From the Departments of Biochemistry,
Radiation Oncology, and § Medicine, Division
of Hematology/Oncology, MCP Hahnemann School of Medicine, Philadelphia,
Pennsylvania 19102, ** Schering-Plough Research Institute, Kenilworth,
New Jersey 07033, the
Department of
Pathology and Laboratory Medicine, University of North Carolina, Chapel
Hill, North Carolina 27599, and the
§§ Departments of Molecular and Experimental
Medicine and of Vascular Biology, The Scripps Research Institute,
La Jolla, California 92037
Received for publication, September 8, 2000, and in revised form, March 29, 2001
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ABSTRACT |
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Thrombin plays a central role in
normal and abnormal hemostatic processes. It is assumed that
Despite advances in anti-platelet and anti-thrombotic treatment
regimens, cardiovascular diseases remain the leading cause of death in
the United States (1). Clinically employed anti-platelet and
anti-thrombotic agents include heparin, aspirin (2), integrilin (3),
and anti-GP1 IIb/IIIa
antibodies (c7E3 Fab, abciximab, or, ReoPro) (4, 5). We demonstrate here the existence of two thrombin receptors on human
platelets that respond to Materials--
The Platelet Preparation--
Blood was drawn by venipuncture into
plastic tubes that contained Platelet Aggregation--
Platelet aggregations were performed
on a dual channel Chronolog lumiaggregometer (Chronolog Corp.) as
previously described (31). Aggregations were conducted with 480 µl of
washed platelets or a 50-µl sample of the concentrated platelets
added to 430 µl of Hepes-Tyrode buffer with a final platelet count of
2-3 × 105/µl. Agonists and inhibitors were added
as detailed throughout with the concentration of the reagents noted. In
some experiments, the Calcium Mobilization--
The mobilization of internal stores of
calcium, [Ca2+]i, was monitored with a Hitachi
F-2000 fluorescence spectrophotometer as previously reported (32) in
the presence of extracellular EGTA to chelate extracellular
Ca2+. Platelets, as PRP, were preloaded with 1 µM Fura-2/AM for 45-60 min and then washed and
resuspended in Hepes-Tyrode buffer as described above, at a 10×
concentration. Samples were incubated, at room temperature, with or
without 10 µM SCH203099 for 1 h prior to analysis. A
50-µl sample of platelets was added to 430 µl of Hepes-Tyrode
buffer in a special quartz microcuvette with a 4.5-mm path length with
stirring at 37 °C. Agonists were added through an injection port at
the levels described. Excitation wavelengths were 340 and 380 nm, and
emission was measured at 505 nm. Calibration and conversion of raw data
were performed exactly as reported (32).
ATP Release--
The platelet release reaction was monitored
simultaneously, in some experiments, with aggregation as previously
reported (33). The release of dense granule ATP from aggregating
platelets was detected as light emission in the Chronolog
lumiaggregometer produced by the reaction of ATP with luciferin
catalyzed by luciferase (Chrono-lume).
Fluorescence-activated Cell Sorting Analysis--
Platelets were
washed in cold phosphate-buffered saline containing 0.1% (w/v) bovine
serum albumin and 15 mM NaN3 (Buffer) for 5 min
at 1200 × g. Pellets were resuspended and washed once, and the pellet was resuspended in 1 ml of room temperature Buffer. Platelets were incubated with 20 µl of antibody at 20 µg/ml for 40 min at 4 °C, washed once in Buffer, and incubated with 20 µl of
secondary fluorescein isothiocyanate-conjugated goat anti-mouse antibody (1:40 dilution) for 30 min at 4 °C. Then platelets were washed as above, resuspended in 1 ml of count solution, and analyzed for fluorescence in a FACSort flow cytometer (Becton Dickinson, San
Jose, CA) using the Lysis II software program. An acquisition in a live
gate containing the platelets and excluding red blood cells and
debris was performed.
Electron Microscopy--
Platelet aggregations were performed as
described above and monitored in the aggregometer. A 300-µl
sample of Trump's EM fixative (1% glutaraldehyde plus 4%
formaldehyde) was added to the 500-µl aggregated platelet sample and
incubated for 5 min. Samples were then pelleted at 4000 rpm for 5 min
in a microcentrifuge, and the pellet was fixed with Trump's EM
fixative. Samples were postfixed with OsO4 and
embedded in epon, and sections were stained with uranyl acetate and
lead citrate.
The experiments described here indicate that an -thrombin activates platelets by hydrolyzing the protease-activated
receptor (PAR)-1, thereby exposing a new N-terminal sequence, a
tethered ligand, which initiates a cascade of molecular reactions
leading to thrombus formation. This process involves cross-linking of
adjacent platelets mediated by the interaction of activated
glycoprotein (GP) IIb/IIIa with distinct amino acid sequences,
LGGAKQAGDV and/or RGD, at each end of dimeric fibrinogen molecules. We
demonstrate here the existence of a second
-thrombin-induced
platelet-activating pathway, dependent on GP Ib, which does not require
hydrolysis of a substrate receptor, utilizes polymerizing fibrin
instead of fibrinogen, and can be inhibited by the Fab fragment of the
monoclonal antibody LJIb-10 bound to the GP Ib thrombin-binding site or
by the cobra venom metalloproteinase, mocarhagin, that hydrolyzes the
extracellular portion of GP Ib. This alternative
-thrombin pathway
is observed when PAR-1 or GP IIb/IIIa is inhibited. The recognition
sites involved in the cross-linking of polymerizing fibrin and surface integrins via the GP Ib pathway are different from those associated with fibrinogen. This pathway is insensitive to RGDS and anti-GP IIb/IIIa antibodies but reactive with a mutant fibrinogen,
407, with
a deletion of the
-chain sequence, AGDV. The reaction is not due to
simple trapping of platelets by the fibrin clot, since ligand binding,
signal transduction, and second messenger formation are required. The
GP Ib pathway is accompanied by mobilization of internal calcium and
the platelet release reaction. This latter aspect is not observed with
ristocetin-induced GP Ib-von Willebrand factor agglutination nor
with GP Ib-von Willebrand factor-polymerizing fibrin trapping of
platelets. Human platelets also respond to
-thrombin, an
autoproteolytic product of
-thrombin, through PAR-4. Co-activation
of the GP Ib, PAR-1, and PAR-4 pathways elicit synergistic responses.
