(Received for publication, September 10, 1996, and in revised form, November 27, 1996)
From the Platelet responses to thrombin are at least
partly mediated by a G-protein-coupled receptor whose NH2
terminus is a substrate for thrombin. In the present studies we have
examined the location of thrombin receptors in resting platelets and
followed their redistribution during platelet activation. The results
reveal several new aspects of thrombin receptor biology. 1) On resting platelets, approximately two-thirds of the receptors were located in
the plasma membrane. The remainder were present in the membranes of the
surface connecting system. 2) When platelets were activated by ADP or a
thromboxane analog, thrombin receptors that were initially in the
surface connecting system were exposed on the platelet surface,
increasing the number of detectable receptors by 40% and presumably
making them available for subsequent activation by thrombin. 3)
Platelet activation by thrombin rapidly abolished the binding of the
antibodies whose epitopes are sensitive to receptor cleavage and left
the platelets in a state refractory to both thrombin and the agonist
peptide, SFLLRN. This was accompanied by a 60% decrease in the binding
of receptor antibodies directed COOH-terminal to the cleavage site
irrespective of whether the receptors were activated proteolytically by
thrombin or nonproteolytically by SFLLRN. 4) The loss of antibody
binding sites caused by thrombin was due in part to receptor
internalization and in part to the shedding of thrombin receptors into
membrane microparticles, especially under conditions in which
aggregation was allowed to occur. However, at least 40% of the cleaved
receptors remained on the platelet surface. 5) Lacking the ability to
synthesize new receptors and lacking an intracellular reserve of
preformed receptors comparable to that found in endothelial cells,
platelets were unable to repopulate their surface with intact receptors
following exposure to thrombin. This difference underlies the ability
of endothelial cells to recover responsiveness to thrombin rapidly
while platelets do not, despite the presence on both of the same
receptor for thrombin.
In its proteolytically active form, thrombin evokes responses from
a number of cells located in and around the vascular space, including
endothelial cells, vascular smooth muscle, and platelets. Although the
existence of an unknown second thrombin receptor in mouse platelets has
recently been inferred from knockout studies (1), many of the responses
of human platelets to thrombin are thought to be mediated by a
previously identified G-protein-coupled receptor whose NH2
terminus is a substrate for thrombin (2). The evidence that this
receptor is responsible in part for thrombin responses in human
platelets is compelling: RNA encoding the receptor is present in
platelets and in megakaryoblastic cell lines (2); antibody binding
studies show that it is expressed on the platelet surface (3, 4); and
peptide agonists based on the receptor's tethered ligand domain are
able to activate human platelets, mimicking many of the effects of
thrombin (e.g. 2, 5-8). This is in contrast to mouse
platelets, which respond to thrombin but not to the agonist peptides
(9, 10).
One of the properties that sets thrombin receptors apart from most
other G-protein-coupled receptors is their inability to be activated by
thrombin more than once. This is thought to be due in part to receptor
phosphorylation and in part to the apparent inability of thrombin to
reactivate cleaved receptors (11-14). Therefore, for cells to recover
responsiveness to thrombin, cleaved thrombin receptors have to be
replaced with intact receptors, a process that is usually preceded by
the clearance of at least some of the cleaved receptors from the cell
surface (15). Recent studies in cells other than platelets have
demonstrated two general mechanisms for accomplishing this. In the
megakaryoblastic HEL and CHRF-288 cell lines, where >90% of the
cleaved thrombin receptors are internalized within 5 min, recovery is a
slow process dependent on the synthesis of new receptors (13, 14, 16).
Endothelial cells and fibroblasts, on the other hand, contain a large
intracellular reserve of preformed receptors (17-19). These receptors
can quickly repopulate the cell surface after the addition of thrombin,
allowing recovery to occur more rapidly than it can when dependent upon receptor synthesis alone.
Platelets normally need to respond to thrombin only once, in contrast
to endothelial cells, which may encounter thrombin repeatedly. We were
interested in determining whether this difference between platelets and
endothelial cells would be reflected in differences in thrombin
receptor biology between the two cell types. We were particularly
interested in determining the distribution of thrombin receptors on
resting platelets, identifying any intracellular pools of receptors
which might exist and tracing the movements of thrombin receptors when
platelets are activated, either by thrombin or by agonists that
activate platelets via receptors other than the thrombin receptor.
