induced by vWf binding to GPIb/IX in CHOIX and
K562VIX cells, DNase I inhibition assays were performed on whole
cell lysates from aggregated cells. BvWf-induced aggregation of
CHOIX and K562VIX resulted in an overall increase in the
cellular content of F-actin from ~30 to 45% (Fig.
7). In general the rate and extent of
aggregation as well as the increase in F-actin in transfected cells
were less than that observed in platelets (Figs 3 and 4), probably
reflecting lower receptor density on these cells. The increase in
F-actin was dependent on the vWf-GPIb interaction in that it was
abolished by pretreating the cells with AK2 and was not observed in
CHO/IX cells (Fig. 7B). Similar to platelets, the
vWf-induced increase in F-actin was not inhibited by EDTA but was
completely abolished by latrunculin B (Fig. 7B).
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Fig. 7.
vWf-induced actin polymerization in
GPIb/IX-transfected CHO and GPIb/V/IX-transfected K562 cells.
A, CHOIX cells (3 × 106/ml) were
stirred for the indicated time with BvWf (20 µg/ml) to induce cell
aggregation. Cells were then lysed, and the level of F-actin in the
whole cell lysates was determined using the DNase I inhibition assay.
Results are from one experiment representative of four performed in
duplicate. B, CHOIX, CHOIX, and K562VIX cells
(3 × 106/ml) were stirred in the presence of buffer
(Rest) or BvWf (20 µg/ml) for 20 min. Where indicated,
CHOIX cells were pretreated with the anti-GPIb mAb
(AK2) (5 µg/ml), EDTA (2 mM), or latrunculin B
(LB) (500 ng/ml) for 10 min prior to the addition of BvWf.
Results represent the mean ± S.E. from four independent
experiments performed in duplicate. ***, p < 0.0001.
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Signaling Processes Regulating vWf-induced Actin
Polymerization--
It is now well established that vWf binding to
platelets induces multiple signaling events, including calcium influx
(28, 29), protein tyrosine phosphorylation (12, 19, 34, 35), prostaglandin metabolism (8), activation of PKC (31), and PI 3-kinase
(19). To investigate the potential importance of these signaling events
in vWf-induced actin polymerization and cytoskeletal reorganization, we
pretreated washed platelets with a range of well characterized signal
transduction inhibitors. These included pharmacological inhibitors of
tyrosine kinases (genistein, tyrphostin or erbstatin), PI 3-kinase
(wortmannin or LY294002), PKC (calphostin C or bisindolylmaleimide),
prostaglandin metabolism (aspirin), and a chelator of extracellular
calcium (EDTA). The effects of these inhibitors on vWf-induced actin
polymerization and cytoskeletal reorganization are summarized in Fig.
8. None of these inhibitors prevented
platelet shape change and filopodial extension in rolling platelets
(Fig. 8B) or inhibited platelet aggregation and actin
polymerization in response to 20 µg/ml HvWf (Fig. 8A). In
control studies, each inhibitor was demonstrated to be
pharmacologically active and capable of inhibiting other functional
platelet responses (see "Experimental Procedures"). Consistent with
our earlier studies on rolling platelets (Fig. 2), the signaling
processes responsible for vWf-induced actin polymerization appeared to
be sensitive to the inhibitory effects of PKA, as pretreating platelets
with PGE1 and theophylline led to an ~70% reduction in
actin polymerization (Fig. 8A) without affecting vWf-induced
aggregation. Taken together, these studies suggest the existence of
alternative signaling pathways linking the vWf-GPIb interaction to
cytoskeletal reorganization.
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Fig. 8.
Signaling processes regulating vWf-induced
actin polymerization in aggregated and rolling platelets.
A, washed platelets (3 × 108/ml) were
treated with either buffer (Control), EDTA (2 mM), aspirin (1 mM), PGE1 (200 ng/ml), and theophylline (10 mM)
(PGE1/Theo), or the indicated inhibitors of PKC
(PKC), tyrosine kinases (TK), or PI 3-kinase
(PI 3-K), as detailed under "Experimental Procedures."
Platelets were stirred in the presence of buffer (Resting),
or aggregated with HvWf (20 µg/ml) in the presence of ristocetin (1 mg/ml) for 10 min, and F-actin content was determined using the DNase I
inhibition assay. Results represent the mean ± S.E. from five
individual experiments performed in duplicate. ***, p < 0.0001. B, the treated platelets were also perfused
through vWf-coated microcapillary tubes (100 µg/ml) in the presence
of c7E3 Fab (20 µg/ml) as described under "Experimental
Procedures." Images of rolling platelets were obtained as described
in Fig. 3. The images presented are from one experiment, representative
of three.
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Calcium Mobilization in Rolling Platelets--
In further studies
we investigated the possibility that the vWf-GPIb interaction may
induce mobilization of calcium from intracellular stores, independent
of calcium influx. Cytosolic calcium plays a key role in activating the
actin filament severing mechanism as an essential step for platelet
sphering, normal filopodial formation, and lamellipodial development
(23). To investigate changes in the cytosolic concentration of calcium
during platelet rolling, studies were performed on washed platelets
loaded with the calcium indicator dye, Oregon Green 488 BAPTA-AM-1.