The presence of the GP Ib pathway may explain why
anti-
-thrombin/anti-platelet regimens fail to completely abrogate
thrombosis/restenosis in the cardiac patient.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Thrombin,
generated at the site of vessel injury, is generally assumed to
catalyze the hydrolysis of an N-terminal peptide from the human
platelet seven-transmembrane thrombin receptor, protease-activated
receptor 1 (PAR-1), which initiates a cascade of molecular reactions
leading to thrombus formation. Thrombin-induced activation of PAR-1, as
for other agonist-activated platelet receptors, results in an
outside-in signal transduced process followed by the alteration of the
surface integrin, GP IIb/IIIa, by an inside-out signal (6). The
conformational change of GP IIb/IIIa leads to the
Ca2+-dependent binding of the bifunctional
fibrinogen molecule (7). The fibrinogen-GP IIb/IIIa binding sites
recognize RGDX sequences on the fibrinogen
-chains
and an LGGAKQAGDV sequence on the
-chains (8). Potential competing
peptides of RGDS and peptides including the
sequence LGGAKQAGDV
were found to be effective antagonists of platelet aggregation (9).
Anti-GP IIb/IIIa antibodies such as c7E3 Fab (4, 5) and LJ-CP8 (10) are
also potent inhibitors of fibrinogen binding to this glycoprotein
complex in activated platelets. Early studies of the cellular thrombin
receptor indicated that more than one species exist in platelets (11,
12). Many questions related to the identity and mechanism(s) of action
of the platelet thrombin receptor(s) were resolved with the cloning and
sequencing of PAR-1 (13). Human platelets appear to respond to PAR-1
and a second minor receptor PAR-4 (14-16), while the recently cloned
PAR-3 (17) is either absent or present in only trace amounts. Mouse
platelets, on the other hand, respond to
-thrombin primarily through
PAR-3 and, secondarily, PAR-4, with no involvement of PAR-1 (14). Other
important issues still remain unresolved with regard to the PARs.
Another platelet membrane protein, GP Ib, may also function, in part,
as a thrombin receptor (11, 12, 18-21). A major role of GP Ib,
complexed with GP IX, is the specific interaction with
subendothelium-bound von Willebrand factor (vWF) under high shear rates
to facilitate platelet adhesion to injured vascular walls (22). The
expression on the plasma membrane of the vWF receptor, GP Ib, requires
the stable expression of GP Ib
, GP Ib
,
and GP IX (23). The GP Ib-IX complex associates with the cytoskeletal
actin-binding protein via the cytoplasmic domain of GP
Ib
(24, 25). This GP Ib
-actin-binding protein
association is initiated by the binding of vWF to GP Ib and appears to
be linked to vWF-induced transmembrane signaling (25). Signal
transduction appears to be regulated, at least in part, by one form of
the 14-3-3
protein (26) and its association with the GP Ib-IX-V
complex (27). The GP Ib receptor also possesses a thrombin binding site
that may respond to lower concentrations of thrombin than required to
activate the PARs (11, 12). The GP Ib-thrombin complex may serve to
prime the activation of PAR-1 as the thrombin levels rise (11, 18). The
physiologic roles of the three purported platelet thrombin receptors
have yet to be clearly defined. While an in vitro functional
role of PAR-1 has been demonstrated for
-thrombin-induced platelet
aggregation (13), no comparable response has ever been described for
PAR-4 or GP Ib with a natural thrombin agonist.
-thrombin. One is the PAR-1 receptor, and
the second is GP Ib. Unlike the activation of platelets via PAR-1,
activation by the GP Ib pathway does not require thrombin hydrolysis of
the substrate receptor, utilizes polymerizing fibrin instead of
fibrinogen, and is inhibited by the Fab fragment of the monoclonal
antibody LJIb-10 that specifically binds to the GP Ib thrombin-binding
site (28). This alternative pathway is readily observed in the presence
of PAR-1 or GP IIb/IIIa inhibitors. Human platelets also respond to
-thrombin, the autoproteolytic product of
-thrombin, through
activation of a second protease-activated receptor, PAR-4.
Co-activation of the GP Ib, PAR-1, and PAR-4 pathways elicits
synergistic responses.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-thrombin was initially obtained from
Ortho Diagnostic Systems (Raritan NJ), as Fibrindex. Production of
Fibrindex was discontinued, and
-thrombin was subsequently obtained
from Chronolog Corp. (Havertown, PA). The two products at 0.05-0.1
units/ml gave identical results. The
-thrombin was obtained from
Hematologic Technologies. The thrombin receptor-activating peptide for
PAR-1 (TRAP-1), SFLLRNP, was synthesized by TANA Laboratories (Houston, TX), and peptides for PAR-4, GYPGQV (TRAP-4) and AYPGKF (TRAP-4A), were
supplied from the Schering-Plough stocks. The anti-PAR-1 drug,
SCH203099, was also supplied by Schering-Plough. Human fibrinogen, RGDS, GPRP-amide, and the water-soluble serine protease inhibitor, 4-[2-aminoethyl]-benzene sulfonyl fluoride (AEBSF) were purchased from Sigma. The thrombin substrate CBS 34.47 came from Diagnostica Stago-American Bioproducts. Fura-2 AM was purchased from Molecular Probes, Inc. (Eugene, OR). The ristocetin and Chrono-lume
(luciferin-luciferase) were obtained from Chronolog Corp. Anti-PAR-1
antibodies were kindly supplied by Drs. Greco and Jamieson (polyclonal
antibody that recognizes the sequence LLRNPNDKYEPF) and Dr. Brass
(monoclonal antibody ATAP-2). The anti-GP IIb/IIIa antibody c7E3 Fab
was a kind gift from Dr. Coller. All other anti-GP Ib and anti-GP
IIb/IIIa antibodies employed along with the recombinant fibrinogens
were from our laboratories. The cobra metalloproteinase, mocarhagin, from Naja mocambique mocambique was kindly supplied by Dr.
