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(Received for publication, September 11, 1995; and in revised form, December 21, 1995) From the
Previously, we showed that a subpopulation of the major platelet
integrin,
Integrins are a family of transmembrane glycoproteins that bind
adhesive molecules and play important roles in mediating cell-cell and
cell-matrix interactions(1) . Several integrins are unable to
bind their ligand unless the cells are
activated(2, 3, 4, 5, 6) .
One such integrin is the glycoprotein IIb-IIIa complex
( The cytoplasmic
face of the plasma membrane of platelets is coated by a membrane
skeleton that associates with the cytoplasmic domains of transmembrane
glycoproteins and with underlying cytoplasmic actin
filaments(18, 19, 20) . When platelets are
lysed with Triton X-100, the cytoplasmic actin filaments can be
sedimented by low speed centrifugation. The membrane skeleton separates
from the cytoplasmic filaments; the fragments of membrane skeleton and
associated membrane glycoproteins require higher g forces to
be sedimented(19) . Recently, we showed that some
Our
previous study (21) was based on the co-sedimentation of the
integrin with detergent-insoluble fractions. In the present study, we
provide evidence that membrane skeleton-associated
In dual-label experiments, platelets that had been activated in the
presence of PAC-1, A5G8, or fibrinogen (a kind gift of Dr. Leslie
Parise of the University of North Carolina, Chapel Hill, NC) were fixed
and permeabilized with Triton X-100. Platelets that had been activated
in the presence of PAC-1 or A5G8 were incubated for approximately 16 h
with rabbit polyclonal antibodies against talin, or
To determine whether
ligand-occupied
Figure 1:
Immunofluorescence confocal images
showing the distribution of ligand-occupied
The platelets shown in Fig. 1had been activated
with thrombin at a platelet concentration of 1
Figure 2:
Immunofluorescence images showing the
distribution of fibrinogen in activated platelets. A platelet
suspension was agitated gently with 1.0 unit/ml thrombin and 200
µg/ml fibrinogen for 5 min. Platelets were then fixed with 4%
paraformaldehyde and 0.1% glutaraldehyde in the presence of detergent.
Fibrinogen was detected with polyclonal antibodies (A) and
To
determine whether the clustering and centralization of
Figure 3:
Electron micrographs showing the selective
disruption of the cytoplasmic actin filaments by cytochalasin B.
Platelets were preincubated alone (A) or in the presence of
2.5
Figure 4:
Immunofluorescence confocal images showing
the distribution of ligand-occupied
Figure 5:
Immunofluorescence images showing the
distribution of ligand-occupied
Figure 6:
Electron micrographs showing the
distribution of ligand-occupied
Figure 7:
Effect of cytochalasin on the PAC-1
binding to ADP-activated platelets. Suspensions of platelets were
incubated for 20 min with 0.1% Me
The binding
of PAC-1 was quantitated by flow cytometry. As shown in Fig. 8,
cytochalasin inhibited the binding of PAC-1 to thrombin-activated
platelets in a dose-dependent manner. Cytochalasin also inhibited
binding of the natural ligand, fibrinogen, as shown by an inhibition of
the thrombin-induced aggregation of a stirred platelet suspension (Fig. 9, top panel). This was a specific inhibitory
effect, as shown by the lack of an inhibitory effect on the secretion
of ATP (Fig. 9, bottom panel). The concentrations of
cytochalasins required to inhibit aggregation (Fig. 9) were
comparable to those required to inhibit PAC-1 binding (Fig. 8).
Figure 8:
Effect of cytochalasin E (CE) on
the PAC-1 binding to platelets. Suspensions of platelets (1
Figure 9:
Dose-dependent inhibition of aggregation
by cytochalasin E. Suspensions of platelets (3
The binding of ligand to
Figure 10:
Effect of cytochalasin E on the
reversible (A) and irreversible (B) binding of PAC-1
to thrombin-stimulated platelets. Suspensions of gel-filtered platelets
were preincubated with 0.2% Me
The transmembrane signaling that occurs across integrins is
of critical importance in a variety of events such as inflammation,
embryonic development, arterial thrombosis, and hemostasis. The
mechanisms involved in the two-way signaling across integrins are not
well understood. In a previous study(21) , we provided evidence
that the integrin The goal of the present study was to determine
whether
In our previous study, we found that
Others
have used fibrinogen-coated gold beads or soluble fibringen to study
movements of
Immunofluorescence experiments have revealed that
in cultured cells, binding of an integrin to its ligand in the
extracellular matrix causes it to cluster and become incorporated into
complexes of cytoskeletal proteins and signaling molecules known as
focal contacts(54) . The selective clustering of
ligand-occupied
Volume 271,
Number 12,
Issue of March 22, 1996 pp. 7004-7011
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
and Its Ligand and Induces
Selective Movements of Ligand-occupied Integrin (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, co-sediments from
detergent lysates with talin and other membrane skeleton proteins. Once
has bound adhesive ligand in a
platelet aggregate, the detergent-insoluble
redistributes (along with the
detergent-insoluble membrane skeleton proteins and a variety of
signaling molecules) to a fraction that contains cytoplasmic actin
filaments. Concomitantly, certain signaling molecules are activated.
