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J. Biol. Chem., Vol. 277, Issue 4, 3011-3019, January 25, 2002
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From the Department of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania 19104
Received for publication, October 9, 2001
The platelet response to collagen is a primary
event in hemostasis and thrombosis, but the precise roles of the
numerous identified platelet collagen receptors remain incompletely
defined. Attention has recently focused on glycoprotein VI (GPVI), a
receptor that is expressed on platelets in association with a signaling
adapter, the Fc receptor gamma chain (Fc R The response of platelets to vessel wall injury is a primary event
in arterial thrombosis (1). Platelets respond to vessel wall injury via
surface receptors that recognize exposed subendothelial matrix
proteins, the most abundant of which is collagen. Collagen is unusual
among platelet ligands because it mediates both platelet adhesion and
platelet activation (2). Platelet adhesion to collagen initiates the
thrombotic response, and platelet activation in response to collagen
accelerates the thrombotic response through release of granule contents
and activation of platelet integrins. The ability of collagen to
mediate both of these steps suggests that the platelet response to
collagen may be a key step in the regulation of arterial thrombosis. A
molecular understanding of the platelet receptors that mediate collagen
responses is therefore likely to shed light on the pathogenesis of
common vascular diseases such as myocardial infarction and stroke.
The receptors for collagen on the surface of platelets may be divided
into those which interact indirectly through collagen-bound von
Willebrand factor (vWF),1
including GPIb To address the role of GPVI we have expressed the receptor in RBL-2H3
cells, a basophilic leukemia cell line that lacks GPVI and In the present study we have used a novel anti-GPVI monoclonal antibody
to directly measure GPVI receptor density on human platelets and on
both previously described and newly generated GPVI-expressing RBL-2H3
cells. Our results demonstrate that GPVI expression is sufficient to
confer both adhesive and signaling responses to collagen independent of
other collagen receptors but that GPVI-collagen responses are strictly
dependent on receptor density. A 5-fold difference in GPVI receptor
density separates RBL-2H3 cells that exhibit no collagen responses (14)
from those that exhibit both adhesion and signaling responses.
Comparison of the GPVI receptor density required to confer collagen
responses in RBL-2H3 cells with that on the surface of human platelets
reveals that platelets have a GPVI receptor density equivalent to that required to confer a full collagen response in RBL-2H3 cells. To
determine whether the GPVI expression level on human platelets modulates collagen responses we measured GPVI levels on the platelets of a small but genetically diverse group of individuals and found only
minimal variation compared with those of Materials and Animals--
Fibrillar collagen and convulxin used
in the studies are from the same sources as previously described (14).
Fluorescein isothiocyanate (FITC)-conjugated anti-FLAG (Clone M2) was
purchased from Sigma. Anti-human GPVI monoclonal antibody (clone HY101; mouse IgG1, Generation of GPVI-expressing RBL-2H3 Cells--
RBL-2H3 cells
stably expressing FLAG-GPVI were generated as previously described. A
total of 177 G418-resistant stable clones were screened for FLAG
epitope expression using M2 anti-FLAG antibody and for collagen
signaling and adhesion as described below; selected clones were chosen
for further analysis.
Generation of the Anti-GPVI Monoclonal Antibody
HY101--
To generate monoclonal antibodies versus the
human GPVI receptor we expressed FLAG-GPVI R272L (14) on the surface of
3T3 fibroblasts derived from BALB/c mice (ATCC) that were injected intraperitoneally as immunogens. The GPVI R272L mutant, which has a
wild-type extracellular domain, was expressed to ensure that surface
GPVI expression was not limited by lack of co-expressed Fc R Intracellular Calcium Studies--
Increases in cytoplasmic
calcium were measured using the calcium-sensitive dye Fura-2 as
previously described (14).
Measurement of RBL-2H3 Cell Adhesion--
2 × 105 cells were allowed to adhere to the plate coated
overnight with 1 µg/well of CVX or type I collagen for 5', 15', and 30', respectively. The plate was washed with PBS several times. The number of cells bound to the plate was correlated to intracellular Platelet Aggregation Assays--
Mouse blood was drawn into
citrate buffer from the inferior vena cava of mice anesthetized by
pentobarbital. Platelet-rich plasma was separated from the rest of the
blood cells by centrifugation of 1-2× diluted blood with modified
Tyrode's buffer at 220 g for 10 min at room temperature.