Berndt (Baker Medical Research Institute, Victoria,
Australia). The concentrations and conditions employed with all
reagents are described throughout.
-thrombin plus fibrinogen was added to the
platelets, while in others the order of addition was reversed. This
reversal of order allowed for the generation of polymerizing fibrin
prior to the addition of the platelets.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-thrombin-GP Ib
interaction may induce a distinct pathway of platelet aggregation and,
along with PAR-1 and PAR-4, may be functionally relevant.
-Thrombin
(0.05-0.1 units/ml) and the peptide SFLLRNP, the PAR-1 TRAP-1, induced
platelet aggregation with similar kinetics (Fig. 1, A (a) and
B (a)). An equivalent amount of
-thrombin
(10-20 nM, comparable with the activity of 0.05-0.1
units/ml
-thrombin) induced platelet aggregation with distinctly
slower kinetics but a similar end point (Fig. 1C
(a)), as did the PAR-4 thrombin receptor-activating peptide
(TRAP-4), GYPGQV (Fig. 1D (a)). Platelets that
had been preincubated with an anti-PAR-1 antibody (either a polyclonal antibody that recognizes the sequence LLRNPNDKYEPF or the monoclonal antibody ATAP-2 (34); only results with the latter are shown) or
treated with a chemically defined PAR-1 inhibitor, SCH203099 (35), had
a delayed aggregation profile relative to controls when stimulated with
-thrombin but ultimately reached a similar level of aggregation
(Fig. 1, A (e) and E (a),
respectively). SCH203099 at 5-10 µM did not inhibit
platelet activation induced by 1 mM TRAP-4 or 10-30
nM
-thrombin (Fig. 1, G and H);
however, TRAP-1-induced aggregation was completely inhibited but not
platelet shape change (Fig. 1F). Total inhibition of
platelet aggregation was maintained for greater than 15 min, at which
time monitoring ceased. Platelets treated with SCH203099 (7.5 µM) plus ATAP-2 (50 µg/ml) have the PAR-1 receptor
blocked at two levels: at the tethered ligand, blocking hydrolysis by
-thrombin, and at the PAR-1 receptor site for the tethered ligand or
synthetic TRAP-1. These platelets still responded to
-thrombin after
a delay period (Fig. 1E (e)), suggesting the
presence of another receptor. The complete inhibition of platelet
aggregation upon the addition of the Fab fragment of the anti-GP Ib
antibody, LJIb-10, (binds to the GP Ib thrombin binding site) to the
SCH203099 plus ATAP-2-treated platelets indicates that GP Ib is the
second
-thrombin receptor (Fig. 1E (g)).
View larger version (28K):
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Fig. 1.
Platelet aggregation induced by
-thrombin,
-thrombin, or
TRAPs in the presence or absence of specific inhibitors. Washed
platelets were treated or untreated with 5-10 µM
SCH203099, a PAR-1 inhibitor, for 1-2 h. Representative curves of
several experiments are shown for each test condition. Curves labeled
a in panels A-D are with control
platelets (50 µl) added to 430 µl of Hepes-Tyrode buffer, plus 5 µl of fibrinogen (final concentration 100 µg/ml) plus
-thrombin
(
-T, 0.05 NIH units/ml) (A); TRAP-1 (7.6 µM) (B);
-thrombin (
-T, 10 nM) (C); and TRAP-4 (1 mM)
(D). Curves labeled a in panels
E-H are with SCH203099-treated platelets, using the same
conditions as in A-D. Listed below each
panel for the curves labeled a are the percent
mean aggregation (Agg), slope measured for the major portion
of the aggregation curve (grid divisions/min; 1 grid division equals
1.25% aggregation), and t5O (min). S.D. and
number of determinations (in parentheses) are shown for each
measurement. The t50 for control samples is the
time required to reach 50% of maximal aggregation; for treated
samples, it is the time required to reach a level of aggregation
equivalent to the control t50. Representative
results obtained with different inhibitors of platelet function are
shown in selected panels. Control platelets (without
antibody or drug) were preincubated with subaggregating concentrations
(350-700 µM) of TRAP-4 (GYPGQV) to desensitize PAR-4
prior to the addition of
-thrombin (curve b in
panel A),
-thrombin (curve b in
panel C), or TRAP-4 (curve b in
panel D). Results with control platelets treated
simultaneously with TRAP-1 plus 200 µM RGDS
(c) or 1.2 mM GPRP (d) are presented
in panel B. In panel E,
SCH203099-treated platelets were incubated, prior to the addition of
-thrombin, with ATAP-2 (50 µg/ml) (e), ATAP-2 plus a
subaggregating concentration of TRAP-4 (f), or ATAP-2 plus
50 µg of the Fab fragment of the anti-GP Ib antibody, LJIb-10
(Fab-10) (g). Control platelets treated with ATAP-2 are
shown in panel A (e). Curves labeled
with the same lowercase letter represent an
equivalent experimental condition with each agonist.