The present study shows that, in intact platelets,
forms clusters when occupied by
ligand and is selectively moved into the open canalicular system;
that has not bound ligand remains
diffusely distributed at the periphery of the cell. When cytoplasmic
actin filaments are depolymerized by cytochalasins, the ability of
to bind ligand is decreased, and
the movement of ligand-occupied is
prevented. Together with the previous findings, these results suggest
that (i) membrane skeleton-associated is selectively induced to bind ligand in activated platelets,
(ii) ligand-induced transmembrane signaling causes an altered
association of membrane skeleton-associated
with the cytoplasmic component of
the cytoskeleton, (iii) ligand-induced cytoskeletal reorganizations
stabilize the interaction between ligand and integrin, and (iv)
ligand-occupancy triggers cytoskeletal reorganizations that result in
selective movements of occupied ligand.
) on platelets(7) . When
platelets are activated, unidentified intracellular events act on
to induce binding of fibrinogen to
the extracellular domain of the receptor; by cross-linking
molecules on adjacent platelets,
fibrinogen is thought to mediate the formation of platelet aggregates.
Although binding of the adhesive ligand is initially reversible, it
becomes irreversible after several minutes(8, 9) . The
binding of adhesive ligand to in a
platelet aggregate induces a number of intracellular events, including
phosphorylation of specific proteins on tyrosine
residues(10, 11, 12) , activation of
calpain(13) , the calpain-induced hydrolysis of cytoskeletal
proteins(13, 14, 15, 16) , and
activation of Na/H
exchange(17) . How
is induced to bind adhesive ligand, how binding is rendered
irreversible, or how binding of adhesive ligand to the extracellular
domain of activates intracellular
signaling molecules are all unanswered questions. sedimented with fragments of the
membrane skeleton from lysates of unstimulated platelets(21) .
Tyrosine kinases (pp60 and
pp62
) were also recovered in this
detergent-insoluble fraction(21) . Once
had bound to fibrinogen in a
platelet aggregate, along with
membrane skeleton proteins(21) , tyrosine kinases(21) ,
tyrosine-phosphorylated proteins(21) , and a variety of
additional signaling molecules (e.g. phosphoinositide 3-kinase
and protein kinase C) (22) were recovered in the
detergent-insoluble fraction that contained the cytoplasmic component
of the cytoskeleton. We suggested that the membrane skeleton may play a
role in regulating the activation of
, that binding of ligand to
membrane skeleton-associated causes the integrin-skeleton complexes to undergo an altered
association with cytoplasmic actin forming ``focal
contact-like'' structures, and that these focal contact-like
structures may in turn play an important role in positioning signaling
molecules and in regulating the ability of such molecules to mediate
integrin-induced signaling events(21, 23) . binds ligand and is incorporated
into complexes with cytoplasmic actin filaments in intact cells, that
the formation of these integrin-cytoskeletal complexes results in
stabilization of integrin-ligand interactions and a selective movement
of ligand-occupied integrin into the surface-connected open canalicular
system.
Antibody Production and Characterization
PAC-1
and A5G8 are both monoclonal antibodies of the immunoglobulin (Ig) (
)M class that recognize epitopes on
. PAC-1 is similar to fibrinogen in
that it binds only to activated platelets(4) . A5G8 binds to
both unstimulated and activated platelets and does not inhibit
fibrinogen binding. (
)Polyclonal and monoclonal antibodies
against were provided by Dr. David
Phillips of COR Therapeutics (South San Francisco, CA), polyclonal
antibodies against fibrinogen were from Calbiochem (San Diego, CA) and
those against glycoprotein Ib were raised as described
previously(18) .
