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Prepublished online as a Blood First Edition Paper on June 21, 2002; DOI 10.1182/blood-2002-02-0514.
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Blood, 15 October 2002, Vol. 100, No. 8, pp. 2793-2800
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Sequential cytoplasmic calcium signals in a 2-stage platelet
activation process induced by the glycoprotein Ib
mechanoreceptor
Mario Mazzucato,
Paola Pradella,
Maria Rita Cozzi,
Luigi De
Marco, and
Zaverio M. Ruggeri
From the Servizio Immunotrasfusionale e Analisi
Cliniche, Centro di Riferimento Oncologico, Aviano, Italy; and the Roon
Research Center for Arteriosclerosis and Thrombosis, Division of
Experimental Hemostasis and Thrombosis, Departments of Molecular and
Experimental Medicine and of Vascular Biology, Scripps Research
Institute, La Jolla, CA.
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Abstract |
We found that the interaction of platelets with immobilized von
Willebrand factor (VWF) under flow induces distinct elevations of
cytosolic Ca++ concentration
([Ca++]i) that are associated with sequential
stages of integrin  IIb 3 activation.
Fluid-dynamic conditions that are compatible with the existence of
tensile stress on the bonds between glycoprotein Ib (GPIb) and
the VWF A1 domain led to Ca++ release from intracellular
stores (type / peaks), which preceded stationary platelet
adhesion. Raised levels of cyclic adenosine monophosphate (cAMP) and
cyclic guanosine monophosphate, as well as membrane-permeable calcium
chelators, inhibited these [Ca++]i
oscillations and prevented stable adhesion without affecting the
dynamic characteristics of the typical platelet translocation on VWF
mediated by GPIb. Once adhesion was established through the integrin
IIb3, new
[Ca++]i oscillations (type  ) of greater
amplitude and duration, and involving a transmembrane ion flux,
developed in association with the recruitment of additional platelets
into aggregates. Degradation of released adenosine diphosphate (ADP) to
AMP or inhibition of phosphatidylinositol 3-kinase (PI3-K)
prevented this response without affecting stationary adhesion and
blocked aggregation. These findings indicate that an initial signal
induced by stressed GPIb-VWF bonds leads to
IIb3 activation sufficient to support localized platelet adhesion. Then, additional signals from ADP receptors and possibly ligand-occupied
IIb3, with the contribution of a pathway
involving PI3-K, amplify platelet activation to the level required
for aggregation. Our conclusions modify those proposed by others
regarding the mechanisms that regulate signaling between GPIb and
IIb3 and lead to platelet adhesion and
aggregation on immobilized VWF.
(Blood. 2002;100:2793-2800)
© 2002 by The American Society of Hematology.
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Introduction |
Platelets adhere at sites of vascular injury to
prevent hemorrhage1 but may also form occluding arterial
thrombi that cause disease.2,3 In vessels in which rapid
blood flow creates high wall-shear rates, such as arterioles in the
normal circulation or atherosclerotic arteries with restricted lumen,
platelet thrombus formation depends on von Willebrand factor (VWF)
immobilized on extracellular matrix components, particularly
collagens.4 Binding of glycoprotein Ib (GPIb), a
constituent of the GPIb-IX-V complex, to the VWF A1 domain (A1VWF)
initiates platelet tethering to these surfaces but by itself can only
support translocation with stop-and-go motion.5 Once
tethered, however, platelets rapidly achieve irreversible adhesion
mediated by different integrins, including IIb3 bound to the Arg-Gly-Asp (RGD) motif
in the VWF C1 domain.4,5 Activated
IIb3 serves also to immobilize on the
surface of adherent platelets the plasma proteins, mainly VWF and
fibrinogen, that mediate the recruitment of additional platelets into
the forming thrombus.6
Platelet activation, necessary to promote the ligand-binding function
of IIb3, is coupled to the interactions
that establish initial platelet-surface contacts, as shown by the fact
that VWF binding to GPIb leads to aggregation.7,8
Sustained elevations of intracellular calcium concentration
([Ca++]i), a marker of activation, occur in
association with shear-induced platelet aggregation dependent on VWF
and GPIb9 and may be the consequence of a transmembrane
ion flux.10 Oscillations of
[Ca++]i have also been observed to accompany
platelet adhesion to VWF, but this finding has been given discordant
interpretations.11,12 Some results13 suggest
that the VWF-GPIb interaction may induce transient elevations
(spikes) of [Ca++]i that activate
IIb3 in an initially reversible manner
and influence the dynamic aspect of platelet-surface contacts before stable adhesion is established. This is in contrast to the idea that
transient tethering to immobilized VWF depends only on GPIb, whereas
activation of IIb3, like that of other
integrins on leukocytes,14 leads to irreversible
adhesion.4,5 Moreover, it has been proposed that
phosphatidylinositol 3-kinase (PI3-K) plays an essential role in
the activation of IIb3 required for stable platelet adhesion.15 To evaluate these conclusions,
we concurrently analyzed the instantaneous velocity and
[Ca++]i in single platelets interacting with
immobilized VWF. We identified a sequence of distinct cytosolic
Ca++ elevations associated with a 2-step process of
IIb3 activation. The first signal
involves release from intracellular stores and always precedes
stationary adhesion. The second signal, which is coupled to adenosine
diphosphate (ADP)-receptor stimulation and is inhibited by wortmannin,
follows stationary adhesion but precedes the initiation of platelet
aggregation on the surface. These results challenge the current
interpretation13,15 of the mechanisms leading to the
intracytoplasmic Ca++ transients linked to stable platelet
adhesion and aggregation.
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Materials and methods |
Preparation of blood samples
These studies were approved by an institutional review board.
Venous blood from healthy volunteers who were not taking any medication
and who gave informed consent according to the Declaration of Helsinki
was collected into a 1:6 final volume of citric acid/citrate/dextrose (pH 4.5) or 400 units/mL (final concentration) of the -thrombin inhibitor hirudin (Iketon, Milan, Italy). Fifty milliliters of blood
was centrifuged at 800g for 50 seconds, and the supernatant platelet-rich plasma (PRP) was collected. The procedure was repeated twice to obtain approximately 15 mL PRP. Platelets were incubated for
20 minutes at 37°C with the calcium fluorescent probe fluo-3 acetoxymethyl ester-AM (Fluo 3-AM; 8 µM final
concentration; Molecular Probes, Eugene, OR). In selected experiments,
platelets were loaded simultaneously with Fluo 3-AM and
1,2-bis(o-aminophenoxy)ethane-N, N, N',
N'-tetraacetic acid tetra(acetoxymethyl) ester
(BAPTA-AM; 80 µM final concentration; Molecular Probes).