The potential presence of three distinct thrombin receptors on human
platelets could be defined with combinations of inhibitors and PAR-4
desensitization. Platelets preincubated for 1 h with 350-700
µM TRAP-4, under nonstirring conditions, could not be activated by subsequent additions of 10 nM -thrombin or
TRAP-4 at mM concentrations (Fig. 1, C
(b) and D (b)). Identical results were
obtained with the more reactive PAR-4-activating peptide, AYPGKF
(TRAP-4A) (36). Control platelets aggregated optimally with 100 µM TRAP-4A. Platelets incubated for 20 min with
suboptimal concentrations of TRAP-4A (30 µM) were totally
unreactive with 100 µM TRAP-4A or 10 nM
-thrombin but were fully aggregated upon the addition of 0.1 units/ml
-thrombin (data not shown). Furthermore, control platelets
and platelets treated with SCH203099 plus ATAP-2 that were preincubated
with TRAP-4 still aggregated upon the addition of
-thrombin (Fig. 1,
A (b) and E (f),
respectively). This evidence indicates that TRAP-4 and
-thrombin
activate PAR-4, while
-thrombin activates PAR-1 and a third receptor.
Further evidence for the presence of a thrombin-GP Ib platelet
aggregation pathway comes from studies with the cobra venom metalloproteinase, mocarhagin, which has been shown to hydrolyze the
extracellular portion of GP Ib that contains the vWF and thrombin binding domains (37). Concentrated platelet samples were incubated with
5-20 µg/ml mocarhagin for 60-90 min at 37 °C in the absence or
presence of SCH203099 and/or Fab LJ Ib-10 (Fab-10). Mocarhagin (20 µg/ml for 90 min) alone did not alter the slope or extent of platelet
aggregation induced by the PAR-1 or PAR-4 agonists, TRAP-1 or
-thrombin, respectively (Fig.
2A). However, in a dose- and
time-dependent fashion, mocarhagin significantly inhibited
-thrombin-induced aggregation of platelets simultaneously incubated with the PAR-1 inhibitor, SCH203099. Fig. 2B is
representative of three different experiments where 10 µM
SCH203099 plus 20 µg/ml mocarhagin inhibited
-thrombin-induced
platelet aggregation ~100% for the first 3 min with the eventual
slow aggregation phase occurring at the 3-6-min point after the
addition of
-thrombin. The delayed aggregation appears to be due to
residual intact GP Ib molecules on the platelet surface as demonstrated
by fluorescence-activated cell sorting analysis (Fig. 2C).
The combined addition of SCH203099 plus Fab-10 and mocarhagin
essentially abrogated any delayed aggregation phase in the first 7 min
(Fig. 2B).
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We reasoned that GP Ib may be the third thrombin receptor that, along
with PAR-1, is involved in the -thrombin-induced activation of
platelets. In this regard, we hypothesized that the 1-2-min delay in
aggregation observed with platelets treated with anti-PAR-1 antibody or
SCH203099 may correspond to the time required for the generation of
polymerizing fibrin, which could then participate in platelet
aggregation in a manner different from fibrinogen, as previously
reported (38 39). This alternative
-thrombin pathway would normally
be obscured by the action of the rapidly acting PAR-1 pathway. We
further hypothesized that
-thrombin-induced aggregation via
the PAR-1 pathway, as depicted in Fig. 1A, is entirely, or
predominantly, dependent upon fibrinogen-platelet interactions.
Initial studies with TRAP-1-induced platelet aggregation demonstrated
that the PAR-1 pathway was blocked by RGDS but little affected by
GPRP-amide (Sigma), an inhibitor of fibrin polymerization (Fig. 1,
B (c) and B (d),
respectively). Thus, we tested the hypothesis that an alternate
thrombin-induced pathway is associated with platelet aggregation
mediated by polymerizing fibrin in lieu of native fibrinogen. Washed
platelets (50 µl), as a 10× concentrate, were added to 0.05-0.1
units/ml -thrombin plus fibrinogen preincubated for 2 min
(polymerizing fibrin) in 430 µl of buffer. The kinetics of
aggregation with thrombin plus polymerizing fibrin (Fig.
3B (a)) was
essentially the same as that seen with thrombin plus fibrinogen added
simultaneously to platelets (Fig. 3A (a)).
Aggregation in the presence of polymerizing fibrin was little affected
by RGDS (Fig. 3D (a)). In contrast, the rapid
onset
-thrombin-induced PAR-1 pathway, seen in Fig. 3A
(a), was inhibited by the fibrinogen-competing peptide RGDS
for the first few minutes, a time sufficient for the generation of
polymerizing fibrin (Fig. 3C (a)), after which aggregation ensued via the GP Ib pathway, overriding the RGDS inhibition. The addition of GPRP along with RGDS completely blocked platelet aggregation (Fig. 3, C (b) and
D (b)), while the addition of GPRP alone had
little effect on
-thrombin-induced aggregation (Fig. 3A
(b)). A recombinant mutant fibrinogen (
407), lacking the
AGDV sequence in the
-chain required for GP IIb/IIIa-fibrinogen interactions (40, 41), still supported
-thrombin-induced platelet
aggregation with normal kinetics, as did recombinant wild type
fibrinogen (data not shown). Aggregation via the PAR-1 pathway was
again initially blocked by the addition of RGDS when
407 replaced
normal fibrinogen, until
407 presumably began to polymerize (Fig. 3,
compare E (a) and C (a)).
Residual adhering/endogenous fibrinogen cannot account for the observed
aggregation, since none occurred in the presence of RGDS without added
fibrinogen (Fig. 3E (c)). Aggregation occurred
with polymerizing
407 via the GP Ib pathway even in the presence of
RGDS (Fig. 3F) although at a reduced level.