Plasma-free platelets were obtained by filtering platelet-rich plasma
through a Sepharose 2B column (Amersham Biosciences) in modified
Tyrode's buffer (137 mM NaCl, 20 mM HEPES, 5.6 mM Glucose, 1 mg/ml BSA, 1 mM
MgCl2, 2.7 mM KCl, and 3.3 mM
NaH2PO4, pH 7.4). The platelets were counted in
a Coulter Counter and adjusted to 5 × 108/ml in
modified Tyrode's buffer. In each aggregation test, 0.25 ml of
filtered platelets was stimulated with 20 µM ADP or
collagen ranging from 1.25 to 10 µg/ml. Platelet aggregation was
monitored in a Chrono-log aggregometer.
Western Blotting--
RBL cells and GPVI-expressing RBL cells
were lysed in radioimmune precipitation (RIPA) buffer, and Western
blotting was performed as previously described (14).
Live Cell Immunoprecipitation--
Ten million cells were
incubated with 1 ml of HY101 supernatant for 2 h at 4 °C with
rocking. The treated cells were then washed with cold PBS three times
and lysed in radioimmune precipitation buffer. 20 µl of protein
G-agarose was added into each type of lysate supernatant and incubated
at 4 °C for 1 h with rocking. The protein G-agarose pellets
were washed with radioimmune precipitation buffer three times, mixed
with 50 µl of 2× sample buffer supplemented with 10%
2-mercaptoethanol, and boiled for 5 min. The supernatant of protein
G-agarose was analyzed in SDS-PAGE and Western blotting as described
above except that the primary antibody was 1 µg/ml anti-FLAG M2
antibody instead of the hybridoma supernatant.
Generation of FITC-HY101 and 125I-HY101--
HY101
was covalently modified with FITC using the FluoReporter labeling kit
according to the manufacturer's instructions (Molecular Probes
F-6434). FITC-conjugated HY101 with 5.1 FITC molecules per antibody was
obtained. Iodination of HY101 was carried out using Iodo-tubes
according to the manufacturer's instructions (Pierce).
Determination of GPVI Receptor Density Using
125I-HY101--
Human platelet-rich plasma and
GPVI-expressing RBL cells were incubated with 0-3 µg/ml
125I-HY101 for 1 h at room temperature. The
cell/antibody mixtures were loaded on top of a sucrose solution (500 µl of 20% w/v sucrose in 5 mM EDTA and 0.1 M
PBS, pH 7.4) in microcentrifuge tubes and centrifuged for 5 min at
12,000 g for platelets and 15 min at 200 g for RBL cells. The
supernatants were aspirated, and radioactivity of cell pellets was
counted for determination of the total 125I-HY101 bound.
Nonspecific binding was determined in the presence of 50-fold excess of
unlabeled HY101 and was subtracted from the values for the total
125I-HY101 bound. 125I-HY101-specific binding
was converted to the number of GPVI receptors per cell based on the
specific activity of 125I-HY101 and the assumption that one
antibody binds to one GPVI molecule. To determine the percent platelet
GPVI receptor density of different GPVI-expressing RBL-2H3 cells, we
used an average platelet diameter of 1 µM (17) and an
average RBL-2H3 cell diameter of 10 µM. The RBL-2H3 cell
diameter was estimated using the "three-quarter size method" with a
single threshold Coulter Z series counter (Coulter, Hileah, FL).
Briefly, values for the mean and median of the cell population
distribution were obtained by taking a value representing 75% of the
modal particle diameter; modal value was determined after current, and
gain was optimized using the "plateau" method. Latex beads (10.05 µm) were used to precalibrate the machine. The mean/median bead size
was estimated to be 10.10 µm ± 0.35 by the three-quarter size
technique (n = 6). The RBL cell population had a skewed
distribution with median 10.80 µm ± 1.07 µm and mode 12.26 µm ± 0.78 µm (n = 6). These results agree with published data using direct biophysical methods (18, 19). Cell
diameter was used to obtain a ratio of 100:1 for RBL-2H3:platelet surface area (based on the surface area as a function of
r2). The percent platelet receptor density was therefore
calculated as RBL-2H3 receptor/1260.