Erythrocytes separated from the same blood were washed 3 times in a
divalent cation-free HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid)-Tyrode buffer (10 mM HEPES, 140 mM sodium chloride [NaCl], 2.7 mM potassium chloride [KCl], 0.4 mM monobasic sodium phosphate
[NaH2PO4], 10 mM sodium bicarbonate, and 5 mM
dextrose [pH 6.5]) and finally resuspended in the same buffer with
1.75 mM probenecid (Sigma, St Louis, MO). Probenecid was used to
prevent leakage of Fluo 3-AM from cells.16 An
adequate volume of PRP containing 2 × 108 to
8 × 108 platelets loaded with Fluo 3-AM was
mixed with an aliquot of erythrocyte suspension (0.50 hematocrit) and 5 U/mL apyrase (grade 3; Sigma). The mixture
was centrifuged at 800g for 15 minutes, the supernatant was
discarded, and the cell pellet was suspended in HEPES-Tyrode buffer
containing 1.75 mM probenecid to obtain a hematocrit value of 0.42 to
0.45. If needed, a suspension of erythrocytes in HEPES-Tyrode buffer
containing 1.75 mM probenecid and with a hematocrit level of 0.42 to
0.45 was added to obtain a platelet count between 5000 and 20 000
µ/L. Divalent cations (2 mM Ca++ and 1 or 2 mM magnesium
[Mg++]) were added before perfusion. In some experiments,
autologous plasma containing 400 U/mL hirudin and 1.75 mM probenecid
was used instead of HEPES-Tyrode buffer to prepare the final cell suspension. ADP plus epinephrine and collagen (Chrono-Log, Havertown, PA) induced normal aggregation of platelets loaded with Fluo
3-AM. The mean percentage (± 95% confidence interval
[CI]) of platelets with surface expression of P-selectin, a marker of
activation, was 4.84% ± 0.86% before and 6.66% ± 1.22% after
labeling; the difference was not statistically significant.
Perfusion experiments
Human plasma VWF and a recombinant VWF fragment containing the
A1 domain (residues 445 to 733 of the mature protein) were prepared as
described previously.7,17 The 2 proteins were diluted in
phosphate-buffered saline (20 mM dibasic sodium phosphate, 20 mM
NaH2PO4, 2.7 mM KCl, and 0.15 M NaCl [pH
7.4]) to a final concentration of 100 µg/mL, and 100 µL of the
solution was used to coat glass coverslips for 60 minutes at 22 to
25°C.5 These were then washed with the coating buffer
and kept in a moist environment until assembled in a modified Hele-Shaw
flow chamber.4,5 The chamber was positioned in an inverted
microscope equipped with epifluorescent illumination (Diaphot-TMD;
Nikon Instech, Kanagawa, Japan), an intensified charge-coupled digital
video camera (C-2400-87; Hamamatsu Photonics, Shizuoka, Japan), and appropriate filters. The total area of an optical field corresponded to
approximately 0.007 mm.2 Blood cells were aspirated
through the chamber with a syringe pump (Harvard Apparatus, Boston,
MA), at flow rates calculated to obtained wall-shear rates between 500 and 20 000 seconds 1. When indicated, various substances
were added to the blood cell suspension before perfusion. These
included prostaglandin E1 (PGE1), EGTA (ethyleneglycoltetraacetic
acid), dibutyryl cyclic adenosine monophosphate (cAMP), 8Br-cyclic
guanosine monophosphate (cGMP), theophylline, wortmannin (all from
Sigma), sodium nitroprusside (Merck, Sharp and Dome, Whitehouse
Station, NJ), and sildenafil (Pfizer, New York, NY). To prepare a
solution of sildenafil, a tablet (50 mg) was stripped of the outside
coating, crushed, and mixed with 0.5 mL water and 0.5 mL ethanol 95%;
the suspension was centrifuged at 1000g for 5 minutes; and
the clear supernatant was centrifuged again at 11 000g for
5 minutes to eliminate insoluble particles. Monoclonal IgGs, used when
indicated, were prepared and characterized as described previously;
LJ-Ib1 blocks the VWF-binding function of GPIb18 and
LJ-CP8 blocks the ligand-binding function of
IIb3.5,19 Experiments were
recorded in real time on videotape at the rate of 25 frames/second,
which resulted in a time resolution of 0.08 second. Selected sequences
were also digitized in real time by using a TARGA-2000 Plus board
(Truevision, Indianapolis, IN).
Measurement of motion and Ca++ mobilization in
platelets
Image analysis was performed either on recorded experiments or
online with custom-made software (Casti Imaging, Venice, Italy). The
program tracked the area of single platelets and determined the
position of the corresponding centroid on all the frames collected at a
sampling rate of 25/second, then calculated instant velocity and
variations of light intensity measured as the total of all the pixels
in a platelet. Thus, information on the measured variables was obtained
every 0.04 second. Instantaneous velocity was calculated according to
the general equation v = s/t, where s is the distance separating the
centroid of a platelet in 2 successive frames, and t is the time
interval between the 2 frames (0.04 second). To minimize the effects of
variations in centroid position resulting from changes in platelet
shape or orientation, the instantaneous velocity at time x
(tx) was obtained by applying a smoothing algorithm according to the following equation:
where Vx is the instantaneous velocity at time
tx (calculated from the change in position between frames
x 1 and x), vx1 is the instantaneous velocity at time
tx1 (calculated from the change in position between
frames x2 and x1), and vx+1 is the instantaneous
velocity at time tx+1 (calculated from the change in
position between frames x and x+1). When the instantaneous velocity was
less than 0.5 mm/second, the platelet was considered to be transiently
arrested (zero velocity), provided that any changes in centroid
position between frames x2 and x+1 occurred in aberrant directions
(as determined by a test of biunivocal correspondence) and the distance
between the initial and final position was less than a platelet
diameter. Arrest times were calculated as the sum of all frames in
which a platelet had zero velocity. Stable adhesion was defined as zero
velocity for 30 seconds or more. Thus, in this case, the surface
imprint of a platelet in the first frame of the observation period
overlapped at least partly with the imprints in all subsequent 749 frames.
The variations in intensity of the Fluo-3 AM fluorescence
were converted into [Ca++]i by
using the following equation:
[Ca++]i =Kd F Fmin/Fmax F, where Kd is the
dissociation constant of Fluo-3 AM for the interaction with
Ca++, corresponding to 864 nM at 37°C16; F
is the measured fluorescence intensity of a single platelet; Fmax is the fluorescence intensity of a single platelet
treated with the Ca++ ionophore A23187 (10 µM; Sigma) in
the presence of 2 mM calcium chloride; and Fmin is the
fluorescence intensity of an unstimulated single platelet. The baseline
[Ca++]i of the resting state was calculated
in single platelets that translocated on the VWF surface without
showing changes in fluorescence for at least 10 consecutive frames
(that is, the fluorescence intensity in each frame was within 15% of
the value in the first frame and < 200 nM). The baseline
[Ca++]i was also determined in platelets
treated with PGE1 and sodium nitroprusside, which did not undergo any
change in cytosolic Ca++ on interacting with
immobilized VWF (Figure 5). The mean (± SD) baseline value was
calculated for each platelet examined in detail. A platelet was
considered activated when all of the following conditions were met: a
change of [Ca++]i was more than 3 SDs above
the resting state value in at least 3 consecutive frames, and at least
200 nM, and the [Ca++]i oscillation showed an
identifiable peak and returned to baseline in a discrete period
of time.
