(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, , 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.
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
() 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.
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
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) .
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
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.
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
; 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.
To determine whether
ligand-occupied 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.
Figure 1:
Immunofluorescence confocal images
showing the distribution of ligand-occupied
(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.
The platelets shown in Fig. 1had been activated
with thrombin at a platelet concentration of 1 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.
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
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.
To
determine whether the clustering and centralization of
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.
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 10
M 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.
Figure 4:
Immunofluorescence confocal images showing
the distribution of ligand-occupied and total
in
cytochalasin-treated platelets. A platelet suspension was incubated
with 10
M 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.
Figure 5:
Immunofluorescence images showing the
distribution of ligand-occupied 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.
Figure 6:
Electron micrographs showing the
distribution of ligand-occupied 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.
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% MeSO (Panels A and B) or with 10
M 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.''
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
10
platelets/ml) were incubated with 0.1% Me
SO
or with the indicated concentrations of cytochalasin E in the presence
of 0.1% Me
SO 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.
Figure 9:
Dose-dependent inhibition of aggregation
by cytochalasin E. Suspensions of platelets (3 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% Me
SO, 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).
The binding of ligand to 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.
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% MeSO or 10
M 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.
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 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.
The goal of the present study was to determine
whether 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.
In our previous study, we found that
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.
Others
have used fibrinogen-coated gold beads or soluble fibringen to study
movements of 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.
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 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.