Information about the distribution of thrombin receptors on human
platelets is limited, in part because of the small size of platelets
and the small number of receptors per cell. Based upon antibody
binding, resting platelets express 1,500-2,000 copies of the thrombin
receptor per cell on the cell surface (3, 4). Data on the
redistribution of thrombin receptors following platelet activation is
limited to a study in which Norton and co-workers (4) found no decrease
in the binding to cleaved receptors of at least one antithrombin
receptor antibody, leading them to propose that in contrast to
endothelial cells and the megakaryoblastic cell lines, cleaved thrombin
receptors on platelets remain on the platelet surface.
In the present studies we have used electron microscopy and flow
cytometry to examine the initial distribution of thrombin receptors on
and within human platelets and follow the redistribution that occurs
when platelets are activated. Since thrombin can interact with more
than one protein on the platelet surface (20-22), peptide-directed monoclonal and polyclonal antibodies were used to detect the receptor. The results show that in resting platelets only two-thirds of the total
number of thrombin receptors are located on the plasma membrane. The
remainder are initially present in the membranes of the intracellular
surface connecting system, a structure contiguous with the platelet
plasma membrane that is exposed during platelet activation. In the case
of platelet activation by ADP and the thromboxane A2 analog
U46619, this leads to a net increase in the number of cell surface
thrombin receptors by exposing receptors that were initially in the
surface connecting system. Platelet activation by thrombin or the
peptide agonist SFLLRN, on the other hand, caused a net decrease in the
number of receptors that could be detected by all of the antibodies
that were tested. When platelet aggregation occurred, this decrease
appeared to be at least partly due to receptor internalization and the
shedding of thrombin receptors into membrane microparticles. We found
no evidence, however, for an intracellular reserve of thrombin
receptors in platelets comparable to that found in endothelial cells
and no evidence that platelets could mobilize intact receptors from
sites other than the surface connecting system. This accounts for the
observed inability of platelets to recover responsiveness to thrombin
or SFLLRN following an initial exposure to thrombin.
Monoclonal antibodies SPAN11, SPAN12, ATAP2, and
WEDE15 were prepared against peptides corresponding to overlapping
regions of the human thrombin receptor NH2 terminus (Fig.
1) (3, 14, 16). Antibody 1047 is the purified IgG
fraction from a polyclonal antibody prepared in rabbits immunized with
the peptide YEPFWEDEEKNESGLTEYC conjugated via the cysteine to keyhole
limpet hemocyanin (23). Monoclonal antibody A2A9 is directed against
the platelet glycoprotein IIb-IIIa (integrin
Blood (60 ml) was obtained from healthy
volunteers and anticoagulated with acid-citrate-dextrose (8.6 ml).
Platelet-rich plasma was prepared by centrifugation for 15 min at
169 × g with the centrifuge brake off and incubated
with prostaglandin E1 (1 µM) for 5 min at
room temperature. Afterwards, the platelets were sedimented at
1,200 × g for 15 min, resuspended in 10 ml of
HEPES-Tyrode buffer (129 mM NaCl, 2.8 mM KCl,
0.8 mM KH2PO4, 8.9 mM
Na2HCO3, 0.8 mM MgCl2,
5.6 mM glucose, 10 mM HEPES, pH 7.4)
supplemented with 1 µM prostaglandin E1 and 1 mM EGTA, sedimented a second time at 1,200 × g for 15 min, and then resuspended at 2 × 108 platelets/ml in HEPES-Tyrode buffer. RGDS (200 µM) was added to inhibit platelet aggregation. When
indicated, the platelets were incubated with thrombin, SFLLRN, ADP, or
U46619 for 15 min at room temperature, after which the desired
monoclonal antibody was added (10 µg/ml of purified protein), and the
platelets were incubated for a further 15 min at 4 °C. Afterwards, 1 ml of HEPES-Tyrode buffer containing 2 mM EGTA and 2 mM EDTA was added, and the platelets were sedimented
(1,200 × g for 5 min) and resuspended in 50 µl of
fetal calf serum plus 50 µl of a 1:40 dilution of fluorescein isothiocyanate-labeled goat anti-mouse IgG (BioSource International, Camarillo, CA). After a 15-min incubation with the secondary antibody at 4 °C, the platelets were diluted with 500 µl of HEPES-Tyrode buffer containing 2 mM EGTA and 2 mM EDTA,
sedimented at 1,200 × g for 5 min, resuspended in 500 µl of HEPES-Tyrode buffer, and analyzed on a FACScan flow cytometer
(Becton Dickinson, Mountain View, CA).