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Results |
Two types of [Ca++]i elevations occur in
platelets interacting with immobilized VWF under flow
Platelets loaded with Fluo-3 AM and suspended with red
cells in plasma were perfused over immobilized VWF at different
wall-shear rates. Use of a low platelet count
(2 × 107/mL) reduced the number of interactions on the
surface and facilitated the analysis of single-cell events while still
permitting the formation of small thrombi. We found that all aggregates
formed around single platelets that displayed transient
[Ca++]i elevations, first during
translocation and then after stationary adhesion to immobilized VWF
(Figure 1). We identified 2 types of
peaks that differed in the [Ca++]i level
reached, the duration of the elevation, and the relation to motion on
the surface. One type, designated /, appeared while platelets
were translocating on the VWF surface and was characterized by a rapid
increase to concentrations as high as 1 to 2 µM (Figure 1). This peak
was arbitrarily designated when the
[Ca++]i level was above 0.4 µM and when
it was lower. In experiments performed with a wall-shear rate of 3000 seconds1, approximately 20% of the translocating
platelets showed at least one / Ca++
peak, and 9% established stationary adhesion within the limited observation period of 30 seconds (Table
1). Approximately 30% of the firmly
adherent platelets had a distinct type of Ca++ elevation,
designated , which reached levels higher than 2 to 3 µM in most
cases, had a total duration of several seconds, and showed a pulsing
behavior (Figure 1 and Table 1). After a type Ca++
increase, platelets were likely to promote the arrest of additional platelets that translocated in their vicinity, and these in turn showed
pronounced cytosolic Ca++ elevations and started to form
aggregates (Figure 1). Once established, these aggregates could grow
quickly, displaying periodical and synchronous
[Ca++]i pulses (Figure 1).
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| Figure 1.
Real-time analysis of
[Ca++]i during platelet translocation and
aggregate formation on immobilized VWF.
Platelets loaded with Fluo-3 AM (2 × 107/mL)
were suspended with washed erythrocytes in homologous plasma and
perfused over immobilized VWF for 3 minutes at a shear rate of 1500 seconds1. Panel A shows an example of aggregate
formation. At 0 second, platelet 1 appears in the optical field; by 10 seconds, it has moved in the direction of flow by approximately 20 µm; at 20 seconds, it has moved by an additional few millimeters; and
at 30 seconds, it is in the same position and 2 new platelets (2 and 3)
are attached in close proximity, forming a small aggregate. Panel B
shows [Ca++]i and instant velocity of
platelets 1, 2, and 3. The translocation of platelet 1 occurs mostly
during a few seconds of relatively rapid movement, coincident with the
appearance of transient [Ca++]i peaks
(/); a higher and longer lasting increase in
[Ca++]i () develops while the platelet is
stationary. Cytosolic Ca++ oscillations also appear when
platelets 2 and 3 arrest on the surface, without a clear sequence from
/ to . Panel C, captured between 60 and 63 seconds after the
appearance of platelet 1 in the field, shows the long-lasting
synchronous increase of [Ca++]i in platelets
forming a large aggregate. The 3-dimensional diagrams below each image
show the measurement of [Ca++]i in all
platelets in the field.
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Table 1.
Selected variables characteristic of platelet interaction
with immobilized VWF and Ca++ signaling under different
conditions
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To evaluate more extensively the signals elicited during initiation of
platelet adhesion to VWF, we performed experiments with platelets
suspended in buffer at a count of 5 × 106/mL to minimize
aggregate formation. On perfusion over immobilized VWF at a wall-shear
rate of 3000 seconds1, surface-interacting platelets
showed all 3 types of cytosolic Ca++ peaks, with typical
magnitude and duration (Figure 2, left
panel). Addition to the cell suspension of a function-blocking
anti-IIb3 monoclonal antibody had no
effect on the appearance of / peaks but obliterated peaks
(Figure 2, middle panel; and Table 1). Moreover, when A1VWF was used as
the immobilized substrate instead of native multimeric VWF, only
/ peaks occurred, even when platelets were perfused without
IIb3 inhibition (Figure 2, right panel). Platelets interacting with A1VWF, which lacks the RGD sequence, and
those with blocked IIb3 interacting with
native VWF could roll but could neither adhere irreversibly nor
aggregate; the absence of prolonged arrest of motion was reflected in
an increase in the average translocation velocity (Table 1).
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| Figure 2.
Distinct [Ca++]i elevations
mediated by GPIb or IIb3 in platelets
interacting with immobilized VWF.
Platelets loaded with Fluo-3 AM were suspended at a count
of 5 × 106/mL with homologous washed red cells in a
buffer containing 2 mM Ca++ and 1 mM Mg++ (the
same results were obtained with 2 mM Mg++). The suspension
was perfused over immobilized multimeric VWF or A1VWF at the indicated
shear rates for 90 seconds, after which the
[Ca++]i of all surface-interacting platelets
was monitored in real time for the next 30 seconds during translocation
or stationary adhesion. In some experiments, the
anti-IIb3 monoclonal antibody LJ-CP8 was
added at the final concentration of 100 µg/mL. Experiments with A1VWF
were performed at the shear rate of 2000 seconds1 to
obtain comparable numbers of surface-tethered platelets under all
conditions. Arrows indicate typical Ca++ peaks, which
were present only in untreated platelets interacting with VWF.
Comparable results were obtained in 4 different experiments.
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Ca++ signals induced by GPIb binding to VWF
differentially depend on release from intracellular stores or
transmembrane ion flux and are initiated under shear stress
Platelets translocating on VWF in the presence of the
extracellular Ca++ chelator EGTA (2 or 10 mM) showed only
/ peaks (no peaks; Figure 3A).
In contrast, when cytoplasmic Ca++ was chelated with
BAPTA-AM, all Ca++ oscillations in
translocating platelets were obliterated (Figure 3A). These results
indicate that / peaks involve release from intracellular stores,
whereas peaks depend on a transmembrane ion flux. The latter idea
was also supported by the demonstration that platelets resuspended in
the absence of added extracellular Ca++ and with 2 mM
Mg++ did not show peaks but could still establish
irreversible adhesion, thereby indicating that
IIb3 function was preserved under these conditions (not shown). The number of platelets tethered to VWF was
influenced by hydrodynamic flow conditions and reached a plateau at
3000 to 6000 seconds1 wall-shear rate. The interaction
was approximately 50% lower at 20 000 seconds1 and 80%
lower at 500 seconds1 (Figure 3B). Type / cytosolic
Ca++ oscillations occurred in an increasing proportion of
translocating platelets as the shear rate increased, reaching a maximum
at 6000 seconds1 that was unchanged up to 20 000
seconds1. In contrast, less than 2% of the
surface-translocating platelets showed cytosolic Ca++
oscillations at the shear rate of 500 seconds1 (Figure
3B). The peak [Ca++]i of a type elevation
was also directly related to the shear rate, reaching an average value
of 1.8 µM at 6000 seconds1 (Figure 3C).