These cells were perfused over a vWf matrix at low (150 s1), intermediate (600 s1), or high (1500 and 3000 s1) shear rates, and in all experiments the
platelets were resuspended in EGTA-containing buffers to examine
specifically the contribution of intracellular calcium mobilization to
changes in cytosolic calcium. As demonstrated in Fig.
9A, real-time confocal imaging of platelets exposed to low shear (150 s1) demonstrated
that 2-3% of cells exhibited a high level of fluorescence intensity
during surface translocation. In contrast, exposing these cells to
thrombin (1 unit/ml) induced high fluorescence emission in >80% of
the cells examined, consistent with the ability of this agonist to
induce calcium mobilization from intracellular stores. This
thrombin-induced increase in cytosolic calcium was prevented by
pretreating platelets with PGE1 (data not shown), a finding
consistent with the ability of PKA to antagonize agonist-induced mobilization of intracellular calcium. Exposing platelets to increasing shear in the absence of thrombin resulted in individual cells exhibiting intermittent calcium pulsing during the rolling process (Fig. 9, B and C). As demonstrated in Fig.
9B, the percentage of cells pulsing at any one time
increased as a function of wall shear rate with 3-5% pulsing at 600 s1, 10-13% at 1800 s1, and 18-24% at
3000 s1 (n = 4). As with
thrombin-stimulated platelets, the shear-induced increase in cytosolic
calcium was abolished by pretreating platelets with PGE1
(Fig. 9B). To confirm that the vWf-GPIb interaction was
sufficient to induce mobilization of intracellular calcium independently of integrin IIb3, studies
were performed on Oregon Green 488 BAPTA-AM-1-loaded CHOIX cells
resuspended in EGTA-containing buffers. These cells exhibited a low
basal level of fluorescence when exposed to serum-blocked
microcapillary tubes (Fig. 9D). Following adhesion to BvWf
under static conditions, a small percentage of cells exhibited high
fluorescence emission (Fig. 9D). As with platelets, the
percentage of cells fluorescing at any one time increased as a function
of wall shear rate with 5-8% of cells exhibiting high fluorescence
intensity at 150 s1 and 18-23% at 1500 s1.
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Fig. 9.
Shear-dependent Ca2+
mobilization in rolling platelets and GPIb/IX-transfected CHO
cells. Washed platelets were loaded with the Ca2+
indicator dye, Oregon Green 488 BAPTA-AM-1, and perfused through HvWf
(100 µg/ml)-coated microcapillary tube at the indicated shear rates
in the presence of 1 mM EGTA and c7E3 Fab (20 µg/ml), as
described under "Experimental Procedures." Changes in fluorescence
intensity of individual cells were monitored by confocal microscopy
(63× objective) at a scanning rate of 1 frame/s. A,
Ca2+ mobilization in platelets at 150 s1 in
the presence or absence of thrombin (1 unit/ml). The relative extent of
Ca2+ mobilization in rolling platelets is indicated by the
accompanying look-up table (inset). B,
shear-dependent increase in cytosolic calcium in rolling
platelets and its inhibition by PGE1. C, changes
in cytosolic calcium in rolling platelets at 3000 s1. The
indicated platelets (numbered 1-3) were imaged at 1.7-s intervals over 6.8 s. D, CHOIX
(1 × 106/ml) cells were loaded with Oregon Green 488 BAPTA-AM-1 and applied to serum-coated microcapillary tubes under
static conditions or, alternatively, perfused through BvWf (20 µg/ml)-coated microcapillary tubes at 150 s1 or 1500 s1 in the presence of EGTA (1 mM), as
described under "Experimental Procedures." The fluorescent images
(top panel) demonstrate a shear-dependent
increase in cytosolic calcium. DIC images of the same cells are
demonstrated in the bottom panels. The images in this figure
are from one experiment, representative of four independent experiments
performed in duplicate.
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Comparative imaging of unloaded platelets by DIC and Oregon Green 488 BAPTA-AM-1-loaded platelets by fluorescence microscopy highlighted the
close relationship between shear-induced calcium pulsing and
cytoskeletal reorganization. For example, time-lapse imaging of
platelet shape change and filopodial extension under static conditions
demonstrated that these morphological changes occur slowly over a
5-10-min time period, consistent with the weak calcium response under
these experimental conditions. In contrast, the rate of platelet shape
change increased as a function of shear with the majority of platelets
undergoing morphological changes within the first 15-30 s of tethering
at high shear rates.