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RGDS completely blocked aggregation induced by U46619 and ADP, two
agonists that cannot generate polymerizing fibrin (Fig. 3, G
and H, without (a) versus with
(b) RGDS) like -thrombin. When the thrombin inhibitor,
hirudin, was added to platelets (1-2 units/ml) followed by the
addition of thrombin plus fibrinogen, aggregation was completely
inhibited (Fig. 5I). If this inhibitor was added 2 min after
fibrinogen was preincubated with thrombin and then platelets were added
30 s later, hirudin continued to prevent aggregation completely
(Fig. 3B (d)), indicating that polymerizing
fibrin alone could not account for the observed aggregations as
platelet "trapping." Electron micrographic analysis was conducted with control platelets aggregated by
-thrombin (Fig.
4A) and with 10 µM SCH203099-treated platelets added to polymerizing
fibrin (fibrinogen plus
-thrombin preincubated for 2 min) (Fig.
4B) as described in experiments above. The platelet
aggregates of both samples are indistinguishable and indicate true
platelet aggregation of the SCH203099-treated platelets in the presence of polymerizing fibrin as opposed to platelet trapping.
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Further studies were conducted to distinguish between
polymerizing fibrin-dependent platelet aggregation
versus platelet trapping. True platelet aggregation would
presumably involve signal transduction, second messenger formation with
the subsequent mobilization of internal calcium, and the platelet
release reaction. On the other hand, platelet trapping by the
polymerizing fibrin should not involve any of these secondary
responses. Control and SCH203099-treated platelets preloaded with
Fura-2 were both shown to mobilize internal calcium,
[Ca2+]i, in response to -thrombin, while only
control platelets responded to TRAP-1 (Fig.
5A). The mobilization of
[Ca2+]i was delayed in the SCH203099-treated
platelets, as compared with controls, in a fashion similar to that
observed with the kinetics of aggregation. The platelet release
reaction was monitored by release of dense granule ATP. Here the
release reaction was followed simultaneously with aggregation. Fig. 5, C and G, demonstrates that drug-treated platelets
aggregated under conditions dependent upon polymerizing fibrin with
equal amounts of ATP released to the corresponding control samples that
presumably can aggregate independent of polymerizing fibrin (Fig. 5,
F and G; compared with control, Fig. 5,
C and D). However, conditions that induced
platelet adhesion/polymerizing fibrin-trapping are not associated with
any ATP release (Fig. 5, H-J). The ristocetin-induced adhesion/agglutination between platelets was dependent upon residual vWF present in the washed platelet preparation. In some experiments, the amount of residual vWF was too low to support this reaction but
could be replaced by the addition of 25 µl of PPP. In all cases, no
ATP was released (Fig. 5H). It has previously been shown that polymerizing fibrin interacts with vWF-GP Ib (42, 43). Results in
Fig. 5J clearly demonstrate that this interactive process resulted in an almost immediate trapping of platelets in a clot without
any release of ATP where hirudin was added after polymerizing fibrin
was generated. The 1 unit/ml hirudin was sufficient to totally block
-thrombin-induced platelet aggregation when added prior to fibrin
formation (Fig. 5I).
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The serine protease inhibitor, AEBSF, completely inhibited aggregation
when added to thrombin prior to the addition of fibrinogen and
platelets (Fig. 6B). However,
AEBSF did not inhibit aggregation when added after the formation of the
thrombin-fibrin complex but prior to the addition of platelets (Fig.
6C). These results imply that thrombin-induced aggregation
via the GP Ib pathway requires thrombin interaction with GP Ib but is
independent of proteolytic activity at the receptor site as long as
polymerizing fibrin is present. AEBSF blocks the catalytic site but not
the GP Ib-binding site of thrombin, as previously demonstrated with its
analogue, phenylmethlylsulfonyl fluoride (44). Platelets were
treated under identical conditions with AEBSF plus the thrombin substrate, CBS 34.47 (7.5 µM) in order to determine if
AEBSF remains active in the presence of the polymerizing
fibrin-thrombin complex. After a 5-min incubation, the platelets were
pelleted in a microcentrifuge for 4 min at 14,000 rpm, and the
generation of product was monitored at 405 nm (30). AEBSF inhibited
thrombin activity in excess of 90%.
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Significant synergistic responses were observed with suboptimal doses
of -thrombin plus TRAP-4, as well as
-thrombin plus
-thrombin
or TRAP-1 plus TRAP-4 (Fig. 7,
A-C). In all cases, the sum of the aggregation induced by
the individual agonists at suboptimal concentrations varied between 0 and 15% (agonist concentrations varied between 0.01 and 0.005 units/ml
for
-thrombin; between 0.1 and 2 nM for
-thrombin;
between 0.38 and 0.76 µM for TRAP-1; and between 175 and
350 µM for TRAP-4). Presumably, the low dose of
-thrombin employed is below the level required to function/associate
with PAR-1 or PAR-4 but adequate to activate/bind to GP Ib. TRAP-4 and
-thrombin appear to function only at PAR-4. To demonstrate a
synergistic mechanism between GP Ib and PAR-4 and to exclude a
role for PAR-1, platelets were incubated with SCH203099 to inactivate
PAR-1 (see Fig. 7D). The SCH203099-treated platelets had a
robust response with low dose
-thrombin plus TRAP-4 (Fig.
7E), but this synergistic response was totally abrogated by
the presence of Fab-10 (Fig. 7F).