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| Figure 3.
Effect of chelating agents on
[Ca++]i elevations dependent on the
interaction between GPIb and A1VWF.
A blood cell suspension prepared as described in the legend for Figure
2 was perfused over immobilized VWF at the indicated shear rates for 90 seconds. Interacting platelets were identified and analyzed as
described in the legend for Figure 2. (A) After addition of the
membrane-impermeable Ca++ chelator EGTA (2 mM), no type [Ca++]i oscillations (Figure 1) occurred, but
/ peaks were unchanged. In contrast, after addition of
membrane-permeable BAPTA-AM, all
[Ca++]i oscillations were obliterated. The
same results were obtained in 3 different experiments. (B) After
addition of EGTA (2 mM) and the anti-IIb3
monoclonal antibody LJ-CP8 (100 µg/mL), all the platelets interacting
with the surface in a 30-second period after an initial 90-second
perfusion were enumerated ( ); the proportion of these showing
/ [Ca++]i elevations was calculated
( ). There were no peaks under these conditions. Results are the
mean ± 95% CI from 3 separate experiments performed at the
indicated shear rates between 500 and 20 000 seconds1.
(C) The peak [Ca++]i of type oscillations
was measured as a function of the shear rate during perfusion. Each
point shows the mean ± 95% CI of at least 10 peaks and is
representative of the results obtained in 3 different experiments using
the same conditions described for panel B.
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Measurements of the instantaneous velocity of individual platelets
revealed a stop-and-go motion, with alternating rapid deceleration and
acceleration (Figure 4A). The peak of all
type [Ca++]i elevations (by
definition > 0.4 µM) was coincident with a temporary arrest
that occurred, on average, 2.06 seconds (range, 0.2 to 8.12 seconds)
after the preceding velocity peak and 0.61 seconds (range, 0.24 to 1.12 seconds) before the subsequent one (n = 30; Figure 4A). Therefore,
the arrest during which a platelet could generate a signal through
GPIb had a variable duration, but the resumption of motion occurred
after a consistent time interval. Of note, the relation between type
[Ca++]i elevations and platelet motion
was similar regardless of whether IIb3
was functionally blocked (Figure 4A) or not. The type / pattern
of cytosolic Ca++ elevations was distinctly different from
type , which developed during several seconds only in a motionless
platelet that had already established irreversible adhesion to VWF
(Figure 4B). The observation of Ca++ elevations in relation
to platelet motion allowed a reproducible discrimination between type
/ and peaks by different observers. Moreover, the shape of
all type peaks (by definition > 0.4 µM) was represented
with good statistical parameters (R2 = 0.71; coefficient
of variation, < 6%) by a mathematical formula that yielded the value
of 1.5 second for the typical duration of the oscillation, with a lag
time of 0.2 second from beginning to maximum value of the
[Ca++]i increase.
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| Figure 4.
Relation between instantaneous velocity and
[Ca++]i elevations during platelet
interaction with immobilized VWF.
(A) A blood cell suspension was prepared as described in the legend for
Figure 2. After addition of the anti-IIb3
antibody LJ-CP8 (100 µg/mL) and 2 mM EGTA to chelate extracellular
Ca++, the suspension was perfused over immobilized VWF at a
shear rate of 3000 seconds1 for 90 seconds. Interacting
platelets were analyzed during the successive 30 seconds. Panel Ai
shows movement and fluorescence changes of a representative platelet
interacting with the surface during the indicated time interval. The
diagrams on the right depict [Ca++]i changes
(Aii) and instantaneous velocity (Aiii) of the same platelet observed
for 7 seconds. Vertical arrows identify the peak of a type /
Ca++ elevation (Ca) as well as the preceding
(Vb) and following (Va) instantaneous velocity
peaks. The mean ± 95% CI of the time intervals between these
events, calculated for 15 separate measurements, is shown. In panel
Aiii, the horizontal arrow indicates the time interval corresponding to
the images in panel Ai. (B) The blood cell suspension contained 2 mM
Ca++ and 1 mM Mg++, but no
anti-IIb3 antibody and no
EGTA. The results, presented as in panel A, show a type Ca++ oscillation in a motionless platelet analyzed for 7 seconds. Similar results were observed in more than 100 separate
experiments.
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Stimulation through GPIb leads to sequential stages of
IIb3 activation that distinctly mediate
stable platelet adhesion and aggregation
When blood cells were perfused over immobilized VWF with a
wall-shear rate of 3000 seconds1, apyrase, which contains
an adenosine diphosphatase activity, and wortmannin, at concentrations
that inhibit PI3-K, had no effect on the frequency and amplitude of
type / [Ca++]i elevations in platelets
but completely prevented the appearance of type peaks (Figure
5). Apyrase and wortmannin did not
significantly affect the average translocation velocity, and a normal
proportion of platelets achieved stationary adhesion, but no platelet
aggregation was observed on the surface (Table 1). In previous studies,
we showed that PGE1, which increases cAMP
levels,20 interferes with
IIb3 activation and blocks the
irreversible adhesion of flowing platelets interacting with immobilized
VWF, as well as thrombus formation.5 Sodium nitroprusside,
which acts as a caged nitric oxide and activates cGMP-dependent protein
kinases,20 has the same effect (data not shown). Both
inhibitors, as exemplified here by sodium nitroprusside (Figure 5),
completely prevented both / and Ca++ oscillations
in platelets interacting with immobilized VWF under shear stress. In
this case, the average translocation velocity was increased and
platelets could not establish irreversible adhesion (Table 1). Two
distinct phosphodiesterase (PDE) inhibitors, the nonspecific
theophylline and the PDE5-specific sildenafil,21 also
inhibited in a dose-dependent manner all cytoplasmic Ca++
elevations induced by platelet interaction with VWF, as did the cAMP
analog dibutyryl-cAMP, and the cGMP analog 8Br-cGMP (concentration that
inhibits 50%, 21.7 µM and 1.7 µM, respectively; Figure
6). These results show that the
GPIb-mediated pathway of Ca++ release from cytoplasmic
stores is susceptible to regulation by cAMP and cGMP levels.
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| Figure 5.
Distinct inhibition of [Ca++]i
elevations in platelets interacting with immobilized VWF under flow.
A blood cell suspension prepared as described in the legend for Figure
2 was perfused for 90 seconds over immobilized VWF at a shear rate of
3000 seconds1. The diagrams show the
[Ca++]i changes in all surface-interacting
platelets during translocation or stationary adhesion for 30 seconds.