A Critical Role for Cytoplasmic Calcium in Mediating vWf-induced
Shape Change--
To investigate the functional importance of
intracellular calcium mobilization in inducing cytoskeletal changes in
platelets adherent to vWf, washed platelets were incubated with the
membrane-permeable calcium chelators, EGTA-AM or BAPTA-AM, and examined
for morphological changes under static or flow conditions. As
demonstrated in Fig. 10, chelating
cytosolic calcium prevented the normal disc-to-sphere transformation in
platelets adherent under static conditions and flow. Many of the
rolling platelets lost their smooth surface appearance and developed
irregular margins (Fig. 10, upper and middle
panel). Moreover, there was a marked reduction in the number and
size of filopodia extending from the cell surface. In general, the
filopodia were short and had a thickened bulbous appearance, consistent
with the role of calcium in inducing actin filament severing during
normal filopodial development. EGTA-AM loading of CHO-Ib/IX cells
resulted in a similar defect in filopodial formation with the majority
of membrane extensions having a short irregular appearance (Fig. 10,
bottom panel).
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Fig. 10.
vWf-induced calcium mobilization is required
for cytoskeletal reorganization in rolling platelets and
GPIb/IX-transfected cells. Washed platelets (3 × 108/ml) were incubated with Me2SO
(Control) or EGTA-AM (50 µM) for 30 min in the
presence of 1 mM EGTA and c7E3 Fab (20 µg/ml). The cells
were then allowed to adhere to HvWf (100 µg/ml)-coated coverslips for
60 min (Static). Alternatively, the washed platelets were
perfused through HvWf (100 µg/ml)-coated capillary tubes at a shear
rate of 1500 s1 (Flow). The cell images were
obtained using DIC microscopy (63× objective) and recorded real-time
on video tape. CHOIb/IX cells (1 × 106/ml) were
preincubated with EGTA-AM (80 µM) for 30 min in the
presence of EDTA (1 mM). The cells were adhered to a HvWf
(10 µg/ml)-coated coverslip for 60 min at 37 °C, in the presence
of botrocetin (1 µg/ml). The adherent cells were then fixed,
permeabilized, stained with FITC-phalloidin, and subjected to
fluorescence microscopy. Images presented are from one experiment,
representative of five experiments performed in duplicate.
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DISCUSSION |
The results presented here demonstrate that platelets tethering to
a vWf matrix under physiological flow conditions undergo rapid
cytoskeletal reorganization during the rolling process. Studies using
Glanzmann thrombasthenic platelets and GPIb/IX-transfected cells have
demonstrated that the vWf-GPIb interaction is sufficient to induce
these cytoskeletal changes independent of integrin
IIb3. Cytoskeletal reorganization in
rolling platelets is a shear-dependent event that does not
involve several well characterized signaling processes previously
linked to vWf-induced platelet activation, including calcium influx,
protein tyrosine phosphorylation, prostaglandin metabolism, and
activation of PKC and PI 3-kinase. In contrast, our studies suggest
that the vWf-GPIb interaction activates a shear-sensitive signaling
process linked to intracellular calcium mobilization. This signaling
pathway is sensitive to the inhibitory effects of PKA and is
functionally linked to cytoskeletal reorganization in rolling platelets.
Our studies with Glanzmann thrombasthenic platelets have confirmed
previous reports suggesting a critical role for integrin IIb3 in inducing cytoskeletal
reorganization as a necessary event for platelet spreading. Despite the
presence of at least four other integrins on the platelet surface,
including receptors for collagen (21),
laminin (61), vitronectin
(v3), and fibronectin (51), integrin
IIb3 appears to play an indispensable
role in the normal spreading process even when platelets are exposed to
a heterogeneous mix of adhesive substrates in the subendothelium (14,
15). A previous report has demonstrated that GPIb/IX-transfected CHO
cells extend filopodia and spread on a human vWf matrix in the absence
of integrin IIb3 (16). However a
follow-up study by the same group suggested that vWf binding to GPIb/IX
was not sufficient to induce these cytoskeletal changes independently of endogenous CHO cell integrins (17). Whereas our studies have also
suggested an important role for integrins in mediating platelet and
CHO-Ib/X cell spreading on a vWf matrix, several lines of evidence
indicate that binding of vWf to GPIb was sufficient to induce actin
polymerization and cytoskeletal reorganization independently of other
major platelet adhesion receptors. First, Glanzmann thrombasthenic platelets and normal platelets treated with inhibitors of integrin IIb3 were able to change shape and extend
filopodia under static and flow conditions. Second, vWf-induced
aggregation of Glanzmann thrombasthenic platelets or normal platelets
pretreated with EDTA, RGDS, or c7E3 Fab was associated with a marked
increase in the level of F-actin. Third, vWf was able to induce actin
polymerization in cells expressing GPIb, Ib, and IX but not in
cells expressing GPIb and GPIX alone. Fourth, adhesion of
GPIb/IX-transfected cells to a vWf matrix resulted in actin filament
reorganization and the extension of numerous membrane projections, in
the presence of inhibitors of endogenous integrins.
Our studies define a key role for intracellular calcium in mediating
cytoskeletal changes in rolling platelets. Chelating intracellular
calcium prevented the normal disc-to-sphere transition in rolling
platelets and resulted in a substantial reduction in the number and
size of filopodia extending from the surface of platelets and
GPIb/IX-transfected CHO cells. These observations are in keeping with
previous studies demonstrating an indispensable role
TOP
ABSTRACT
INTRODUCTION