For the studies on resting platelets,
10 ml of human blood was dripped directly from intravenous tubing into
a beaker containing 90 ml of 4% paraformaldehyde in McLean and
Nakane's buffer for 1 h (25). The mixture was spun at 150 × g for 20 min at 20 °C (26). The supernatant was then spun
at 1,000 × g for 10 min, producing a cell pellet that
was embedded in 2.1 M sucrose, frozen, and stored in liquid
nitrogen. Frozen thin sections were processed as described previously
(27-29). The primary antibody (1047) was used at a dilution of 1:100.
This was followed by the gold label, protein A-10, obtained from the
Department of Cell Biology, University of Utrecht (Utrecht, the
Netherlands) at 1:50 dilution. The grids were then stained with uranyl
acetate and embedded in methyl cellulose. As a control, normal rabbit
serum was also tested. For the studies of activated platelets, washed
platelets were prepared from human blood anticoagulated with citrate as
described previously (30) then activated with thrombin (1 unit/ml) and
processed in the same way as the resting platelets.
Washed platelets were resuspended at
3 × 108/ml in HEPES-Tyrode buffer with 1 µM prostaglandin E1 and incubated with 5 µM Fura-2/AM for 20 min at 37 °C. Afterwards, 2 mM EGTA and 1 µM prostaglandin E1
were added, and the platelets were sedimented and resuspended in
HEPES-Tyrode buffer containing 1 mM CaCl2 at
1 × 108/ml. Changes in the cytosolic free
Ca2+ concentration were measured using a SLM/Aminco AB-2
spectrophotometer (31).
Washed platelets were resuspended at
2 × 108/ml in the presence of 200 µM
RGDS and stimulated for 15 min at room temperature with either 0.25 unit/ml thrombin or 100 µM SFLLRN without stirring. Platelets were sedimented by centrifugation at 1,200 × g for 15 min. The supernatants were then recentrifuged at
27,000 × g for 30 min. Both fractions were resuspended
and processed as follows. Samples were stained for 15 min at 4 °C
with either the thrombin receptor antibodies or EH1, washed with
HEPES-Tyrode containing 2 mM EGTA and 2 mM
EDTA, resuspended in fetal calf serum, and stained with a 1:40 dilution
of a fluorescein isothiocyanate-labeled anti-mouse secondary antibody
for 15 min at 4 °C. After a further wash samples were incubated
under the same conditions with biotinylated AP-1, a monoclonal antibody
that recognizes glycoprotein Ib Human We
have shown previously that monoclonal antibodies directed against the
thrombin receptor NH2 terminus can be used as probes to
detect thrombin receptor cleavage and internalization and to discriminate between receptor recycling and receptor replacement (14,
16-18, 32, 33). Four such antibodies were used in the present studies.
Two, designated SPAN11 and SPAN12, are directed at the site of cleavage
and were selected for their ability to recognize intact receptors but
not cleaved receptors (Fig. 1). A third antibody, ATAP2, is directed
against an epitope within the tethered ligand domain and has been shown
to recognize cleaved as well as intact thrombin receptors, as does
antibody WEDE15, which is directed against the domain of the receptor
NH2 terminus thought to interact with the thrombin
anion-binding exosite. The fifth antibody shown in Fig. 1, 1047, is a
rabbit polyclonal antibody that was raised against a peptide that
partially overlaps the peptide used to prepare antibody WEDE15
(23).
Fig. 2 is an electron micrograph of human platelets that
were fixed and then stained with polyclonal antibody 1047. The results show that on resting platelets thrombin receptors are present on the
both the plasma membrane and the membranes of the surface connecting
system (SC in the figure). Receptors were not detectable in
The effect of platelet activation on the distribution
of thrombin receptors was studied with platelets incubated with
thrombin, SFLLRN, ADP, or the thromboxane analog U46619. In the initial studies, platelet aggregation was inhibited by removing
Ca2+ and adding RGDS to inhibit fibrinogen binding to the
A different result was obtained when the platelets were activated with
ADP or U46619 rather than thrombin. Both of these agonists caused an
increase, rather than a decrease, in thrombin receptor antibody binding
(Fig. 4). This increase averaged approximately 40% and was similar in
magnitude to the increase observed in the binding of an antibody, A2A9,
which recognizes the integrin We have shown
previously that thrombin receptor activation on endothelial cells,
fibroblasts, and several megakaryoblastic cell lines is followed by
receptor internalization. This process was shown by colocalization
studies and electron microscopy in the megakaryoblastic cell lines to
involve coated vesicles and endosomes (16). Coated vesicles have been
observed in platelets (40, 41) and could provide a mechanism for
thrombin receptor internalization in platelets, but other alternatives
were considered as well.