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DISCUSSION |
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Our studies reveal that three different thrombin
receptors exist on the surface of human platelets. Each appears to
respond to - or
- thrombin under distinct conditions specific to
the individual receptor. Our results indicate that PAR-1 is
not functional in the presence of the anti-PAR-1 drug,
SCH203099, assuming that the physiologic tethered ligand and
TRAP-1 have similar affinities for the receptor. Since drug
reversal is minimal in the time period studied, the
-thrombin response observed with platelets preincubated with
SCH203099 or the monoclonal anti-PAR-1 antibody, ATAP-2, cannot be
explained by displacement of the antibody/drug and appears to be
mediated by another receptor. PAR-4 is not likely to be the alternate
receptor, because it is known to be insensitive to the low levels of
-thrombin employed in our studies (14, 15). GP Ib, with its specific
-thrombin binding site, is a probable candidate. Since SCH203099 and
anti-PAR-1 antibodies had no effect on
-thrombin-induced platelet
aggregation and
-thrombin cannot bind to or activate GP Ib (45),
PAR-4 appears to be the receptor sensitive to
-thrombin. These
conclusions are in concert with another recent report (46) that
implicates the presence of three thrombin platelet receptors. Smith and
Owens (46) demonstrated that
-thrombin interacts with an
unidentified receptor other than PAR-1 and that
-thrombin (an
autoproteolytic intermediate between
- and
- thrombin) interacts
with a receptor other then PAR-1. Our preliminary studies indicate that
-thrombin, at least in part, acts at PAR-4 as we demonstrate here
with
-thrombin.
The proof that GP Ib is a functional thrombin receptor has been
difficult to establish (11, 12, 18-21). More conclusive evidence comes
from our current studies, which demonstrate that the venom
metalloproteinase, mocarhagin, that hydrolyzes the extracellular portion of GP Ib, abrogates
-thrombin-induced aggregation under appropriate conditions, and the Fab fragment of the anti-GP Ib antibody, LJ Ib-10, that selectively binds to the thrombin binding site
completely blocks
-thrombin-induced platelet aggregation under
conditions where the PAR-1 and PAR-4 receptors are nonfunctional. Platelet activation via the thrombin-GP Ib pathway is quite distinct from the PAR pathways. Thrombin must be bound to GP Ib; however, based
upon our results with AEBSF-inactivated thrombin, platelet activation
occurs in the absence of hydrolysis of the receptor. This pathway
requires polymerizing fibrin and not the parent molecule, fibrinogen.
Therefore, the
-thrombin, bound to GP Ib, must remain catalytically
active to hydrolyze fibrinogen, but not the receptor itself, in order
to induce platelet aggregation via this pathway.
The requirement for polymerizing fibrin is clearly defined by the
series of studies where results are compared with thrombin plus
fibrinogen added to platelets versus platelets added to
polymerizing fibrin. The initial inhibition observed in Fig. 3,
C (a) and E (a), is in
agreement with previous evidence that RGD and the -chain AGDV are
required for fibrinogen binding to GP IIb/IIIa activated via the PAR-1
pathway. The reversal of inhibition with time (delayed platelet
aggregation) indicates that the alternate
-thrombin pathway, the GP
Ib pathway, is independent of RGD/
-peptide sequences. Platelet
aggregation, under our conditions, absolutely requires polymerizing
fibrin based upon the fact that fibrin monomers generated in the
presence of GPRP followed by the addition of RGDS do not support
aggregation, while RGDS alone can only delay the reaction. Platelets
preincubated with the potent inhibitors of fibrinogen binding to GP
IIb/IIIa, anti-GP IIb/IIIa antibody c7E3 Fab or LJCP-8 (Refs. 5 and 10,
respectively), aggregated with the same kinetics as those
treated with RGDS (data not shown). These results further substantiate
the independence of the GP Ib pathway on the RGD/
peptide sequences.
The conclusion that thrombin-GP Ib-induced aggregation, in the presence
of RGDS, involves polymerizing fibrin instead of fibrinogen is also in
agreement with the observation that RGDS irreversibly blocks
aggregation induced by U46619 or ADP, since neither agonist can
generate polymerizing fibrin.
The thrombin inhibitor, hirudin, completely blocks platelet
aggregation, whether it is added first to thrombin plus fibrinogen, preventing the formation of polymerizing fibrin, or if it is added after the formation of polymerizing fibrin but prior to the addition of
platelets. These results demonstrate that generation of polymerizing fibrin alone under our experimental conditions is insufficient to
induce aggregation or trapping of platelets. Also, aggregation will not
occur if -thrombin cannot bind to GP Ib, since hirudin blocks both
the proteolytically active and GP Ib-binding sites on thrombin, even
when thrombin is complexed with fibrin (47). Further evidence that
platelet aggregation is not "platelet trapping" by the low levels
of polymerizing fibrin employed here comes from the fact that these
activated platelets release ATP and mobilize internal stores of
Ca2+. Both of these responses are coupled to ligand-induced
signal transduction, indicating that the polymerizing fibrin is binding to a selective receptor. This is in sharp contrast to the case where
ristocetin-treated platelets are agglutinated/trapped by vWF alone or
by vWF plus polymerizing fibrin. No ATP was released. Also, exposure of
platelets to
-thrombin plus fibrinogen in the absence of stirring
was insufficient to induce significant aggregation or ATP release
although the thrombin presumably was bound to the thrombin receptors
(data not shown). This excludes the possibility that the observed ATP
release in our experimental samples was due to
-thrombin binding to
a thrombin receptor(s) at the same time platelets were being trapped in
a fibrin clot. Some may argue that the slower
-thrombin response to
[Ca2+]i mobilization in the presence of SCH203099
is PAR-4 and not GP Ib. However, the level of
-thrombin used in our
experiments is 10-100-fold lower than necessary to elicit a response
presumably via PAR-4 in platelets pretreated with 30 mM
SFLLRNP (16). Finally, electron microscopic analysis of aggregated
platelets under our experimental conditions demonstrates that platelets
are not trapped in a fibrin network but undergo spreading and
degranulation expected in agonist-induced aggregation.