Isolation and Analysis of Platelet Suspensions
The
venous blood of healthy adult donors was collected into acid
citrate/dextrose solution, and platelet-rich plasma was
prepared(19) . Platelets were isolated by gel filtration (24) or by centrifugation (19) and resuspended at a
concentration of 1 
10
to 1 10
platelets/ml in Tyrode's buffer(19) . Platelets
were activated with thrombin (a gift of Dr. John W. Fenton II of the
New York Department of Health, Albany, NY) or ADP (Sigma). Unless
otherwise stated, activation was induced without stirring. In some
experiments, platelets were preincubated with cytochalasins (Sigma),
which were added in a final concentration of 0.1% dimethyl sulfoxide
(MeSO). Control incubations also contained
MeSO. Platelet aggregation and secretion of ATP were
assessed in a lumiaggregometer (Chrono-Log Corporation, Havertown, PA) (25) . Western blotting was performed by the method of Towbin et al.(26) . Antigen-antibody complexes were detected
with I-labeled anti-IgG (Dupont NEN).
Binding of PAC-1 to Platelets
Monoclonal antibody
PAC-1 was labeled with fluorescein isothiocyanate (FITC) (24) and incubated with platelets in the presence of an
agonist. At intervals, platelets were diluted into suspension buffer,
and the amount of bound PAC-1 was determined by flow cytometry (24) (Becton Dickinson FACS 440 cytometer, San Jose, CA). For
analysis of the reversible and irreversible components of PAC-1
binding, 45-µl aliquots were added to 50 µl of buffer or to the
same buffer containing 100 mM EDTA; after 10 min, samples were
diluted 3-fold, and the amount of bound PAC-1 was determined.Localization of Proteins by
Immunofluorescence
Platelets were activated in the presence of
monoclonal antibody PAC-1 or A5G8. Incubations were terminated by
addition of 9 volumes of a solution containing 4% paraformaldehyde in
150 mM sodium chloride, 10 mM Tris-HCl, pH 7.4, and
platelets were allowed to settle onto poly-L-lysine-coated
glass slides(19) . Fixed platelets were permeabilized by
addition of a buffer containing 0.5% Triton X-100, 0.1% Carnation milk,
150 mM ammonium acetate, 150 mM sodium chloride, 10
mM Tris-HCl, pH 7.4, then washed three times in a buffer that
contained a 1:50 dilution of sheep serum (Sigma). Specimens were then
incubated with biotinylated sheep anti-mouse IgM (Amersham Corp.) for 3
h, washed five times, incubated with Texas Red-labeled streptavidin
(Amersham) for 30-60 min, washed another five times, and mounted. ; those that had been incubated
with fibrinogen were incubated with polyclonal antibodies against
fibrinogen and a monoclonal antibody against
(that binds to a site other than
that occupied by fibrinogen). Specimens were washed five times,
incubated with biotinylated sheep anti-mouse IgM to label the
monoclonal antibody, washed five times, and incubated with FITC-labeled
streptavidin to detect monoclonal antibody and with Texas Red-labeled
anti-rabbit IgG to detect polyclonal antibodies. Specimens were
examined with a Zeiss Universal microscope or with a Bio-Rad MRC-600
confocal microscope (both equipped with dual fluorescence dichroic
filters) and photographed.Electron Microscopy of Activated
Platelets
Platelets were activated with thrombin in the presence
of PAC-1. After 15 min, suspensions were fixed with 4% paraformaldehyde
in 0.1 M sodium cacodylate buffer, pH 7.4, and centrifuged.
The pellets were dehydrated in a graded ethanol series, embedded in LR
White resin, and 60-90-nm thick sections collected on
carbon-Formvar-coated nickel grids. Sections were incubated for 4 h
with goat anti-mouse IgG and IgM conjugated to 5-nm diameter gold
colloid (Janssen Life Sciences Products, Beerse, Belgium). The
colloidal gold was silver-enhanced (27) . The sections were
stained with uranyl acetate and lead citrate and viewed in a Philips
301 transmission electron microscope.Electron Microscopy of Platelet
Cytoskeletons
Platelets were lysed by the addition of a Triton
X-100 containing buffer and anti-glycoprotein Ib (13
µg/ml) and Protein A coupled to 15-nm diameter gold colloid (200
µl) (Janssen) were added(19) . Lysates were incubated at 37
°C for 60 min and then fixed in glutaraldehyde. Specimens were
processed for electron microscopy and photographed in a JEM 100 CX II
microscope (JEOL, Peabody, MA) (19) .