Noteworthy are the presence of / peaks and peaks (arrows) in
the control experiment, the selective absence of peaks in the
presence of wortmannin (100 nM) or apyrase (10 U/mL), and the absence
of any Ca++ elevation in the presence of sodium
nitroprusside (5 µM). Similar results were obtained in 4 different
experiments.
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| Figure 6.
Effect of intracellular cAMP and cGMP modulation
on GPIb-mediated [Ca++]i elevations in
platelets interacting with immobilized VWF.
A blood cell suspension (see the legend for Figure 2) was supplemented
with the anti-IIb3 antibody LJ-CP8 (100 µg/mL) and 2 mM EGTA to block [Ca++]i
elevations not mediated by GPIb. Perfusion and analysis were
performed as described in the legend for Figure 5. Before perfusion,
samples were incubated at 37°C with either theophylline for 15 minutes (A), sildenafil for 20 minutes (B), or dibutyryl cAMP or
8Br-cGMP for 10 minutes (C). Theophylline was used at a high
concentration to offset adsorption by erythrocytes.22 The
number of platelets with at least one [Ca++]i
elevation of type / (no peaks were observed in these
experiments) was measured, and results are shown normalized to the
values observed with control blood cell suspensions left untreated. The
data represent the mean ± 95% CI from 5 different
experiments.
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| |
Discussion |
In this study, we showed that the interaction of platelet GPIb
with VWF leads to 2 distinct types of [Ca++]i
elevations linked to sequential stages of integrin
IIb3 activation (Figure
7). The first Ca++ peaks in
the temporal sequence appear to have been initiated by mechanical
stimulation, since their frequency increased as a function of shear
stress above 2 Pa (the value in blood flowing with a wall-shear rate of
500 seconds1). Within this group, the distinction between
type and peaks was based solely on intensity and the fact that
peaks, which by definition reach a
[Ca++]i level above 0.4 µM, have a
reproducible shape that facilitates the analysis of their relation to
platelet motion. Indeed, type peaks a reached maximum level while
platelets were transiently arrested but at a predictably short time
before detachment from the surface, when the tensile stress on the
GPIb-VWF bonds may be greatest. These findings support the concept
that GPIb has a mechanoreceptor function, although the proximal
events responsible for transducing force into a biochemical signal
remain unknown. Linkage of the GPIb cytoplasmic tail to the membrane
skeleton through filamin-a24,25 and to the  isoform of 14.3.3,26 a regulatory molecule in cellular
signaling,27 may be relevant in this regard. Neither
association is needed for the GPIb-dependent induction of
IIb3 activation in heterologous
cells,28 but a role in flowing platelets cannot be
excluded. These uncertainties notwithstanding, it is clear that type
/ peaks are the consequence of rapid Ca++ release
from intracellular stores. Such cytoplasmic Ca++ elevations
are likely mediated by inositol-1,4,5-trisphosphate29 generated with diacylglycerol through the action of
phosphatidylinositol-specific phospholipase C (Figure 7); a similar
response has also been observed in endothelial cells30,31
and osteoblasts32 subjected to shear stress.
View larger version (49K):
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| Figure 7.
Schematic representation of the mechanisms of platelet activation by
immobilized VWF under shear stress.
A1 and A3 are the VWF domains that interact with platelet GPIb and
fibrillar collagen, respectively. The A1 domain also interacts with the
nonfibrillar collagen type VI.23 The sequence RGD in the
VWF C1 domain interacts with activated
IIb3. PIP2 indicates
phosphatidyl-inositol-4,5-bisphosphate; IP3,
inositol-1,4,5-trisphosphate; DG, diacylglycerol; PKC, protein
kinase C; and PI3-K = phosphatidylinositol 3-kinase. ADP is
shown interacting with two 7-transmembrane-domain, G-protein-coupled
receptors: P2Y1, linked to Ca++ release from internal
stores, and P2Y12, linked to the regulation of adenylyl cyclase
activity. The position of / and [Ca++]i peaks relative to GPIb-IX-V
engagement by A1VWF and signal amplification by ADP and PI3-K,
respectively, differs from the previous interpretation by Yap et
al.15 Also, an increase in cAMP and cGMP levels blocked
the first Ca++ response linked to GPIb-IX-V stimulation
(that is, / peaks) and consequently other downstream activation
events.
|
|
The first level of IIb3 activation
induced by platelet interaction with VWF under shear stress leads from
transient to stable adhesion and is regulated by the cellular levels of
cAMP and cGMP that control type / Ca++ signals.
However, thrombus formation cannot progress at this stage, possibly
because IIb3 molecules are activated only
in the vicinity of stimulated GPIb and they can bind immobilized VWF
but not soluble VWF and fibrinogen as required for
aggregation.4,6 A second level of
IIb3 activation must be reached for
aggregation to occur, and this appears to require signal
amplification associated with type Ca++ elevations
induced by ADP released in response to the initial GPIb stimulation
(Figure 7). Release of ADP in the microenvironment of firmly adherent
platelets may also explain why other platelets that transiently arrest
in their vicinity become rapidly activated (Figure 1). These
conclusions are in agreement with the idea that secreted ADP is
necessary for shear-induced platelet aggregation initiated by soluble
VWF binding to GPIb33 and that inhibition of the P2Y1
and P2Y12 ADP receptors reduces platelet aggregation after adhesion to
collagen-bound VWF.34 In other experiments, we found that
concurrent blockage of P2Y1 and P2Y12 has the same effect as apyrase in
preventing type [Ca++]i elevations and
platelet aggregation on immobilized VWF (data not shown). Progression
to the second stage of IIb3 activation involves one or more signaling molecules inhibited by wortmannin, possibly including PI3-K. Activation of PI3-K may be
linked directly to the VWF-GPIb interaction35 or follow
further cytoplasmic Ca++ mobilization induced by the P2Y1
receptor.36 Both ADP stimulation and activation of a
wortmannin-sensitive pathway are needed for the appearance of a second
Ca++ signal, a type peak, which precedes the onset of
platelet aggregation and is prevented by monoclonal antibodies that
block ligand binding to activated
IIb3.11,13 The effect of
IIb3 blockage on the appearance of peaks may be explained by the fact that only firmly adherent
platelets can be activated locally by ADP, or it may indicate a
previously suggested role of IIb3 in
mediating Ca++ transport across the platelet
membrane.37 The characteristics of type [Ca++]i elevations are compatible with a
mechanism of store-mediated Ca++ entry,38
which is known to be inhibited by wortmannin.39
Results similar to ours were reported previously,13,15 but
some differences are notable both with respect to experimental findings
and interpretation of the mechanisms that mediate stable platelet
adhesion and aggregation on a VWF surface. A distinctive aspect of our
studies was the discrimination between type / and Ca++ oscillations, which was supported by experiments
performed with a recombinant VWF A1 domain fragment and may be
explained by the methods used. Measurements performed every 0.04 second
provide a sufficient time resolution to identify the rapid
Ca++ peaks related to GPIb function that, as type oscillations, have a 0.2-second average interval between onset and peak
Ca++ concentration. Monitoring of calcium dynamics in
individual platelets every 0.586 second, as was done
previously,15 may have concealed the occurrence of type
/ Ca++ oscillations, even though they appear in 6 times as many platelets as type peaks (Table 1). The biased
overestimation of type peaks may explain the conclusion that a flux
of extracellular Ca++ contributes to all signals linked to
the interaction of platelets with immobilized VWF.12 In
fact, as shown here, type / Ca++ oscillations
directly related to the engagement of GPIb by surface-bound VWF
under shear stress are insensitive to extracellular Ca++ concentration.