Others have also shown that fibrin-induced procoagulant (thrombin generation) activity in platelets requires a GP IIb/IIIa independent fibrin-integrin interaction (42). Loscalzo et al. (43) reported that GP Ib could serve as a fibrin-vWF receptor involved in the incorporation of platelets into fibrin thrombi. The reaction was independent of GP IIb/IIIa. However, comparisons between our work and this group's work are difficult, if not impossible, since their experimental conditions were so different from ours. They were not measuring platelet aggregation but rather platelet association with the fibrin thrombi in the presence of active protease (bovine, not human thrombin, or Bothrops atrox venom) and a calcium chelator (10 mM EDTA). Their reaction took place over a 20-30-min period at 25 °C. It is clear from our work that the association of polymerizing fibrin with the ristocetin-induced GP Ib-vWF complex can be almost immediate under the appropriate conditions, resulting in the complete trapping of platelets in a clot. Therefore, while there are interesting similarities to our observations, it is not currently clear if this is just coincidence or perhaps overlapping mechanisms. Other reports have observed "deviant" binding of fibrinogen/fibrin presumably to the GP IIb-IIIa complex (48).
It is apparent that thrombin binding alone to GP Ib is not sufficient
to induce aggregation in the presence of fibrinogen, as demonstrated by
the total inhibition by AEBSF added prior to the formation of
polymerizing fibrin. AEBSF blocks the catalytic site but not the
binding site of thrombin. The fibrinogen must be hydrolyzed by thrombin
to produce polymerizing fibrin. Thrombin bound to the extracellular
portion of GP Ib, glycocalicin, remains catalytically active (49, 50).
It is possible that under normal conditions this thrombin activates
platelets via a nonprotelolytic GP Ib pathway in conjunction with the
presentation of bound thrombin to activate platelets proteolytically
via PAR-1 (18, 50) and/or PAR-4 pathways. The results of our studies
may explain observations made nearly a quarter of a century ago with
phenylmethlylsulfonyl fluoride-inactivated thrombin, showing
that the phenylmethlylsulfonyl fluoride-thrombin complex bound
to a platelet membrane site potentiated the activity of a second
proteolysis-dependent site (51). Others have shown that
catalytically inactive thrombin (chemically modified) bound to GP Ib
inhibits the PAR-1 pathway (46). This implies a spatial arrangement of
the two receptors such that the entry of a second, active thrombin
molecule is blocked from interacting with PAR-1, although antibody
studies might argue against this. The ability of -thrombin to bind
to and activate GP Ib and simultaneously activate PAR-1 and/or PAR-4
may explain the synergistic potential of these three thrombin receptors
at both the extracellular level in addition to cross-talk between
intracellular second messenger pathways.
Thrombin-induced activation of PAR-1 results in an outside-in signal
transduction process followed by the alteration of the surface
integrin, GP IIb/IIIa, by an inside-out signal (6). The conformational
change of GP IIb/IIIa leads to the
Ca2+-dependent binding of the bifunctional
fibrinogen molecule (7, 8). It is hypothesized that the outside-in
and/or inside-out signals initiated by thrombin binding to GP Ib are
different than the thrombin-induced PAR-1 or PAR-4 signals. One
possibility is that the GP Ib-associated pathway induces a
conformational alteration of GP IIb/IIIa that binds polymerizing fibrin
in preference to fibrinogen in a Ca2+-dependent
fashion at peptide sites other than those commonly observed. This is
consistent with the concept that integrin affinities for ligands can be
modified by altered inside-out signals (52). A second, perhaps more
likely case is that the thrombin-GP Ib complex activates a
signal-transducing pathway that activates a different integrin that
selectively binds polymerizing fibrin. This latter case is supported by
studies with platelets derived from Glanzmann thrombasthenic patients.
These platelets lack a functional GP IIb/IIIa complex yet were shown to
effectively bind polymerizing fibrin in a
Ca2+-dependent manner (39). It is well
established that GP Ib is coupled to a different signal-transducing
pathway than PAR-1/4, at least in response to vWF (25-27).
Furthermore, it was shown that GP IIb/IIIa could be activated in a
transfected cell system, by an interaction of GP Ib with vWF in the
absence of GP V and actin-binding protein and with GP Ib lacking
the domain that binds 14-3-3 (53). While the mechanism of
activation is unresolved, it is concluded that the vWF-GP Ib signaling
pathway is different from the thrombin-induced pathway.
The dogma that fibrinogen and polymerizing fibrin both bind to GP
IIb/IIIa via the same peptide sequences during platelet aggregation is
based more on assumptions than proof. The evidence that fibrinogen
peptide sequences selectively associate with GP IIb/IIIa is
irrefutable; however, this is not the case for polymerizing fibrin. It
is clear that polymerizing fibrin plays a role in platelet aggregation
and adhesion/agglutination. It was presumed that its interaction was
the same as fibrinogen, and this presumption slowly became "fact"
without proof. Our studies demonstrate that polymerizing fibrin plays
some role in platelet aggregation via a pathway that is independent of
fibrinogen. The polymerizing fibrin apparently can bind at different
amino acid sequences and/or integrins from fibrinogen based on our
studies with several different antibodies and peptide inhibitors.