Ligand-induced Association of
Based on studies in detergent-lysed platelets,
we have previously suggested that with Cytoplasmic Actin in the
Intact Platelet can associate with a submembranous component of the cytoskeleton
and that binding of adhesive ligand to leads to altered association of the integrin and membrane
skeleton proteins with cytoplasmic actin filaments. If this model is
correct, then that had bound its
adhesive ligand would be expected to show a different distribution
within intact platelets from that of that had not bound ligand. Further, any ligand-induced change in
distribution that occurred as a consequence of association of the
integrin with cytoplasmic actin would be prevented by disrupting the
network of cytoplasmic actin filaments. had a different
distribution from unoccupied integrin, platelets were incubated with
the fibrinogen-mimetic monoclonal antibody, PAC-1. At intervals
following thrombin addition, platelets were fixed, permeabilized with
Triton X-100, and incubated with a polyclonal
antibody that bound to both
ligand-occupied and -unoccupied .
The distribution of PAC-1 -occupied and total
was visualized by dual-label
confocal microscopy. As reported previously(4) , virtually no
PAC-1 bound to unstimulated, discoid platelets (Fig. 1A) even though was over all of the surface of the platelets (Fig. 1B). As the platelets were activated with
thrombin, PAC-1 binding occurred. At early times after platelet
activation (e.g. 60 s), PAC-1-occupied
was clustered in a few discrete
areas (Fig. 1C). However,
antibodies revealed that the rest
of the was still present over all
of the surface of the activated platelet (Fig. 1D). At
later times after platelet activation, clusters of PAC-1-occupied
became concentrated toward the
center of the platelet (Fig. 1E). Polyclonal antibodies
revealed that that had not bound
PAC-1 was still present in a relatively uniform distribution at the
periphery of these platelets (Fig. 1F). The diameters
of two representative platelet profiles (1 and 2) in Fig. 1, E and F, have been marked by pairs of arrows. When comparing, profiles 1 + 2 (PAC-1
distribution) in Fig. 1E with profiles 1 + 2
( distribution) in Fig. 1F, it is obvious that the PAC-1 labeling pattern
has a much smaller diameter than the labeling pattern in each case, small enough, in fact, to fit
within the peripheral labeling
pattern.
(left column) and total
(right column) in
thrombin-stimulated platelets. Platelet suspensions were incubated with
40 µg/ml PAC-1 for 2 min and then with 1.0 unit/ml thrombin for the
indicated times. Incubations were terminated by the addition of
paraformaldehyde. Platelets were permeabilized and incubated with
polyclonal anti- antibodies. The
distribution of PAC-1 and polyclonal antibodies were detected by
confocal microscopy as described under ``Materials and
Methods.'' The images in the right-hand panels show the
same platelets as are shown in the corresponding left-hand
panels. In Panel F the outline of the platelets is
revealed by labeling with antibodies; in Panel E, the location of PAC-1 in the
same platelets is shown; comparison of the platelets indicated with arrows in the two panels shows that PAC-1 is concentrated
toward the center of the platelets.
10 platelets/ml. A similar distribution of PAC-1-occupied
was detected on platelets that
were activated at a concentration of 1 10 platelets/ml (data not shown). Another monoclonal antibody of the
IgM class, A5G8, binds to a site on other than that to which adhesive ligand binds; it recognized
on both unstimulated and activated
platelets and showed a distribution similar to that detected with the
polyclonal antibody (data not
shown). Thus, the clustering and centralization of PAC-1-occupied
presumably results from occupancy
of the ligand binding site rather than from the binding of an IgM.
Further evidence for this came from experiments in which platelets were
incubated with the natural ligand for
, fibrinogen. As with PAC-1,
fibrinogen had a patchy distribution in activated platelets (Fig. 2A). The simultaneous use of an
antibody to reveal the outline of
the platelet in a dual label experiment (Fig. 2B)
showed that, like PAC-1, fibrinogen (shown in Fig. 2A)
was concentrated toward the center of activated platelets.
with a monoclonal antibody that
binds to a site other than that occupied by fibrinogen (B). Panel A shows immunolabeled fibrinogen clustered in each
individual platelet. Panel B shows the same field of platelets
immunolabeled for which covers the
entire surface of the cells. Compare the groups of platelets marked
with arrows in the two panels. Clearly the fibrinogen (A) is more central in each platelet while the
(B) extends right to the
peripheries of the platelets.
was induced by cytoplasmic actin
filaments, platelets were preincubated with cytochalasins.