The discrimination between type / and Ca++ peaks
may also explain a different interpretation of the proposed role of
PI3-K in shear-dependent signaling between GPIb and
IIb3.15 We found that
wortmannin had no influence on the frequency and peak concentration of
the EGTA-insensitive and GPIb-dependent type /
Ca++ oscillations but that it obliterated the
IIb3-dependent type Ca++ peaks also abolished by EGTA (Table 1). Thus,
PI3-K, possibly with other signaling molecules inhibited by
wortmannin, appears to have a role in shear-induced platelet activation
by contributing to the ADP-dependent amplification phase required for
aggregation but not to the initial response directly linked to GPIb
(Figure 7). Our results did not confirm that stationary platelet
adhesion to VWF is inhibited by wortmannin15 and occurs
only after IIb3-dependent sustained
Ca++ oscillations.13 To the contrary, we found
that stable adhesion is a prerequisite for, rather than a consequence
of such oscillations. Thus, although our data do support the conclusion
that Ca++ oscillations linked to
IIb3 function (type peaks) are
influenced by wortmannin, our findings indicate that the function of
PI3-K, possibly dependent on signaling through ADP receptors, is
required for platelet aggregation but not irreversible adhesion to VWF. In fact, removal of ADP or treatment with wortmannin obliterated the
IIb3-dependent and sustained type Ca++ peaks without reducing the frequency of stable
platelet adhesion to VWF and had only a small effect on the average
translocation velocity of the entire platelet population (Table 1). It
should be noted that our definition of stable adhesion was stringent, requiring that a platelet remain in the same position for 30 seconds or longer.
Our results indicate that the contribution of initial
IIb3 activation to the dynamic aspects of
platelet interaction with immobilized VWF is more limited than
suggested by others.13 In fact, inhibition of / and
subsequent peaks by chelating cytoplasmic Ca++ or
raising cyclic AMP or cGMP levels had the same effect on platelet translocation velocity as blocking IIb3,
but the blocking did not affect / Ca++ peaks
(Table 1). Thus, transient arrest times are the same regardless of
whether platelets have no intracytoplasmic Ca++
oscillations or have a normal frequency of type / peaks but IIb3 is inhibited (Table 1). Such
findings indicate that the interaction of GPIb with immobilized VWF
is not directly influenced by cytoplasmic Ca++ oscillations
and is solely responsible for initiating transient platelet-arrest
periods that can last several seconds, even under high shear stress.
Subsequent activation of IIb3, linked to the occurrence of / Ca++ peaks, undoubtedly permits
prolongation of these contacts and establishment of stationary
adhesion, in accordance with a previously suggested
mechanism.5 It is worth noting that the average platelet translocation velocities of 30 to 60 µm/second1
measured by Nesbitt et al13 after functional blockage of
IIb3 or chelation of intracellular
Ca++ were greatly in excess of the values measured in these
(Table 1) or earlier5 studies under comparable flow
conditions and VWF concentration on the surface. The discrepancy,
barring differences in the qualitative multimeric composition of the
VWF used, may be explained by the use of a low time resolution for
motion analysis (0.576 second, that is, < 2 frames/second), which may
have affected the individual tracking of multiple and identical objects
moving rapidly on the surface.
In conclusion, our results support the definition of a mechanism that
links shear-induced stimulation of GPIb to 2 sequential and distinct
stages of IIb3 activation characterized
by specific cytosolic Ca++ elevations (Figure 7). These
findings provide the basis for a detailed definition of the signaling
pathways initiated by the VWF-GPIb interaction that may regulate
platelet participation in hemostasis and thrombosis. The functional
importance of these signals in relation to those generated by other
thrombogenic substrates, such as collagen, remains to be established.
In this regard, the nature of the vascular lesion evoking a platelet
response may be important in determining what pathway of platelet
activation will be followed. For example, injured endothelial cells
release VWF that, while bound to their surface, may initiate platelet adhesion and activation in the absence of subendothelial
denudation.40 Clarification of this issue is one of the
goals of future studies.
| |
Acknowledgments |
We thank Brian Savage and Marco Cattaneo for stimulating discussion
on many aspects of platelet physiology; Jerry Ware, James R. Roberts,
and Richard A. McClintock for contributing to the preparation of
recombinant A1VWF; and Rachel Braithwaite and Eileen Bristow for
excellent secretarial assistance.
| |
Footnotes |
Submitted February 19, 2002; accepted May 21, 2002.
Prepublished online
as Blood First Edition Paper, June 21, 2002; DOI
10.1182/blood-2002-02-0514.
Supported by grants from the Italian Ministry of Health
(ICS-060.2/RF99.74), the European Space Agency (AO-LS-99-MAP-MED-007), the Agenzia Spaziale Italiana (all to L.D.M.); grants HL-42846, HL-31950, and HL-48728 from the National Institutes of Health (NIH;
Z.M.R.); NIH grant RR0833 to the General Clinical Research Center of
Scripps Clinic and Research Foundation; and the Stein Endowment Fund.
L.D.M. and Z.M.R. are cosenior authors.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Zaverio M. Ruggeri, MEM 175, Scripps
Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037;
e-mail: ruggeri{at}scripps.edu.
| |
References |
1.
Ruggeri ZM, Saldivar E.
Platelets, hemostasis and thrombosis. In:
Ruggeri ZM, ed.
Von Willebrand Factor and the Mechanisms of Platelet Function. Berlin, Germany: Springer; 1998:1-32.
2.
Fuster V, Badimon L, Badimon JJ, Chesebro JH.
The pathogenesis of coronary artery disease and the acute coronary syndromes (1).
N Engl J Med.
1992;326:242-250[Medline]
[Order article via Infotrieve].
3.
Fuster V, Badimon L, Badimon JJ, Chesebro JH.
The pathogenesis of coronary artery disease and the acute coronary syndromes (2).
N Engl J Med.
1992;326:310-318[Medline]
[Order article via Infotrieve].
4.
Savage B, Almus-Jacobs F, Ruggeri ZM.
Specific synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow.
Cell.
1998;94:657-666[CrossRef][Medline]
[Order article via Infotrieve].
5.
Savage B, Saldivar E, Ruggeri ZM.
Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor.
Cell.
1996;84:289-297[CrossRef][Medline]
[Order article via Infotrieve].
6.
Ruggeri ZM, Dent JA, Saldivar E.