Anti-GP IIb/IIIa (IIb/
3) and
anti-
v/
3 antibodies that block the fibrinogen
binding site(s) as well as peptide inhibitors had no effect on the
thrombin-GP Ib-polymerizing fibrin pathway (data both shown and not
shown). These results are similar to those previously reported
(48) for platelet adhesion with flowing blood. Also, the anti-GP Ib
antibody, LJIb1, that binds to the vWF binding site (28) had no effect
on this pathway (data not shown), further indicating that the GP
Ib-polymerizing fibrin pathway does not involve a GP Ib-vWF-fibrin
complex. The site and binding parameters of polymerizing fibrin is
clearly very important but remains obscure at this time. The fact that the anti-integrin antibodies tested to date do not block polymerizing fibrin binding does not exclude these integrins as potential binding sites. Other integrin epitopes not recognized by these antibodies may
remain available for interactions with polymerizing fibrin.
Low concentrations of thrombin bound to fibrinogen/fibrin remain catalytically active, even in the presence of endogenous regulators such as anti-thrombin III and heparin (54). Such conditions may exist at sites of high vascular shear forces that periodically strip endothelial cells from the vascular wall (55). These low levels of active thrombin, with or without other potential platelet agonists, may be sufficient to activate the GP Ib thrombin receptor to repair the localized damaged vessel without triggering the more aggressive PAR-1 response. High doses of hirudin administered in clinical trials to effectively inhibit platelet-dependent arterial thrombosis and restenosis also resulted in unacceptable bleeding episodes. These levels of hirudin would inhibit both the PAR-1- and the GP Ib-thrombin-activating pathways. When the hirudin dose was reduced sufficiently to alleviate bleeding problems, the anti-thrombotic effect was greatly reduced. Studies in a nonhuman primate model demonstrated that hirudin coupled to a fibrin antibody fragment F(ab')2 was more effectively targeted to inhibit thrombin than was the free hirudin (56). The indication is that the fibrin-thrombin complex that we have shown to induce platelet aggregation may be physiologically important. High shear forces enhance platelet-induced hemostasis at sites of vessel wall injury (57). GP Ib-vWF complexes play an important role in platelet adhesion to the vascular wall (58, 59). Given the fact that GP Ib has separate binding sites for vWF and thrombin, it is possible that the binding of one ligand will synergistically facilitate the binding of the other ligand to the same GP Ib molecule or to a different GP Ib molecule. This could enhance platelet activation in the presence of suboptimum levels of vWF and thrombin in a fashion similar to that seen with other agonist pairs (60). Once the platelets are activated, they can express P-selectin, which would facilitate leukocyte adhesion even under high shear forces that would otherwise inhibit leukocyte adhesion (57). The deposition of leukocytes under these conditions along with platelets could also contribute to atherogenesis at localized regions of chronic shear-induced vessel injuries.
Our studies demonstrate that human platelets respond to -thrombin
via PAR-1 and GP Ib receptors through mechanisms that are distinct
in vitro and, presumably, in vivo. A second PAR
receptor, PAR-4 (15), does not appear to respond to physiologic levels of
-thrombin but may be responsive to in vivo generated
-thrombin based upon our in vitro observations.
Furthermore, the synergistic effect of minimally activated thrombin
receptors may have significant implications for in vivo
thrombotic events. The recently cloned PAR-3 (17) is either absent or
present in only trace amounts in human platelets. The physiological
role, if any, of PAR-3 and PAR-4 in human platelets remains to be
established. However, the equal intensity of platelet responses
observed in vitro with low levels of
-thrombin acting on
PAR-1 and GP Ib and the synergy between GP Ib and PAR-1 and/or PAR-4
indicate that these three thrombin receptors are candidates to function
in vivo. Multiple platelet thrombin receptors may reflect
the physiological requirement to respond differentially to varying
concentrations of
-,
-, and
-thrombins and/or to different
presentations of thrombin complexed with other proteins. The presence
of GP Ib as a functional thrombin receptor, independent of fibrinogen,
along with its synergy with the
-thrombin-dependent
PAR-4 receptor may explain why anti-
-thrombin/anti-platelet regimens
fail to completely abrogate thrombosis/restenosis in the cardiac
patient. Moreover, the identification of selected conditions to study
the thrombin response where one pathway predominates over another opens
the door to a more complete understanding of the molecular mechanisms
of action of these pathways. It also affords an opportunity to evaluate
new drugs that may abrogate chronic pathologic responses to thrombin
in vivo.
A recent report (61), submitted and published while our paper was in
review, substantiates two aspects of our work presented here with human
platelets and previously published as an abstract (62). Work with a Gp
V null mouse showed that GP Ib was in fact a functional thrombin
receptor for platelet aggregation. Furthermore, activation did not
require a proteolytically active form of thrombin (61). The mouse GP Ib
pathway appears to be dependent upon an inhibitable fibrin(ogen)-GP
IIb/IIIa interaction as opposed to what we observe with the human GP Ib
pathway. Future studies should help resolve this and other differences
between the GP Ib pathway in the two species.
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ACKNOWLEDGEMENTS |
---|
We thank Jan Novack and Bobbi Jo Graeve for manuscript preparation and Dr. Robert McKenzie, Martha Kaplan, and James Diven for technical help.
![]() |
FOOTNOTES |
---|
* 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.
¶ To whom correspondence should be addressed: Biochemistry/IMS, Mail Stop 344, MCP Hahnemann University, 245 N. 15th St., Philadelphia, PA 19102. Tel.: 215-762-7831; Fax: 215-762-7434.
Published, JBC Papers in Press, March 30, 2001, DOI 10.1074/jbc.M008249200
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ABBREVIATIONS |
---|
The abbreviations used are: GP, glycoprotein; PAR, protease-activated receptor; vWF, von Willebrand factor; AEBSF, 4-[2-aminoethyl]-benzene sulfonyl fluoride; PRP, platelet-rich plasma; TRAP, thrombin receptor-activating peptide.
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