Cytochalasins inhibit the burst of actin polymerization that occurs
when platelets are activated(28, 29) . Thin section
electron microscopy (Fig. 3) revealed that concentrations of
cytochalasins higher than those needed to inhibit actin polymerization
also disrupted the preexisting network of cytoplasmic actin filaments. Fig. 3A shows an untreated platelet with intact
cytoplasmic actin filaments. Fig. 3B shows that the
membrane skeleton remained intact after cytochalasin treatment but that
the cytoplasmic actin network was disrupted. In addition, biochemical
experiments revealed that, while the amount of actin sedimenting from
detergent lysates at low g forces (i.e. networks of
cytoplasmic actin) was markedly reduced in cytochalasin-treated cells,
the amount of membrane skeleton proteins and
that sedimented at high g forces (i.e. in the membrane skeleton fraction) was not
detectably altered (data not shown). To determine the effect of
disruption of the network of cytoplasmic actin filaments on the
ligand-induced redistribution of ,
cytochalasin-treated platelets were incubated with thrombin in the
presence of PAC-1, and the distribution of PAC-1 was determined.
Ligand-occupied still appeared to
be present in clusters in the cytochalasin-treated cells (Fig. 4A). However, in contrast to the
non-cytochalasin-treated cells (as seen in Fig. 1and Fig. 2), the clusters of ligand-occupied integrin did not move
inward but remained at the periphery of the cell (Fig. 4A). Fig. 4B shows the same field
as seen in Fig. 4A, but now the platelet periphery is
delineated by immunolabeled total .
These results suggest that in non-cytochalasin-treated platelets,
ligand-occupied associates with
cytoplasmic actin and that this association leads to the inwards
movement of the occupied integrin.
10M cytochalasin B (B) prior to lysis and preparation of the detergent-insoluble
cytoskeletons for electron microscopy. In Panel A, the
membrane skeleton is shown as a cortex enclosing cytoplasmic actin
cables. This image is representative of about 20 different experiments. Panel B shows that cytochalasin B disrupted the cytoplasmic
component of the cytoskeleton, but left the membrane skeleton
intact.
and total in
cytochalasin-treated platelets. A platelet suspension was incubated
with 10M cytochalasin E for 10 min. PAC-1
was then added to a concentration of 40 µg/ml. After 2 min, 1.0
unit/ml thrombin was added, and incubation was continued for another 5
min. The platelets were then fixed by the addition of paraformaldehyde,
permeabilized, and incubated with polyclonal
antibodies. The distribution of
PAC-1 and the polyclonal antibodies were detected by confocal microscopy as described
under ``Materials and Methods.'' Panel A shows the
distribution of PAC-1, and Panel B shows polyclonal
antibody-labeled in the same field
of platelets as is shown in Panel
A.
Localization of Ligand-occupied
The images shown in Fig. 1and Fig. 2were obtained when platelets were
permeabilized with Triton X-100 prior to the addition of the secondary
antibodies. Although the intensity of the fluorescence was often
weaker, the same clustering and centralization of ligand-occupied
was detected when the
permeabilization step was omitted (Fig. 5) (control experiments
using antibodies to actin-binding protein, a protein known to be
present only on the cytoplasmic side of the membrane, confirmed that
the platelets were not permeabilized inadvertently in these
experiments) (data not shown). These results suggested that the
membranes of the surface-connected open canalicular system were the
most likely location of the centralized receptor since the
immunolabeling antibody could have gained access via the open channels.
To determine if this was the case, thin-section electron microscopy of
platelets that had been activated in the presence of PAC-1 and
subsequently fixed, sectioned, and incubated with anti-mouse antibodies
coupled to 5 nm of colloidal gold was performed. Because the PAC-1 was
added prior to sectioning the amount of label in any one section was
small. However, some PAC-1 was detected on the surface of the cell
(where it would initially bind); any nonperipheral PAC-1 was present in
the open canalicular system (Fig. 6, Panels A and B, arrows). When 125 gold particles were counted
(representing staining of 127 platelets), 21 were present on the
surface of the platelets, 93 in the open canalicular system, and 11 in
intracellular areas. In platelets that had not been activated, a total
of 13 gold particles were counted in 130 platelets; 3 particles on the
surface, 3 in the open canalicular system, and 7 in the cytoplasm (data
not shown). These data suggest that the cytoplasmic labeling represents
background levels. The finding that ligand-occupied
is present primarily in the open
canalicular system, is consistent with the observation by others that
fibrinogen is localized in the open canalicular system of activated
platelets(30) .