One such alternative is that activated thrombin receptors are shed from
the platelet surface in membrane microvesicles. Several studies have
shown that membrane microparticles are formed when platelets are
activated, particularly if they are activated by strong agonists such
as thrombin or collagen under conditions in which aggregation is
allowed to occur (e.g. 42-45). These microparticles include
not only platelet membrane lipids, but also integral membrane proteins
such as glycoprotein Ib. To test whether loss of thrombin receptors
into microparticles could account in part for the observed decrease in
antibody binding sites on activated platelets, platelets were incubated
with thrombin or SFLLRN under the same conditions in which both
agonists caused a decrease in antibody binding to the platelets.
Microparticles were detected by flow cytometry and defined by size and
by the presence of glycoprotein Ib using the glycoprotein
Ib
This suggests that thrombin receptors are present in microparticles,
but since there was no apparent change in the number of receptors per
microparticle, this would not account for the decrease in receptor
number on activated platelets unless there were also an increase in
microparticle number under the conditions in which the platelets were
activated. To determine if this is the case, platelets were activated
with thrombin or SFLLRN, and the number of microparticles was counted.
Under conditions in which aggregation was prevented, microparticles
represented less than 5% of total particles counted and increased
little in number upon platelet activation (Fig. 5B). On the
other hand, when platelet aggregation was allowed to occur, both
thrombin and SFLLRN caused a substantial increase in microparticle
number (Fig. 5C). These results suggest that microparticle
formation could account for part of a decrease in receptor number when
platelets are activated by thrombin or SFLLRN, but only if aggregation
is allowed to occur.
To
examine directly the distribution of thrombin receptors in activated
platelets, platelets were incubated with thrombin and allowed to
aggregate before being studied by immunoelectron microscopy using the
cleavage-insensitive polyclonal antibody 1047. The results are shown in
Fig. 6. Compared with the resting platelet shown in Fig.
2, there was a marked decrease in the staining of the plasma membrane
(pm in the figure), presumably in part because of loss of
thrombin receptors into microparticles under these conditions. The
surface connecting system, which evaginates during platelet activation
by thrombin, was no longer detectable. Instead, large vacuoles were
present which have been shown previously to contain the contents of
fused
Thrombin
receptor activation typically precludes a subsequent response to either
thrombin or SFLLRN unless resensitization or replacement of the
receptors occurs in the interim. However, recent studies have shown
that the extent of thrombin receptor desensitization varies among cell
types, as does the rate of recovery. In the studies shown in Fig.
7, human platelets were loaded with Fura-2, and changes
in the cytosolic free Ca2+ concentration were measured.
Thrombin and SFLLRN both caused a transient rise in cytosolic
Ca2+ and, as was seen with the megakaryoblastic cell lines,
a subsequent addition of either agonist elicited little or no response
when added within a few minutes of the first stimulus (Fig. 7,
A and B), although responses to other agonists
such as U46619 did occur (not shown). To determine whether recovery
would occur after the passage of a longer period of time, platelet
responses to SFLLRN were measured 4 h after briefly incubating
them with thrombin. Despite the passage of time, control platelets
showed a full response to SFLLRN. Thrombin-treated platelets, on the
other hand, continued to show no response. By comparison, HEL cells and
endothelial cells recover 40-60% of their initial response to SFLLRN
over the same time period, due, respectively, to receptor recycling (14) and receptor mobilization (18). Thus, in human platelets receptor
activation by either thrombin or SFLLRN is followed by desensitization
to both which lasts at least 4 h. Antibody binding studies with
ATAP2 and SPAN11 performed under the same conditions show that there
was a continued gradual loss of cleaved receptors from the platelet
surface during this period (Fig. 8). There was no
evidence for either the appearance of new intact receptors (causing a
parallel increase in ATAP2 and SPAN11 binding) or recycled receptors
(causing an increase in ATAP2 binding without a corresponding increase
in SPAN11 binding). These results suggest that the platelets fail to
recover their ability to respond to thrombin because they are unable to
replace cleaved receptors with intact ones.