Characterization of Anti-GPVI Monoclonal Antibody HY101--
To
precisely measure GPVI receptor density on RBL-2H3 cells and human
platelets we raised monoclonal antibodies against mouse BALB/c 3T3
fibroblasts, which express the mutant GPVI receptor FLAG-GPVI R272L on
the surface (GPVI-3T3) as described under "Experimental Procedures." GPVI-3T3 express a large number of FLAG-GPVI R272L on
the cell surface presumably because the R272L mutation uncouples GPVI
receptor expression from that of Fc R GPVI Levels on Human Platelets Vary Less than and Independently of
Comparison of GPVI Receptor Density on GPVI-expressing RBL-2H3
Cells with That on Human Platelets--
We have described previously
GPVI-expressing RBL-2H3 cells that respond to the GPVI ligand CVX but
not to collagen (14). The absence of a GPVI antibody made direct
comparison of GPVI receptor density on these cells with that on
platelets difficult, and the conclusion that the two cell types had
similar GPVI receptor densities was reached on the basis of nearly
identical CVX dose responses (14). The development of the anti-GPVI
antibody HY101 allowed us to directly measure the GPVI receptor number
on the GPVI-expressing RBL-2H3 cells we had originally characterized (GPVI-ori) and to compare the receptor density on the model cell line
with that on human platelets. Our results (summarized in Table
I) revealed that despite robust responses
to CVX the GPVI receptor density on GPVI-ori is only ~20% of that
found on human platelets. These results suggest that the failure to
confer collagen responses in those cells may have been because of
inadequate receptor density, and efforts were made to generate new
GPVI-expressing RBL-2H3 cell lines with GPVI receptor densities closer
to that of human platelets. Of the new GPVI-expressing cell lines
identified, two were chosen for further analysis of collagen adhesive
and signaling responses. GPVI-163 expresses GPVI at approximately half
the receptor density of platelets, and GPVI-173 expresses GPVI at
roughly the same density as platelets (Table I and Fig. 3).
GPVI Expression in RBL-2H3 Cells Confers Collagen Adhesion in a
Receptor Density-dependent Fashion--
To further address
the role of GPVI in platelet adhesion to collagen, static adhesion
assays were performed using GPVI-ori, GPVI-163, and GPVI-173. As
previously reported, GPVI-expressing cells with the lowest density of
GPVI receptors bound CVX but not collagen (Fig.
4). GPVI-163 and GPVI-173, however, both
adhered to collagen-coated surfaces but did so more slowly and to a
lesser extent than to CVX-coated surfaces (Fig. 4). Similar to adhesion to CVX, however, GPVI-mediated adhesion to collagen does not require Mg2+ or Ca2+ (data not shown). These results
demonstrate that GPVI expression is sufficient to confer static
adhesion to collagen but that GPVI-collagen interaction requires a
threshold receptor density that is >20% of that expressed on human
platelets. The role of receptor density for GPVI-collagen adhesive
interaction is further revealed by the slower rate of adhesion observed
using GPVI-163 cells that express half the receptor number of
GPVI-173.
GPVI Expression in RBL-2H3 Cells Confers Collagen Signaling in a
Receptor Density-dependent Fashion--
The ability to
confer collagen adhesion through expression of GPVI at an appropriate
receptor density demonstrates direct GPVI-collagen interaction and
suggests that collagen signaling may also be mediated by GPVI in a
density-dependent fashion. To test the role of GPVI
receptor density for collagen signaling we tested the ability of
GPVI-ori, GPVI-163, and GPVI-173 to mediate calcium signaling to 10 µg/ml fibrillar collagen, a concentration that typically initiates
robust platelet activation (Fig. 6 and data not shown). As previously
described, GPVI-ori responded to CVX but not to collagen (Fig.
5). Interestingly, GPVI-163 also demonstrated no calcium signaling responses to collagen despite being
able to adhere to collagen and expressing GPVI at a receptor density
that is approximately half that in human platelets (Fig. 5). GPVI-173,
however, exhibited a reproducible calcium flux in response to collagen
concentrations as low as 1 µg/ml (Fig. 5 and data not shown). CVX
signaling responses were observed in all three GPVI-expressing cell
lines but were more rapid and greater in magnitude in the
GPVI-expressing cells with higher receptor densities. These results
definitively show that GPVI is capable of mediating collagen signaling
independently of other platelet collagen receptors and that GPVI
signaling in response to collagen, similar to GPVI adhesion to
collagen, is critically dependent on receptor density.