in
thrombin-treated platelets. Platelet suspensions were incubated with 20
µg/ml PAC-1 for 2 min and then incubated alone or with 0.05 unit/ml
thrombin for the indicated times. Incubations were terminated by the
addition of paraformaldehyde and the distribution of PAC-1 detected
with fluorescent secondary antibodies as described under
``Materials and Methods.'' Panels A-C,
platelets were exposed to Triton X-100 prior to the addition of the
fluorescent secondary antibodies; Panels D-F, the platelets
were not exposed to detergent.
in
thrombin-stimulated platelets. Platelet suspensions were incubated with
40 µg/ml PAC-1 for 2 min and then incubated with 0.5 unit/ml
thrombin for 15 min. Platelets were fixed with paraformaldehyde and
embedded in LR White resin. Ultrathin sections were stained with
immunogold to localize PAC-1. Panels A and B represent two different fields of the same sample. The arrows indicate the invaginations of the open canalicular system. Note
the localization of PAC-1 to the open canalicular
system.
Regulation of Fibrinogen Binding by the
Cytoskeleton
To determine whether the cytoplasmic actin
filaments play a role in regulating the binding of ligand to
, platelets were incubated with
cytochalasin under conditions in which the network of cytoplasmic actin
filaments was disrupted and the subsequent ADP-induced binding of PAC-1
to platelets visualized by immunofluorescence (Fig. 7). The
images in Panels A, B, and C were generated
using similar exposures so that the relative amounts of PAC-1 binding
to the platelets in cytochalasin-treated (Panel C) and
-untreated cells (Panel B) could be visualized. Comparison of
these images revealed that cytochalasin decreased the binding of PAC-1
to ADP-activated platelets. Similar results were obtained when
platelets were activated with thrombin (data not shown).
SO (Panels A and B) or with 10M cytochalasin E (CE) (Panel C). PAC-1 was then added to a
concentration of 20 µg/ml. After 2 min, incubation with buffer (Panel A) or with 20 µg/ml ADP (Panels B and C) was initiated. Incubations were terminated after 3 min by
the addition of paraformaldehyde. Platelets were lysed, and the
distribution of PAC-1 detected with fluorescently labeled secondary
antibodies as described under ``Materials and
Methods.''
10 platelets/ml) were incubated with 0.1% MeSO
or with the indicated concentrations of cytochalasin E in the presence
of 0.1% MeSO for 30 min. Platelets were subsequently
incubated in the presence of 20 µg/ml FITC-labeled monoclonal
antibody PAC-1, either with no further addition (Control) or
with the addition of 0.1 unit/ml thrombin. Following an incubation of
60 s, samples were diluted 100-fold with a Tyrode's solution, and
the amount of FITC-labeled PAC-1 bound to the platelets was detected by
flow cytometry. This figure is representative of the results of six
different experiments.
10 platelets/ml) were preincubated for 10 min in the presence or
absence of cytochalasin E at the concentrations shown. Cytochalasin was
added in a final volume of 0.1% MeSO, which was also
present in the control incubation. Luciferin-luciferase reagent was
then added and the platelet suspensions stirred with 1.0 NIH unit/ml
thrombin in an aggregometer. The aggregation of platelets was detected
as an increase in the transmittance of light through the suspension (top panel); secretion of ATP was detected as an increased
luminescence (bottom panel).
initially occurs in a reversible manner (it can be reversed by
addition of EDTA), but with time it becomes
irreversible(8, 9) . To determine whether the
cytoplasmic actin filaments are involved in regulating the reversible
or irreversible component of binding, platelets were preincubated in
the presence or absence of cytochalasins and activated with thrombin in
the presence of PAC-1; after either 5 or 45 min, one aliquot was
diluted into buffer while another was diluted into buffer containing
EDTA. The irreversible component of binding was defined as the PAC-1
that remained bound to the platelets following dilution in EDTA, while
the reversible binding was the difference between that which remained
bound in EDTA and that which remained bound in buffer alone. As shown
in Fig. 10, cytochalasin E inhibited the reversible binding of
PAC-1 by approximately 50%. However, it almost completely inhibited the
irreversible binding of PAC-1 to activated platelets.