Contribution of distinct adhesive interactions to platelet aggregation in flowing blood.
Blood.
1999;94:172-178[Abstract/Free Full Text].
7.
De Marco L, Girolami A, Zimmerman TS, Ruggeri ZM.
Interaction of purified IIB von Willebrand factor with the platelet membrane glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and initiates aggregation.
Proc Natl Acad Sci U S A.
1985;82:7424-7428[Abstract/Free Full Text].
8.
De Marco L, Mazzucato M, Del Ben MG, et al.
Type IIB von Willebrand factor with normal sialic acid content induces platelet aggregation in the absence of ristocetin: role of platelet activation, fibrinogen, and two distinct membrane receptors.
J Clin Invest.
1987;80:475-482[Medline]
[Order article via Infotrieve].
9.
Chow TW, Hellums JD, Moake JL, Kroll MH.
Shear stress-induced von Willebrand factor binding to platelet glycoprotein Ib initiates calcium influx associated with aggregation.
Blood.
1992;80:113-120[Abstract/Free Full Text].
10.
Ikeda Y, Handa M, Kamata T, et al.
Transmembrane calcium influx associated with von Willebrand factor binding to GP Ib in the initiation of shear-induced platelet aggregation.
Thromb Haemost.
1993;69:496-502[Medline]
[Order article via Infotrieve].
11.
Kuwahara M, Sugimoto M, Tsuji S, Miyata S, Yoshioka A.
Cytosolic calcium changes in a process of platelet adhesion and cohesion on a von Willebrand factor-coated surface under flow conditions.
Blood.
1999;94:1149-1155[Abstract/Free Full Text].
12.
Yap CL, Hughan SC, Cranmer SL, et al.
Synergistic adhesive interactions and signaling mechanisms operating between platelet glycoprotein Ib/IX and integrin IIb3: studies in human platelets and transfected Chinese hamster ovary cells.
J Biol Chem.
2000;275:41377-41388[Abstract/Free Full Text].
13.
Nesbitt WS, Kulkarni S, Giuliano S, et al.
Distinct glycoprotein Ib/V/IX and integrin IIb3-dependent calcium signals cooperatively regulate platelet adhesion under flow.
J Biol Chem.
2002;277:2965-2972[Abstract/Free Full Text].
14.
Lawrence MB, Springer TA.
Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins.
Cell.
1991;65:859-873[CrossRef][Medline]
[Order article via Infotrieve].
15.
Yap CL, Anderson KE, Hughan SC, Dopheide SM, Salem HH, Jackson SP.
Essential role for phosphoinositide 3-kinase in shear-dependent signaling between platelet glycoprotein Ib/V/IX and integrin IIb3.
Blood.
2002;99:151-158[Abstract/Free Full Text].
16.
Merritt JE, McCarthy SA, Davies MP, Moores KE.
Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils: loading cells with the dye, calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2+.
Biochem J.
1990;269:513-519[Medline]
[Order article via Infotrieve].
17.
Miyata S, Ruggeri ZM.
Distinct structural attributes regulating von Willebrand factor A1 domain interaction with platelet glycoprotein Ib under flow.
J Biol Chem.
1999;274:6586-6593[Abstract/Free Full Text].
18.
Handa M, Titani K, Holland LZ, Roberts JR, Ruggeri ZM.
The von Willebrand factor-binding domain of platelet membrane glycoprotein Ib: characterization by monoclonal antibodies and partial amino acid sequence analysis of proteolytic fragments.
J Biol Chem.
1986;261:12579-12585[Abstract/Free Full Text].
19.
Niiya K, Hodson E, Bader R, et al.
Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation: relationship to the binding of fibrinogen and platelet aggregation.
Blood.
1987;70:475-483[Abstract/Free Full Text].
20.
Haslam RJ, Dickinson NT, Jang EK.
Cyclic nucleotides and phosphodiesterases in platelets.
Thromb Haemost.
1999;82:412-423[Medline]
[Order article via Infotrieve].
21.
Perry MJ, Higgs GA.
Chemotherapeutic potential of phosphodiesterase inhibitors.
Curr Opin Chem Biol.
1998;2:472-481[CrossRef][Medline]
[Order article via Infotrieve].
22.
Philip-Joet F, Bruguerolle B, Reynaud M, Arnaud A.
Correlation between theophylline concentration in plasma, erythrocytes and cantharides induces blister fluid and peak expiratory flow in asthma patients.
Eur J Clin Pharmacol.
1992;43:563-565[CrossRef][Medline]
[Order article via Infotrieve].
23.
Mazzucato M, Spessotto P, Masotti A, et al.
Identification of domains responsible for von Willebrand factor type VI collagen interaction mediating platelet adhesion under high flow.
J Biol Chem.
1999;274:3033-3041[Abstract/Free Full Text].
24.
Andrews RK, Fox JEB.
Identification of a region in the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX complex that binds to purified actin-binding protein.
J Biol Chem.
1992;267:18605-18611[Abstract/Free Full Text].
25.
Cunningham JG, Meyer SC, Fox JEB.
The cytoplasmic domain of the -subunit of glycoprotein (GP) Ib mediates attachment of the entire GP Ib-IX complex to the cytoskeleton and regulates von Willebrand factor-induced changes in cell morphology.
J Biol Chem.
1996;271:11581-11587[Abstract/Free Full Text].
26.
Du X, Fox JE, Pei S.
Identification of a binding sequence for the 14-3-3 protein within the cytoplasmic domain of the adhesion receptor, platelet glycoprotein Ib.
J Biol Chem.
1996;271:7362-7367[Abstract/Free Full Text].
27.
Fu H, Subramanian RR, Masters SC.
14-3-3 proteins: structure, function, and regulation.
Annu Rev Pharmacol Toxicol.
2000;40:617-647[CrossRef][Medline]
[Order article via Infotrieve].
28.
Zaffran Y, Meyer SC, Negrescu E, Reddy KB, Fox JE.
Signaling across the platelet adhesion receptor glycoprotein Ib-IX induces IIb3 activation both in platelets and a transfected Chinese hamster ovary cell system.
J Biol Chem.
2000;275:16779-16787[Abstract/Free Full Text].
29.
Berridge MJ.
Inositol triphosphate and calcium signalling.
Nature.
1993;361:315-325[CrossRef][Medline]
[Order article via Infotrieve].
30.
Nollert MU, Eskin SG, McIntire LV.
Shear stress increases inositol trisphosphate levels in human endothelial cells.
Biochem Biophys Res Commun.
1990;170:281-287[CrossRef][Medline]
[Order article via Infotrieve].
31.
Bhagyalakshmi A, Berthiaume F, Reich KM, Frangos JA.
Fluid shear stress stimulates membrane phospholipid metabolism in cultured human endothelial cells.
J Vasc Res.
1992;29:443-449[Medline]
[Order article via Infotrieve].
32.
Chen NX, Ryder KD, Pavalko F, et al.