In contrast to most G-protein-coupled receptors, thrombin receptor
activation by proteases involves an irreversible proteolytic event.
This means that to engender a new round of thrombin-responsiveness, cells must bring new receptors to their surface while clearing the old
ones. Several strategies appear to have evolved to accomplish this. In
megakaryoblastic HEL and CHRF-288 cells, essentially all of the cleaved
receptors are rapidly removed and then gradually replaced over a period
of hours by new receptors as they are synthesized (14). In human
umbilical vein endothelial cells and transfected fibroblasts, receptor
replacement initially occurs via a pool of preformed receptors, and
only later do freshly synthesized receptors emerge (17, 18).
The present studies examine the distribution of thrombin receptors on
resting platelets and the redistribution that occurs when platelets are
activated. In several respects the results are different from those
obtained with other types of cells. In resting platelets, thrombin
receptors were detectable in the plasma membrane and the membranes of
the surface connecting system, but not in the membranes of the
Following the addition of thrombin, there was a >90% loss of binding
sites for two antibodies whose epitopes are removed when thrombin
cleaves the receptor. There was also a 60% loss of binding sites for
two monoclonal antibodies whose epitopes are located COOH-terminal to
the thrombin cleavage site. The first of these observations suggests
that thrombin rapidly cleaves all of its available receptors on
platelets, just as it does on other cells. It also suggests that the
newly exposed receptors that were originally in the surface connecting
system have also been cleaved. The net loss of receptors requires
further explanation. We have shown previously in cells other than
platelets that the loss of binding sites for antibodies that bind
COOH-terminal to the thrombin receptor cleavage site can be due to
receptor internalization. However, although coated vesicles have been
observed in platelets, they have never been seen in great numbers (40,
41), and the study by Norton and co-workers (4) which was cited earlier
concluded that cleaved thrombin receptors remain on the platelet
surface. In that study, the insertion of the tethered ligand domain
into a protected environment following receptor cleavage was offered as
the explanation for the observed decrease in the binding of some
thrombin receptors antibodies when platelets are activated by
thrombin.
The present studies suggest that the fate of activated thrombin
receptors on human platelets is at least as complex as it is in other
cells. The fact that nonproteolytic activation of the receptors with
SFLLRN caused the same 60% decrease in antibody binding to the
platelet surface as did activation of the receptors with thrombin
argues against occlusion of the tethered ligand domain as the sole
cause of the reduction in antibody binding. Nevertheless, at least 40%
of the receptors clearly remain on the platelet surface after
activation. Of the remainder, some appear to be internalized, and some
appear to be shed into microparticles along with other integral
membrane proteins, at least under aggregating conditions. As was
suggested previously (4), some of the others may remain in the plasma
membrane but be inaccessible to any of the antibodies. Whether the 40%
of receptors which remain on the platelet surface following exposure to
thrombin are primarily those that were originally within the surface
connecting system cannot be determined from the present data, but it
seems unlikely since these receptors do become cleaved as they
emerge.
In cells other than platelets, thrombin receptor function is restored
over time following an initial exposure to thrombin. In HEL and
CHRF-288 cells this is accomplished by synthesizing new receptors that
are then moved to the cell surface. On endothelial cells and
fibroblasts, cleaved receptors are initially replaced from a large
intracellular store that is eventually replenished by synthesizing
additional receptors. In either case, the return of receptor function
parallels the return to the cell surface of intact receptors. Unlike
the megakaryocytes from which they arise, platelets have only a limited
capacity to synthesize proteins, and we were unable to detect an
intracellular receptor reserve other than the surface connecting
system, which would be exposed to thrombin too rapidly to provide a
useful reserve for future use. This leaves platelets without evident
means of restoring thrombin responsiveness. Conceptually, this makes
sense. Platelets apparently need to respond to thrombin only once,
after which they are incorporated into a growing hemostatic plug. The
structure of the one currently known thrombin receptor appears to be
the same in all cells. However, in platelets strategies have been adopted which maximize responses to thrombin while minimizing the cost
to the cell of maintaining pathways to produce fresh, but perhaps
unneeded, receptors on the cell surface.