Unlike GPVI-expressing RBL-2H3 Cells, Mouse Platelets with a 50%
Reduction in GPVI Receptors Exhibit Only a Minimal Reduction in
Collagen Signaling--
The observation that collagen signaling can be
observed in RBL-2H3 cells that express GPVI at roughly the receptor
density of human platelets but not in cells that express only half that receptor density suggests that platelet collagen responses may be
exquisitely sensitive to a drop in the level of GPVI receptors. To test
this hypothesis we studied mice heterozygous for a deletion in the Fc
R Collagen adhesion and signaling are believed to play early
critical roles in the platelet response to vessel injury, but the molecular events that underlie these responses remain poorly
understood. It is generally accepted that the first platelet response
to exposed collagen is the successive formation and breakage of
platelet GPIb To define the role of GPVI we have expressed GPVI in RBL-2H3 cells, a
basophilic cell line that expresses endogenous Fc R Our observation that GPVI is sufficient for both collagen adhesion and
signaling is supported by the recent report that mouse platelets
deficient in One potential explanation for this discrepancy is suggested by our
finding that GPVI-collagen responses vary markedly with receptor
expression level. It is possible that in the presence of higher levels
of GPVI These studies are the first to clearly confer platelet collagen
responses through expression of GPVI and help resolve some of the
conflicting observations that have been made, because the receptor and
gene encoding it were identified. It is now clear that GPVI is
independently capable of mediating all of the described platelet
responses to collagen, but precisely how it functions in the context of
other platelet collagen receptors and signaling responses remains to be
worked out. Understanding the specific roles of other collagen
receptors such as We thank Dr. Jim Hoxie and Beth Haggarty in
the Hybridoma Core Facility established by National Heart, Lung, and
Blood Institute Grant P01 HL-40387 for help and advice.
*
This work was supported by grants from the W. W. Smith
Charitable Trust and the American Heart Association (to M. L. K.).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.
Published, JBC Papers in Press, November 26, 2001, DOI 10.1074/jbc.M109714200
2
B. Nieswandt, personal communication and
manuscript submitted.
The abbreviations used are:
vWF, von Willebrand
factor;
GPVI, glycoprotein VI;
Fc R
The Platelet Receptor GPVI Mediates Both Adhesion and Signaling
Responses to Collagen in a Receptor Density-dependent
Fashion*
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
). Genetic and
pharmacologic loss of GPVI function results in loss of collagen
signaling in platelets, but studies to date have failed to demonstrate
that GPVI-Fc R expression is sufficient to confer collagen signaling responses. These results have led to the hypothesis that collagen responses mediated by GPVI-Fc R may require the collagen-binding integrin 
2
1 as a co-receptor, but this model has not been
supported by a recent study of mouse platelets lacking 21. In the
present study we have used a novel anti-GPVI monoclonal antibody to
measure the level of GPVI on human platelets and to guide the
development of GPVI-expressing cell lines to assess the role of GPVI in
mediating platelet collagen responses. GPVI receptor density on human
platelets appears tightly regulated, is independent from the level of
21 expression, and significantly exceeds that on previously
characterized GPVI-expressing RBL-2H3 cells. Using newly
generated GPVI-expressing RBL-2H3 cells with receptor densities
equivalent to that on human platelets, we demonstrate that GPVI
expression confers both adhesive and signaling responses to collagen in
a graded fashion that is proportional to the GPVI receptor density.
These results resolve some of the conflicting data regarding
GPVI-collagen interactions and demonstrate that 1) GPVI-Fc R
expression is sufficient to confer both adhesion and signaling
responses to collagen, and 2) GPVI-mediated collagen responses are
receptor density-dependent at the receptor levels expressed
on human platelets.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and the integrin IIb3,
and those which interact directly with collagen, including glycoprotein
VI (GPVI), the integrin 21, CD36, and perhaps others (recently
reviewed in Ref. 3). The present model of platelet responses to
collagen is one of successive receptor interactions mediating rolling, firm adhesion to collagen and platelet activation (4, 5). Although the
receptor actions responsible for collagen-vWF interaction have been
clearly identified (6, 7), those responsible for direct collagen
interactions remain unclear. A recently proposed two-receptor, two-step
model in which adhesion to collagen is first mediated by high affinity
interaction with 21 and platelet activation is subsequently
initiated by low affinity interaction with GPVI (5) is attractive
because it assigns unique roles to each receptor and presents a
stepwise sequence of events that resemble those described for
leukocyte-endothelial interactions during inflammatory responses (8).
Unfortunately, the experimental data reported to date have been
conflicting and have failed to support this model or to demonstrate
clear and sufficient roles for 21 and GPVI. Human 21
deficiency states reportedly result in bleeding disorders and platelets
with severely reduced collagen responses (9, 10), but a recent analysis
of mice lacking platelet 21 demonstrated normal bleeding times,
normal platelet adhesion to collagen, and almost no loss of platelet
signaling responses to collagen (11). Deficiency of GPVI and its
signaling partner Fc R, however, results in loss of platelet
collagen responses in both human and mouse platelets (11-13). Thus
loss of function studies have presented a confusing picture in which
the roles of multiple collagen receptors may be required but are poorly understood. In an effort to more precisely define the roles of individual collagen receptors we have developed a model system with
which to examine collagen receptor function by conferring a gain of
function to collagen-unresponsive cells.