Ca2+ regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts.
Am J Physiol Cell Physiol.
2000;278:C989-C997[Abstract/Free Full Text].
33.
Moake JL, Turner NA, Stathopoulos NA, Nolasco L, Hellums JD.
Shear-induced platelet aggregation can be mediated by vWF released from platelets, as well as by exogenous large or unusually large vWF multimers, requires adenosine diphosphate, and is resistant to aspirin.
Blood.
1988;71:1366-1374[Abstract/Free Full Text].
34.
Turner NA, Moake JL, McIntire LV.
Blockade of adenosine diphosphate receptors P2Y12 and P2Y1 is required to inhibit platelet aggregation in whole blood under flow.
Blood.
2001;98:3340-3345[Abstract/Free Full Text].
35.
Jackson SP, Schoenwaelder SM, Yuan Y, Rabinowitz I, Salem HH, Mitchell CA.
Adhesion receptor activation of phosphatidylinositol 3-kinase: von Willebrand factor stimulates the cytoskeletal association and activation of phosphatidylinositol 3-kinase and pp60c-src in human platelets.
J Biol Chem.
1994;268:27093-27099.
36.
Gachet C.
Platelet activation by ADP: the role of ADP antagonists.
Ann Med.
2000;32(suppl 1):15-20[Medline]
[Order article via Infotrieve].
37.
Brass LF.
Ca2+ transport across the platelet plasma membrane: a role for membrane glycoproteins IIb and IIIa.
J Biol Chem.
1985;260:2231-2236[Abstract/Free Full Text].
38.
Rosado JA, Sage SO.
Phosphoinositides are required for store-mediated calcium entry in human platelets.
J Biol Chem.
2000;275:9110-9113[Abstract/Free Full Text].
39.
Jenner S, Farndale RW, Sage SO.
Wortmannin inhibits store-mediated calcium entry and protein tyrosine phosphorylation in human platelets.
FEBS Lett.
1996;381:249-251[CrossRef][Medline]
[Order article via Infotrieve].
40.
André P, Denis CV, Ware J, et al.
Platelets adhere to and translocate on von Willebrand factor presented by endothelium in stimulated veins.
Blood.
2000;96:3322-3328[Abstract/Free Full Text].
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July 15, 2004;
558(2):
403 - 415.
[Abstract]
[Full Text]
[PDF]
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W. F. Bahou, L. Scudder, D. Rubenstein, and J. Jesty
A Shear-restricted Pathway of Platelet Procoagulant Activity Is Regulated by IQGAP1
J. Biol. Chem.,
May 21, 2004;
279(21):
22571 - 22577.
[Abstract]
[Full Text]
[PDF]
|
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A. Kasirer-Friede, M. R. Cozzi, M. Mazzucato, L. De Marco, Z. M. Ruggeri, and S. J. Shattil
Signaling through GP Ib-IX-V activates {alpha}IIb{beta}3 independently of other receptors
Blood,
May 1, 2004;
103(9):
3403 - 3411.
[Abstract]
[Full Text]
[PDF]
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S. J. Marshall, Y. A. Senis, J. M. Auger, R. Feil, F. Hofmann, G. Salmon, J. T. Peterson, F. Burslem, and S. P. Watson
GPIb-dependent platelet activation is dependent on Src kinases but not MAP kinase or cGMP-dependent kinase
Blood,
April 1, 2004;
103(7):
2601 - 2609.
[Abstract]
[Full Text]
[PDF]
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D. S. Sim, G. Merrill-Skoloff, B. C. Furie, B. Furie, and R. Flaumenhaft
Initial accumulation of platelets during arterial thrombus formation in vivo is inhibited by elevation of basal cAMP levels
Blood,
March 15, 2004;
103(6):
2127 - 2134.
[Abstract]
[Full Text]
[PDF]
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P. R.-M. Siljander, I. C. A. Munnix, P. A. Smethurst, H. Deckmyn, T. Lindhout, W. H. Ouwehand, R. W. Farndale, and J. W. M. Heemskerk
Platelet receptor interplay regulates collagen-induced thrombus formation in flowing human blood
Blood,
February 15, 2004;
103(4):
1333 - 1341.
[Abstract]
[Full Text]
[PDF]
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V. Rathore, M. A. Stapleton, C. A. Hillery, R. R. Montgomery, T. C. Nichols, E. P. Merricks, D. K. Newman, and P. J. Newman
PECAM-1 negatively regulates GPIb/V/IX signaling in murine platelets
Blood,
November 15, 2003;
102(10):
3658 - 3664.
[Abstract]
[Full Text]
[PDF]
|
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P. Wonerow, A. C. Pearce, D. J. Vaux, and S. P. Watson
A Critical Role for Phospholipase C{gamma}2 in {alpha}IIb{beta}3-mediated Platelet Spreading
J. Biol. Chem.,
September 26, 2003;
278(39):
37520 - 37529.
[Abstract]
[Full Text]
[PDF]
|
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S. Feng, J. C. Resendiz, X. Lu, and M. H. Kroll
Filamin A binding to the cytoplasmic tail of glycoprotein Ib{alpha} regulates von Willebrand factor-induced platelet activation
Blood,
September 15, 2003;
102(6):
2122 - 2129.
[Abstract]
[Full Text]
[PDF]
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I. Goncalves, S. C. Hughan, S. M. Schoenwaelder, C. L. Yap, Y. Yuan, and S. P. Jackson
Integrin {alpha}IIb{beta}3-dependent Calcium Signals Regulate Platelet-Fibrinogen Interactions under Flow: INVOLVEMENT OF PHOSPHOLIPASE C{gamma}2
J. Biol. Chem.,
September 12, 2003;
278(37):
34812 - 34822.
[Abstract]
[Full Text]
[PDF]
|
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T. A. Doggett, G. Girdhar, A. Lawshe, J. L. Miller, I. J. Laurenzi, S. L. Diamond, and T. G. Diacovo
Alterations in the intrinsic properties of the GPIb{alpha}-VWF tether bond define the kinetics of the platelet-type von Willebrand disease mutation, Gly233Val
Blood,
July 1, 2003;
102(1):
152 - 160.
[Abstract]
[Full Text]
[PDF]
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H. Shankaran, P. Alexandridis, and S. Neelamegham
Aspects of hydrodynamic shear regulating shear-induced platelet activation and self-association of von Willebrand factor in suspension
Blood,
April 1, 2003;
101(7):
2637 - 2645.
[Abstract]
[Full Text]
[PDF]
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A. Navdaev and K. J. Clemetson
Glycoprotein Ib Cross-linking/Ligation on Echicetin-coated Surfaces or Echicetin-IgMkappa in Stirred Suspension Activates Platelets by Cytoskeleton Modulated Calcium Release
J. Biol. Chem.,
November 22, 2002;
277(48):
45928 - 45934.
[Abstract]
[Full Text]
[PDF]
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Top
Abstract
Introduction