Finally, the existence of additional types of thrombin receptors has
recently been inferred from knockout studies in mice (1, 47). Those
receptors may or may not ultimately prove to exist on human platelets.
However, their mechanism of activation is likely to involve proteolysis
by thrombin. Whether their location preceding activation and their
redistribution following activation are the same as for the one
thrombin receptor that has been identified remains to be determined,
but many of the same limitations and issues inherent in a
"single-shot" receptor that is activated by an irreversible event
are likely to apply to them as well on whatever cell they are
expressed.
We thank N. Blanchard and Yvonne Jacques for
technical assistance.
Departments of Medicine and Pathology and
the Center for Experimental Therapeutics of the University of
Pennsylvania, Philadelphia, Pennsylvania 19104, the
§ Istituto di Ricerche Farmacologiche Mario Negri, Consorzio
Mario Negri Sud, Santa Maria Imbaro 66030, Italy, the
Department of Pathology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
Antibodies
IIb
3) complex (24). EH1, an IgG1 antibody reactive with the human immunodeficiency virus type I nef protein, was
used as an isotype-matched control in the flow cytometry experiments (14). When indicated, the antibodies were purified using a protein A
column (Bio-Rad).
Fig. 1.
Antibodies directed against defined epitopes
within the thrombin receptor NH2 terminus. The figure
shows the sequence of the human thrombin receptor between residues 19 and 69, including the site of cleavage between Arg41 and
Ser42 (2). The numbered lines indicate the
sequences of the peptides used to prepare the antibodies, and except
for the polyclonal antibody (1047), the labels mark the approximate
location of the antibody epitopes.
[View Larger Version of this Image (9K GIF file)]
(a kind gift from T. Kunicki, Scripps Research Institute, La Jolla, CA), washed again,
incubated with a 1:400 dilution of phycoerythrin-labeled streptavidin,
washed, and analyzed by flow cytometry. Microparticles were gated as
AP-1-positive events (red fluorescence) smaller than platelets and
analyzed for the binding of thrombin receptor antibodies (green
fluorescence). In the studies in which aggregation was allowed to
occur, RGDS was omitted, and the platelets were stirred and activated
in the presence of 2 mM CaCl2. Samples were then immediately stained with fluorescein isothiocyanate-labeled AP-1
and the number of microparticles determined without washing.
-thrombin (3,000 units/mg) was provided by J. Fenton (New York State Department of
Health, Albany, NY). ADP and U46619 were obtained from Sigma.
Distribution of Thrombin Receptors on Resting Platelets
-granule membranes, nor was there a detectable intracellular pool of
thrombin receptors within a self-contained organelle such as is present
in endothelial cells and transfected fibroblasts (17, 18). Previous
studies using radioiodinated monoclonal antibodies have shown that
there are 1,500-2,000 copies of the thrombin receptor on the surface
of resting human platelets (3, 4), a number similar to the number of
moderate affinity 125I-thrombin binding sites (34, 35).
Data presented below suggest that the exposure of receptors within the
surface connecting system can increase this number by as much as 40%
during platelet activation, which means that in resting platelets
approximately two-thirds of the thrombin receptors are on the plasma
membrane and one-third are in the surface connecting membrane
system.
Fig. 2.
Electron microscopy of resting
platelets. Transmission electron microscopy of human platelets
illustrates the presence of the thrombin receptor on the plasma
membrane and in the surface connecting canalicular system
(SC). The -granules and other organelles are only rarely
labeled, consistent with background (magnification, × 50,000).
[View Larger Version of this Image (135K GIF file)]
IIb
3 integrin (glycoprotein IIb-IIIa).
Antibody binding to the platelet surface was detected by flow
cytometry. Incubation with thrombin caused a >90% loss of binding
sites for the cleavage-sensitive antibodies, SPAN11 and SPAN12, and a
60% decrease in the binding of the cleavage-insensitive antibodies,
WEDE15 (Figs. 3 and 4) and ATAP2 (see
Fig. 8). The decrease in SPAN12 binding was maximal within 5 min of the
addition of thrombin. The decrease in WEDE15 binding was maximal within 15 min (Fig. 3). These results suggest that thrombin rapidly cleaves all of its receptors on the platelet surface and causes a net decrease
in the number of detectable receptors by as much as 60% (see below).