SO or 10M cytochalasin E for 10 min. FITC-labeled PAC-1 was then
added to a final concentration of 40 µg/ml and thrombin to 0.1
unit/ml. After 5 or 45 min, 45-µl aliquots of each incubation were
added to 5 µl of either buffer or 100 mM EDTA. The amount
of FITC-labeled PAC-1 bound to platelets was analyzed by flow cytometry
10 min later. The amount of PAC-1 that was reversibly bound to
platelets was determined as the amount that was displaced by EDTA (i.e. the difference between plus and minus EDTA samples). The
amount of PAC-1 that was irreversibly bound was defined as that which
remained bound to the platelets following the 10-min incubation in
EDTA. The values shown represent the mean ± S.D. obtained from
three separate incubations.
cosedimented
with membrane skeleton proteins (e.g. spectrin, talin, and
vinculin) from unstimulated platelets, that signaling molecules were
associated with the membrane skeleton, and that binding of ligand to
caused the membrane skeleton
together with associated integrin and signaling molecules to
redistribute to the low-speed detergent insoluble fraction. We
suggested that was associated with
the membrane skeleton, that binding of ligand to
caused the membrane skeleton and
associated integrin to become incorporated into complexes containing
cytoplasmic actin filaments, and that the cytoskeleton might play a
role in regulating the two-way signaling across
. The evidence for these
suggestions came entirely from co-sedimentation of proteins in
detergent lysates. and the membrane skeleton
become associated with cytoplasmic actin filaments in intact platelets
and to determine the consequence of integrin-induced cytoskeletal
reorganizations in intact cells. The studies suggest that
membrane-skeleton associated binds
ligand, clusters, and associates with cytoplasmic actin filaments. The
resulting integrin-cytoskeletal complexes allow
that has bound ligand to be
selectively moved inward into the open canalicular system. Further, the
integrin-cytoskeletal complexes play a role in stabilizing the
integrin-ligand interaction. Taken together with our previous results
in detergent lysates, these studies indicate that the cytoskeleton
plays an important role in regulating the two-way signaling across
in platelets.Association of
It has been known for many years
that a subpopulation of with the
Cytoskeleton in Intact Cells cosediments with cytoplasmic actin filaments from detergent
lysates of platelets that have aggregated(34, 35) . It
has been assumed that this sedimentation results from an association of
with cytoplasmic actin filaments.
The present study shows that this is the case. Thus, a subpopulation of
clustered and moved into the
depths of the open canalicular system in activated platelets; this
movement was induced by the cytoplasmic actin filaments because if
these filaments were disrupted with cytochalasins, the movement was
prevented. only redistributed to the low
speed detergent-insoluble fraction in platelets in which ligand had
bound to the integrin; moreover, ligand binding alone was not
sufficient and the platelets needed to aggregate, presumably because
ligand-induced cross-linking of the integrin was needed(36) .
In the present study, we used the fibrinogen mimetic monoclonal
antibody PAC-1 (which is a pentameric molecule and therefore induces
signaling comparable to that induced by fibrinogen in a platelet
aggregate rather than that induced by fibrinogen in a nonaggregating
suspension)(37) . Dual-labeled immunofluorescence allowed us to
show that only that had bound
ligand moved inward. The rest of the remained at the periphery of the cell. We conclude, therefore,
that the association of an molecule with cytoplasmic actin in aggregating platelets is a
direct consequence of ligand binding to that molecule of integrin. One
can envisage a physiological mechanism in which the selective movement
of ligand-occupied integrin inward into the open canalicular system
allows the externally bound fibrin clot to be pulled inward. on adherent
platelets(38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49) .
The distribution of gold beads on platelets activated in suspension has
also been
studied(40, 41, 50, 51, 52) .