21 but
expresses the GPVI signaling co-receptor Fc R (14). Heterologous
expression of GPVI in RBL-2H3 cells confirmed the critical role of Fc
R for GPVI signaling and demonstrated that both the GPVI
transmembrane domain and C-tail are required for Fc R coupling (14).
Surprisingly, GPVI expression conferred both signaling and adhesive
responses to the high affinity GPVI ligand convulxin (CVX), a snake
venom protein used to biochemically purify the receptor (15), but
failed to confer either adhesive or signaling responses to collagen
(14). These results suggest that GPVI might utilize one or more of the
other platelet collagen receptors as a co-receptor for collagen
interaction. A limitation of this study, however, was the inability to
directly compare GPVI receptor density on RBL-2H3 cells with that on
human platelets because of the lack of an available anti-GPVI antibody.
21. Our findings are
generally consistent with those obtained using 1-deficient mouse
platelets and support a model of platelet collagen responses in which
GPVI plays a central role for both adhesion and signaling. Determining the role of GPVI in the pathogenesis of arterial thrombotic diseases such as stroke and myocardial infarction is an important area of future investigation.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) was produced in our laboratory (see below).
FITC-conjugated anti-rat alpha 2 antibody (clone Ha12/9, hamster) was
obtained from BD PharMingen. RBL-2H3 cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and 10 µg/ml gentamicin. Fc receptor gamma chain (Fc R)-deficient mice
(Taconic Farms) were crossed to BALB/c mice (Ace Animals, Boyertown,
PA) to generate Fc R heterozygous mice, Fc R+/
. All
of the mice used for study were maintained in the animal facility of
the University of Pennsylvania.
(14).
Hybridomas were generated using standard techniques by the monoclonal
antibody core facility at the University of Pennsylvania. Hybridoma
supernatants were pooled (n = 8 or 12) and screened on
GPVI-expressing RBL-2H3 cells for reactivity to GPVI. Reactivity
versus human GPVI was confirmed on human platelets using
fluorescence-activated cell sorter analysis. Pure antibody was prepared
by affinity chromatography on protein G-Sepharose (Mab Trap G II kit,
Amersham Biosciences) from HY101 ascites fluid.
-hexosaminidase activity detected as previously described (14) using
the endogenous RBL-2H3 cell enzyme -hexosaminidase to measure cell
number (16).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, a protein not expressed in
fibroblasts but normally required for GPVI expression (14, 20).
FLAG-GPVI R272L has a wild-type extracellular domain and a molecular
weight indistinguishable from wild-type GPVI (Ref. 14, data not shown).
One antibody (designated HY101) was identified, which bound to the
surface of FLAG-GPVI-expressing RBL-2H3 cells and GPVI-3T3 cells in a
manner identical to the anti-FLAG antibody but did not bind
untransfected RBL-2H3 or 3T3 cells (Fig.
1A), consistent with
recognition of FLAG-GPVI. Western blotting of cell lysate derived from
wild-type and FLAG GPVI-expressing RBL-2H3 cells with HY101 identified
a 60-kDa band in the lysate from GPVI-expressing cells but not
wild-type cells (Fig. 1B). Finally, immunoprecipitation studies using HY101 identified the 60-kDa protein recognized by HY101
as FLAG-GPVI using M2 anti-FLAG secondary antibody (Fig. 1C). The ability of HY101 to recognize wild-type GPVI
on the surface of human platelets was demonstrated by
fluorescence-activated cell sorter analysis (Fig.
2 and discussed below). Thus HY101 is a
novel monoclonal antibody that recognizes the extracellular domain of
human GPVI. Binding of HY101 to human platelets or GPVI-expressing RBL2H3 cells does not interfere with either collagen or CVX signaling (data not shown), suggesting that it binds a region of the
extracellular domain of GPVI distinct from the binding sites of both
ligands.
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Fig. 1.