Notably, incubating the platelets with SFLLRN caused a 60% decrease in
the binding of both the cleavage-sensitive and the cleavage-insensitive
antibodies (Fig. 4). Although it has been proposed that thrombin
receptor cleavage can decrease the binding of antireceptor antibodies
by allowing the insertion of the tethered ligand domain into a
protected environment (4), this explanation alone would not readily
account for the decrease in antibody binding seen when the receptors
were activated by SFLLRN.
Fig. 3.
Time course of the loss of antibody binding
sites from platelets incubated with thrombin. Platelets were
incubated with thrombin (0.25 unit/ml) at room temperature without
stirring for the times indicated after which hirudin (2 units/ml) was
added, and the samples were placed on ice. Antibody binding was
determined by flow cytometry. The results shown are the mean ± S.E. for three studies and are expressed as a percent of the mean
fluorescence intensity in the absence of thrombin.
[View Larger Version of this Image (20K GIF file)]
Fig. 4.
Binding of thrombin receptor antibodies to
human platelets. Washed human platelets were incubated without
stirring for 10 min at room temperature with the agonists shown.
Antibody binding was measured by flow cytometry. Antibodies WEDE15 and SPAN12 are directed against the thrombin receptor NH2
terminus (see Fig. 1). Antibody A2A9 binds to the integrin
IIb
3 complex. The final agonist
concentrations were: thrombin (0.25 unit/ml), SFLLRN (100 µM), ADP (10 µM), and U46619 (10 µM). The results shown are the mean ± S.E. of three
studies and are expressed as a percent of control after subtracting EH1
fluorescence. Mean fluorescence intensity values were: 99 for WEDE15,
51 for SPAN12, 255 for A2A9, and 6 for EH1.
[View Larger Version of this Image (51K GIF file)]
Fig. 8.
Lack of recovery of thrombin receptors on the
surface of platelets activated with thrombin. Platelets were
incubated with 0.25 unit/ml thrombin for 5 min after which 1 unit/ml
hirudin was added. The binding of antibodies ATAP2 and SPAN11 was
measured by flow cytometry at each of the times shown and is expressed as a percent of antibody binding to untreated platelets (mean ± S.E., n = 3).
[View Larger Version of this Image (20K GIF file)]
IIb
3 (Fig.
4).
IIb
3 complexes, like thrombin
receptors, are initially present in both the platelet plasma membrane
and the surface connecting system. Previous studies have shown that
platelet activation increases the amount of
IIb
3 on the platelet surface in part by
exposing complexes that were initially within the surface connecting
system (36-38) (for review, see Ref. 39). By extension, this suggests that the increase in WEDE15 and SPAN12 binding seen in Fig. 4 following
the addition of ADP and U46619 is due to the exposure of thrombin
receptors that were initially in the surface connecting system. It also
suggests that the net decrease in WEDE15 and ATAP2 binding caused by
thrombin and SFLLRN occurs despite the exposure of additional receptors
from the surface connecting system. If so, then the near complete loss
of binding sites for SPAN12 further suggests that the newly exposed
receptors must also be cleaved by
thrombin.1
antibody, AP-1. Thrombin receptors were detected with
antibody WEDE15. In the initial experiments, RGDS was included to
prevent platelet aggregation and limit microparticle formation (45).
The results show that thrombin receptors are present in the membrane
microparticles and that the addition of thrombin under these
conditions causes a decrease in WEDE15 binding to platelets but
has no effect on the binding of the antibody to microparticles (Fig.
5A).
Fig. 5.
Flow cytometry analysis of thrombin receptors
on microparticles. Panel A, platelets were incubated with
thrombin (0.25 unit/ml) or the agonist peptide SFLLRN (100 µM) 15 min at room temperature without stirring in the
presence of 200 µM RGDS. Afterwards, platelet and
membrane microparticles were separated and stained with the thrombin
receptor antibody WEDE15, as described under "Experimental
Procedures." The results shown are the mean ± S.E. for four
studies. Panel B, platelets were stimulated with thrombin or
SFLLRN without aggregation and stained with the glycoprotein Ib antibody, AP-1. The number of microparticles present
in the platelet suspension was determined and is expressed as a percent of total "particles" counted (mean ± S.E., n = 6). Panel C, platelets and microparticles were prepared
and analyzed as in panel B, except that aggregation was
allowed to occur by omitting RGDS, adding Ca2+, and
stirring the samples (average of two studies).