These studies have revealed that that had bound gold beads moves inward. Although the
physiological relevance of the movement of gold beads on the platelet
surface has been questioned (53) and the functional state of
integrins or signaling molecules on surface-activated platelets as
compared to platelets activated with a physiological agonist is
completely unknown, it is of interest that in all cases only the
occupied receptor moves inward. The finding that receptor that has
bound gold beads moves into the open canalicular system if it
exists(40, 41, 42, 43, 45) ,
that it co-localizes with cytoskeletal proteins(38) , and that
cytochalasins can inhibit some of the
movements(42, 44) , suggests that, as with
that has bound soluble ligand in
platelet suspensions, the consequence of receptor occupancy is that the
occupied receptor is selectively pulled by the cytoskeleton toward the
most central regions of the platelet.Selective Association of Membrane Skeleton-associated
In
our previous biochemical experiments(21) , we noticed that in
unstimulated platelets a subpopulation of
with Cytoplasmic Actin co-sedimented with membrane
skeleton proteins at high g forces (about 30% of the total
platelet ). As platelets bound
ligand, it was this population that initially redistributed to the low
speed detergent-insoluble fraction. The present study supports the idea
that only a subpopulation of is
induced to bind ligand and to associate with cytoplasmic actin
filaments in the intact cell. Taken together with the previous
biochemical findings, this suggests that the
that sediments with membrane
skeleton proteins from unstimulated platelets represents a pool of
integrin that can be selectively induced to bind ligand. The finding
that the that bound ligand became
clustered even when the networks of cytoplasmic filaments were
depolymerized with cytochalasins (which did not disrupt the membrane
skeleton) is also consistent with the possibility that it is the
that is associated with membrane
skeleton proteins that is selectively induced to bind ligand. Because
the membrane skeleton is in close contact with the plasma membrane and
associates with both signaling molecules (21) and
(21) , it appears possible
that this structure serves to localize signaling molecules that are
involved in the activation of the integrin. Similarly, the association
of the integrin with this structure may be important in allowing the
selective association of ligand-occupied integrin with underlying
cytoplasmic actin filaments. Future work will be needed to find out how
the integrin associates with submembranous skeletal proteins and to
directly test the hypothesis that this association allows the
activation and ligand-induced association of the integrin with
cytoplasmic actin. and its
association with cytoplasmic actin filaments in platelets is
reminiscent of the formation of focal contacts. Several of the proteins
that co-sediment with from
detergent lysates of unstimulated platelets and redistribute with
into the low-speed
detergent-insoluble fraction from activated platelets are proteins that
have been found to co-localize with integrins in focal contacts (e.g. vinculin, talin, and pp60) (54) . Additional components of focal contacts (e.g. protein kinase C) incorporate into the low speed pellet in
aggregating platelets(22) . Thus, the cytoskeletal
reorganizations that are induced as a consequence of ligand binding to
in platelets may be similar to
those that form as a consequence of integrin-ligand interactions in
cultured cells, and similar signaling mechanisms may be involved.Regulation of Transmembrane
Signaling
can exist in a
number of different affinity states, and several steps may be involved
in the activation of the integrin and subsequent stabilization of
ligand binding(55, 56, 57) . As discussed
above, the present immunofluorescence findings combined with the
previous findings in detergent lysates (21) indicate that the
membrane-skeleton associated is
selectively induced to bind ligand. However, the finding that
concentrations of cytochalasins that disrupted the cytoplasmic actin
filaments inhibited ligand binding suggests that the cytoplasmic actin
filaments also play an important role in regulating ligand binding to
. Previously, variable effects of
cytochalasins on fibrinogen binding(58, 59) ,
aggregation(58) , and fibrinogen-gold distribution (40, 41, 44) have been reported. These
variations may have resulted from the use of cytochalasins at
concentrations that had variable effects on the cytoplasmic actin
filaments. In the present study, we determined concentrations of
cytochalasins that were needed not only to inhibit the increased
polymerization of actin that occurs when platelets were activated (28, 29) but also to disrupt the preexisting networks
of cytoplasmic actin (60) . These concentrations of
cytochalasins almost totally prevented the conversion of the binding of
fibrinogen from reversible to irreversible. In addition, they partially
inhibited the reversible component of the binding. In cultured cells,
the formation of focal contacts plays a role in stabilizing
integrin-ligand interactions(54) . Thus, in platelets, the
formation of the ``focal contact-like'' integrin-cytoskeletal
complexes may be important in rendering the binding of ligand
irreversible. Associations between the cytoplasmic actin filaments and
components of the membrane skeleton probably exist even in the
unstimulated platelets(19, 20) . Thus, one possible
mechanism by which the cytoplasmic actin filaments could exert their
regulatory influence on the earlier, reversible stages of ligand
binding might be their association with membrane skeleton proteins
which in turn might associate with and signaling molecules.Summary
Taken together with the results of our
previous biochemical studies, the present studies indicate that the
subpopulation of that is
associated with the membrane skeleton is preferentially activated, that
it subsequently becomes incorporated into complexes with cytoplasmic
actin filaments, and that the formation of these integrin-rich
cytoskeletal complexes plays a role in stabilizing the ligand-integrin
interactions, inducing a selective redistribution of occupied integrin,
and inducing activation of signaling molecules. Future studies will be
needed to elucidate the molecular nature of the interactions between
and the membrane skeleton and to
identify the mechanisms by which integrin-cytoskeletal complexes
regulate the post-occupancy events in activated platelets.
))
We thank Dr. John W. Fenton II for generously
providing the -thrombin and Dr. Janet Boyles for performing the
electron microscopy shown in Fig. 3. We also thank Dale Newland,
Amy Corder, and Susanne Zuerbig for graphics, Al Averbach for editorial
assistance, and Susanne Zuerbig and Marnie DeReske for technical
assistance.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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