Characterization of a novel anti-human GPVI
monoclonal antibody, HY101. The monoclonal antibody HY101 was
raised against human GPVI expressed on the surface of mouse fibroblasts
as described under "Experimental Procedures." A, HY101
recognizes FLAG-GPVI-expressing RBL-2H3 (GPVI-RBL) and 3T3
(GPVI-3T3) cells in a manner identical to the anti-FLAG
antibody M2. Flow cytometry on cells stably transfected with FLAG-GPVI
(unfilled) is overlaid onto that of the untransfected
parental cell line (filled). B, HY101 recognizes
GPVI by Western blot analysis of lysate derived from GPVI-expressing
RBL-2H3 cells (GPVI-173) but not wild-type RBL-2H3 cells
(wt RBL). C, live cell immunoprecipitation of
GPVI-expressing RBL-2H3 cell lines (GPVI-ori and
GPVI-163) but not wild-type RBL-2H3 cells with HY101
followed by Western blotting with M2 antibody demonstrates that HY101
binds FLAG-GPVI.
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Fig. 2.
Measurement of GPVI receptor density on human
platelets and surface expression of GPVI versus the
integrin 21.
A, 125I-HY101 binding to human platelets. The
receptor number per platelet was calculated as described under
"Experimental Procedures." Data shown are the mean and standard
deviation of triplicate samples and are representative of six
experiments performed on three individuals with similar results.
B, comparison of GPVI receptor levels on six individuals
using FITC-HY101. The six curves shown were obtained with platelets
from three males, three females, three Caucasians, two Asians and one
African-American. This experiment was repeated twice with identical
results. C, comparison of 21 receptor levels on six
individuals using FITC-anti-2 antibody. The individuals studied are
the same as in B, and lines of the same color represent a
single individual. This experiment was also repeated twice with
identical results. Note the significant variability in platelet 2
levels but minimal variability in platelet GPVI levels.
21 Levels--
To quantitate the number of GPVI receptors on
human platelets we used both FITC-HY101 and 125I-HY101
binding. HY101 binding to human platelets is highly specific (Fig.
2A). Analysis of platelets from three individuals with
125I-HY101 (using a full dose-response curve as shown in
Fig. 2A in triplicate for each individual) reveals an
average GPVI receptor density of 1260 ± 190 per platelet (Fig.
2A and data not shown). Virtually identical numbers,
1250 ± 210 receptors per platelet, were obtained using mean
fluorescence and FITC-HY101 analysis of 20 individuals from Caucasian,
Asian, and African-American backgrounds of both sexes (Fig.
2B and data not shown). As reflected by the small standard
deviations, GPVI receptor levels varied by less than 40% in this small
but genetically diverse group of individuals (Fig. 2B). In
contrast, analysis of 21 levels in the same platelets exhibited a
more than 3-fold variation (Fig. 2C). Although these results
are limited by the small size of the group studied, the level of GPVI
receptors on human platelets does not appear to vary as extensively as
or in correlation with that of 21.
GPVI receptor density on three GPVI-expressing RBL-2H3 cell lines and
comparison with that on human platelets
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Fig. 3.
Comparison of GPVI receptor expression levels
in three GPVI-expressing RBL-2H3 cell lines. FLAG-GPVI receptor
level was measured using flow cytometry with FITC-M2 on a previously
characterized GPVI-expressing RBL-2H3 cell line (GPVI-ori)
and on two newly isolated GPVI-expressing RBL-2H3 cell lines expressing
significantly higher levels of GPVI (GPVI-163 and
GPVI-173).
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Fig. 4.
Adhesion of GPVI-expressing RBL-2H3 cells to
convulxin and collagen. Microtiter plates were coated with type I
fibrillar collagen or convulxin, and adhesion of GPVI-expressing RBL
cells was measured at OD405 using a
colorimetric substrate of the endogenous RBL-2H3 enzyme
hexoseaminidase. The results shown are the mean and standard deviation
of a single experiment performed in quadruplicate and are
representative of three distinct experiments. Note that strong adhesion
to CVX was observed for all GPVI-expressing cell lines but that only
GPVI-163 and GPVI-173 demonstrated adhesion to collagen.
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Fig. 5.
GPVI signaling responses to collagen are
receptor density-dependent. Three distinct
GPVI-expressing RBL-2H3 cell lines were exposed to convulxin (10 nM) or collagen (10 µg/ml), and calcium signaling was
measured using the calcium-sensitive dye Fura-2. Note that although CVX
signaling was observed in all GPVI-expressing RBL-2H3 cells collagen
signaling was only observed in those expressing the highest levels of
GPVI (GPVI-173).
gene (21), whose expression is required for GPVI (14).
Heterozygous loss of Fc R (Fc R +/) is predicted to
halve the number of Fc R partners, and measurement of GPVI levels in
Fc R heterozygote platelets using the anti-mouse GPVI antibody JAQ1
does indeed reveal a 50% drop in receptor
level.2 Surprisingly,
platelets from Fc R+/ mice revealed only small
reductions in aggregation responses to collagen (Fig.