[View Larger Version of this Image (29K GIF file)]
-granules (30). In addition, occasional smaller intracellular
vesicles were seen which were stained sparsely with the thrombin
receptor antibody. These vesicles were not present in resting
platelets, and no gold labeling was seen when normal rabbit serum was
substituted for the thrombin receptor antibody as a control.
Fig. 6.
Electron microscopy of platelets activated
with thrombin. Shown is an aggregate of platelets from a sample
stimulated with thrombin (1 unit/ml) and then immunolabeled as in Fig.
2. The platelets (P) have degranulated and formed long
interdigitating extensions or filopodia (f). Most remarkable
is the marked decrease in gold labeled on the plasma membrane
(pm) compared with the resting platelets shown in Fig. 2, as
well as the presence of particles within intracellular vesicles
(arrows) of varying sizes. A few gold particles are also
present on the plasma membrane (arrowheads). The large
intracellular vacuoles (v) seen in platelet P
have been shown previously to contain the contents of fused
-granules, some of which are in contact with the extracellular space
(30). Magnification, × 65,000.
[View Larger Version of this Image (114K GIF file)]
Fig. 7.
Thrombin receptor response and
desensitization in human platelets. The tracings show the changes
in cytosolic Ca2+ which occurred following the addition of
1 unit/ml thrombin, 50 µM SFLLRN, or 5 units/ml hirudin
at room temperature without stirring. Panels A and
B, previously untreated platelets at time zero. Panel
C, platelets were incubated with thrombin for 10 min, after which
hirudin was added. The tracing shows the response when SFLLRN was added
4 h later. Panel D, the response of previously untreated platelets to SFLLRN 4 h after the start of the
experiment. The results are typical of those obtained in five such
studies.
[View Larger Version of this Image (24K GIF file)]
-granules. In contrast to endothelial cells and fibroblasts, there
was no evidence for the existence of an intracellular pool of thrombin
receptors. However, the number of thrombin receptors initially within
the surface connecting system appears to be substantial since the
addition of ADP and U46619, which expose the surface connecting system,
increased the number of binding sites for thrombin receptor antibodies
by 40%. Using the average of 1,500-2,000 plasma membrane thrombin receptors per platelet previously determined with radioiodinated antibodies (3), this suggests that resting platelets have 600-800 thrombin receptors per cell in the surface connecting system. Receptors
in the surface connecting system are not accessible to antibodies
before platelet activation, but they are also not in a self-contained
organelle since the surface connecting system opens on to the plasma
membrane even in resting platelets. The exposure of the surface
connecting system by agonists other than thrombin provides a mechanism
by which such agonists may increase the number of activable thrombin
receptors present on the platelet surface. Since thrombin receptor
responses are directly related to the number of thrombin receptors
activated (32), this may be partially responsible for the synergism
between ADP and thrombin reported 30 years ago by Niewiarowski and
Thomas (46).
*
These studies were supported in part by funds from National
Institutes of Health Grants HL40387 (to L. F. B.), HL31610 (to D. F. B.), and HL44907 (to S. R. C.). Peptide synthesis by the University of Pennsylvania Medical Center Protein Chemistry Facility was supported by National Institutes of Health Grants CA-16520 and
DK-19525. The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Supported by a fellowship from the Fogarty International
Center and by funds from the Italian National Council (Convenzione Consorzio Mario Negri Sud) and Progetto Finalizzato FATMA Contract 94.00951.41).
To whom correspondence should be addressed: University of
Pennsylvania, CRB 678, 415 Curie Blvd., Philadelphia, PA 19104. Tel.:
215-573-3540; Fax: 215-573-2189; E-mail:
brass{at}mail.med.upenn.edu.
1
This conclusion derives from the following
calculation. If there were originally 100 thrombin receptors on the
platelet surface and an additional 40 in the surface connecting system,
then activated platelets would have (transiently) a total of 140, as
was observed with ADP and U46619. If all of the original surface
receptors were cleaved, but the newly exposed receptors were not, then
40 intact receptors would remain on the platelet surface, which would leave 40% of the original number of SPAN12 binding sites, not the 10%
that was observed.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.