6). As expected, the aggregation
responses to ADP were unchanged between Fc R+/+ and Fc
R+/ platelets. Thus although a 50% reduction in GPVI
receptor density virtually eliminates collagen signaling in
GPVI-expressing RBL-2H3 cells, a similar reduction has very little
effect in mouse platelets. Whether this difference reflects
amplification of signaling in platelets or the contribution of other
platelet collagen receptors is uncertain and is discussed below.
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Fig. 6.
Collagen activation of platelets from Fc
R +/ mice. Platelet aggregation responses to
varying concentrations of fibrillar collagen and ADP (20 µM) were measured in wild-type (Fc R+/+)
mouse platelets and those obtained from mice with only a single Fc R
allele (Fc R+/), which express GPVI-Fc R at
50% of wild-type levels. A, platelet aggregation curves
obtained following stimulation with varying concentrations of collagen
or ADP. B, maximum percent aggregation obtained following
collagen or ADP stimulation. C, lag time to onset of
platelet shape change following stimulation with collagen or ADP.
Wild-type platelet responses are shown as black bars, and Fc
R +/ platelet responses are shown as white
bars.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-vWF tethers at high shear (6, 7), an interaction that allows platelets to roll slowly on exposed subendothelial matrix and
facilitates subsequent platelet-collagen interactions. The nature and
sequence of these subsequent platelet interactions with collagen,
however, remain debatable. One model of platelet-collagen interaction
proposes that firm adhesion is (at least in part) mediated by a high
affinity 21-collagen interaction that enables subsequent collagen
signaling via a low affinity GPVI-collagen interaction (5). To date
this model has been tested using loss of function experiments with
conflicting results, and the need for a revised model is evident (22).
Human and mouse GPVI deficiency states support a necessary role for
GPVI-Fc R in collagen signaling in platelets (20, 23), but studies
of human and mouse 21 deficiency states have yielded disparate
results regarding the requirement for 21 for both collagen
adhesion and collagen signaling in platelets (9-11). Two significant
limitations are inherent in these loss of function experiments. First
is the inability to distinguish between loss of function due to
interruption of the action of a single receptor versus
interruption of sequential receptor-receptor interactions
(e.g. loss of collagen adhesion associated with loss of GPVI
may result from the loss of direct GPVI-collagen interaction or from
the loss of GPVI-dependent 21 integrin activation
(11, 24)). Second is the inability to distinguish between loss of
function due to the action of a single receptor versus that
due to the cumulative action of multiple independent receptors
(e.g. GPVI and 21 may act independently, but the
actions of both are required for platelet collagen responses). Given
the large number of platelet collagen receptors and signaling pathways
it is therefore not surprising that loss of function experiments alone
have not provided a clear picture of the events involved in platelet
collagen adhesion and signaling. One way to further clarify the
function of individual collagen receptors is to define sufficient roles
by means of heterologous expression in collagen receptor-deficient cells.
chain and is a
model cell line for studying Fc
RI receptor signaling but does not
express GPVI or 21 (data not shown). As in platelets, Fc R
chain signaling in RBL-2H3 cells results in mobilization of
intracellular calcium and degranulation (25). We have shown previously
that expression of GPVI in RBL-2H3 cells could confer adhesion and
signaling to the GPVI-specific agonist CVX but not to collagen (14).
This was a surprising result given the evidence that GPVI is required
for collagen signaling in platelets, and it suggested that either GPVI
might be necessary but not sufficient for platelet collagen responses
(e.g. perhaps due to a requirement of another collagen
receptor(s) such as 21 for ligand binding) or that our model
system did not completely reproduce GPVI expression or function on
platelets despite platelet-like CVX responses. To examine the latter
explanation we have raised a monoclonal antibody that recognizes human
GPVI and directly measured GPVI receptor levels on human platelets and
on our previously characterized GPVI-expressing cell lines. The finding
that our original GPVI-expressing cell lines have a significantly lower
GPVI receptor density than platelets suggested that the difference in
GPVI receptor density may be responsible for the inability to confer
collagen responses. In the present study we have directly tested this
hypothesis, and our results demonstrate that expression of GPVI in the
absence of other platelet collagen receptors is sufficient to confer
both collagen adhesion as well as signaling and that GPVI-collagen responses are both proportional to and highly dependent on receptor density. This observation has two important implications that are
discussed further below. 1) Under circumstances resembling the
experimental conditions employed for this study, GPVI is sufficient for
some or all platelet collagen responses. 2) Relatively small differences in platelet GPVI receptor expression (e.g. 2- or
3-fold) may significantly alter platelet collagen responsiveness and
thereby raise or lower the threshold for arterial thrombotic responses in vivo.
21 exhibit normal adhesion to collagen under static
and flow conditions as well as near normal aggregation responses to
collagen, unless GPVI function is blocked by blocking antibody (11).
Together these studies place GPVI-Fc R at the center of
platelet-collagen interactions and appear to relegate other collagen
receptors such as 21 to non-essential, accessory roles. It is
difficult to reconcile these observations, however, with the reports of
21-deficient individuals who display bleeding phenotypes and
markedly abnormal platelet collagen responses (9, 10). It has been
suggested that this discrepancy may be caused by additional
uncharacterized platelet defects in the individuals studied or may
reflect a significant species difference in the utilization of collagen
receptors (11). Certainly bleeding phenotypes in mice due to platelet
defects are less likely to appear spontaneously and are more difficult
to detect than human bleeding phenotypes, as illustrated by the
identification of GPVI-deficient individuals through analysis of a
bleeding phenotype (12, 23), whereas the loss of collagen responses in
GPVI-Fc R-deficient mice went virtually undetected (21). The
different aggregation responses to fibrillar collagen in human and
mouse platelets lacking 21, however, are more difficult to
reconcile. In the absence of other identifiable platelet defects in
21-deficient humans this discrepancy raises doubts regarding the
application of genetic studies in mice to understand human platelet
collagen responses.
21 plays a redundant role, but in the presence of lower
levels of GPVI 21 is required for normal platelet collagen
adhesion and signaling. Thus platelets from individuals deficient in
21 who also express lower than normal levels of GPVI may present
deficient platelet collagen responses. Such genetic variability in
receptor expression level is unlikely to be observed in inbred strains
of mice. The ability of 21 to contribute to platelet collagen
responses (even if it is not required for them) is also supported by
studies of individuals with polymorphisms associated with high levels
of platelet 21 who exhibit augmented platelet collagen responses
(26) and appear to be at an increased risk for stroke (27). In an
attempt to test the role of 21 in the presence of lower and
higher levels of GPVI we examined the expression of these two collagen
receptors on a sample population of mixed sex and ethnic background.
Whereas a greater than 3-fold variability in the level of 21 was
observed, GPVI receptor levels varied by less than 40%. Therefore,
although more significant variability in GPVI receptor levels is likely
to be detected in a larger sample population, it appears that GPVI
receptor levels vary significantly less than those of 21 and that
individuals with very low or very high levels of GPVI may be unusual.
In an effort to test the effect of a 50% reduction in GPVI levels in platelets in the presence of unchanged levels of 21, we studied platelets derived from mice in which one of two Fc R alleles was
inactivated by gene targeting (Fc R+/ platelets).
Unlike GPVI-expressing RBL-2H3 cells, however, Fc R+/
platelets exhibited only minimal reductions in collagen-induced aggregation responses. Potential explanations for the difference observed in dropping GPVI receptor levels by 50% in platelets versus RBL-2H3 cells include: 1) direct signaling
contributions by other platelet collagen receptors; 2) indirect
augmentation of signaling responses in platelets (e.g.
release of ADP and TXA2) that are not present in RBL-2H3
cells; 3) a greater than 2-fold miscalculation of the relative GPVI
receptor density on mouse platelets versus RBL-2H3 cells due
to either the methods used or to a real difference in GPVI expression
levels on mouse and human platelets. Thus the hypothesis that
variability in GPVI levels in human platelets modulates the role of
21 and perhaps other platelet collagen receptors remains
incompletely tested.
21 and the relationship to GPVI is likely to
require the generation of more sophisticated ex vivo models,
second-generation genetic experiments in mice, and the study of
collagen receptor expression and function in larger human populations.
Nevertheless, identification of GPVI as a receptor that mediates both
platelet adhesion and activation soon after vessel injury provides an
important new target for therapies to treat common athero-thrombotic
vascular diseases.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: University of
Pennsylvania, 421 Curie Blvd., BRB II/III Room 952, Philadelphia, PA
19104-6100. Tel.: 215-898-9007; Fax: 215-573-2094; E-mail: markkahn@mail.med.upenn.edu.
ABBREVIATIONS
, Fc receptor gamma chain;
CVX, convulxin;
FITC, fluorescein isothiocyanate;
PBS, phosphate-buffered
saline.
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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