Thrombin and Platelet Activation*
Lawrence F. Brass
Chest 2003;124;18S-25S
DOI 10.1378/chest.124.3_suppl.18S
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Thrombin and Platelet
Activation*
Lawrence F. Brass, MD, PhD
A
The accumulation of thrombin at sites of vascular
injury provides one of the chief means for recruiting
platelets into a growing hemostatic plug. Studies
completed over the past 10 years show that platelet
responses to thrombin are mediated by a subset of G
protein-coupled receptors known as protease-activated receptors. These receptors are activated on
cleavage by thrombin, initiating the intracellular
signaling events needed to transform mobile, nonadhesive platelets into cells that can participate in the
growth of an immobile hemostatic plug. How this is
accomplished is the subject of this review.
(CHEST 2003; 124:18S–25S)
Key words: G proteins; G protein-coupled receptors; phospholipase C; platelet; protease-activated receptors; thrombin
Abbreviations: ADP ⫽ adenosine diphosphate; cAMP ⫽ cyclic
adenosine monophosphate; GDP ⫽ guanosine diphosphate;
GP ⫽ glycoprotein; GPCR ⫽ G protein-coupled receptor;
GTP ⫽ guanosine triphosphate; NO ⫽ nitric oxide; PAR ⫽ protease-activated receptor; PGI2 ⫽ prostaglandin I2; TxA2 ⫽ thromboxane A2; VWF ⫽ von Willebrand factor
B
platelets normally circulate in a quiescent state,
H uman
prevented from premature activation by the presence
of the endothelial cell monolayer, by the signal-inhibiting
effects of prostaglandin I2 (PGI2) and nitric oxide (NO),
and by limitations on the local accumulation of platelet
agonists. It is only when these barriers are overcome that
platelets can become activated. That can happen after
local trauma or in response to the rupture of an atherosclerotic plaque. Thrombin plays an essential role in
activating platelets, just as it does in the formation of the
fibrin clot. When added to human platelets in vitro,
thrombin causes platelets to change shape, stick to each
other, and secrete the contents of their storage granules.
How this is accomplished is still not fully understood, but
a major step forward occurred in 19901,2 with the identification of a G protein-coupled receptor (GPCR) that can
be activated proteolytically by thrombin. Until that receptor, now known as protease-activated receptor (PAR)-1,
was identified, there was no clear paradigm for the
initiation of intracellular events by an extracellular protease. In the dozen years since then, a family of proteaseresponsive receptors has been identified and steady
*From the Departments of Medicine and Pharmacology, and the
Center for Experimental Therapeutics, University of Pennsylvania, PA.
Supported by grants HL40387 and HL45181 from the National
Institutes of Health-National Heart Lung, and Blood Institute.
Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail:
[email protected]).
Correspondence to: Lawrence F. Brass, MD, PhD, University of
Pennsylvania, Room 915 BRB-II, 421 Curie Blvd, Philadelphia,
PA 19104; e-mail: [email protected]
18S
progress has been made toward understanding how they
work and how thrombin activates platelets. The results
have provided insights into normal platelet biology and
opened an avenue for the development of new antiplatelet
agents—a promise that has yet to be fulfilled. To place
what is known about the activation of platelets by thrombin into context, this article begins with an overview of the
events encompassing platelet plug formation and then
focuses on the role of thrombin.
Stages in the Formation of a Stable
Platelet Plug
Platelet plug formation requires a coordinated series of
events that can overcome local resistance to platelet
activation long enough for bleeding to stop. This is not a
trivial task, particularly if unwarranted platelet activation is
to be avoided. The barriers to platelet activation are
substantial. Part of the barrier is formed by the endothelial
cell monolayer, which physically separates platelets from
agonists embedded in the vessel wall, especially collagen
and von Willebrand factor (VWF). VWF is a multimeric
protein synthesized by endothelial cells that plays an
essential role in the adhesion of platelets to collagen under
the high-flow conditions found in arteries. It is secreted in
an ultrahigh-molecular-weight form that is normally
cleaved by the metalloprotease, ADAMTS13, preventing
it from binding to platelets spontaneously and causing a
thrombotic microangiopathy.3 The shear stresses produced when blood flows over VWF anchored to collagen
exposes platelet-binding sites, allowing the VWF to support platelet/collagen and platelet/platelet interactions
(Fig 1). In addition to collagen and VWF, tissue factor is
also present in the vessel wall, on the surfaces of activated
endothelial cells and monocytes, and on circulating microvesicles that stick to activated platelets, so injuries that
alter or remove the endothelial barrier result in the local
generation of thrombin as well as the exposure of collagen.
In addition to serving as a physical barrier, endothelial
cells release PGI2 and NO, whose net effect is to globally
depress the intracellular signaling events needed to support platelet activation by raising cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate
levels (Fig 1). The ability of cyclic nucleotides to inhibit
platelet activation has been exploited in the development
of antiplatelet agents, such as dipyridamole, which work by
inhibiting the phosphodiesterases that would otherwise
metabolize cAMP within platelets. The importance of
PGI2 and NO as barriers to platelet activation is indicated
not only by the effectiveness of molecules that mimic PGI2
as antiplatelet agents, but also by the prothrombotic
effects of deleting the gene encoding the platelet PGI2
receptor in mice.4 As a further barrier to platelet activation, some endothelial cells express CD39 on their luminal
surface. CD39 can hydrolyze small quantities of adenosine
diphosphate (ADP) released from damaged red cells and
activated platelets, preventing the ADP from activating
additional platelets.5,6 The use of CD39 as an antithrombotic is being explored in animal models. Other barriers to
platelet activation include the diluting effects of blood
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Figure 1. Stages in platelet plug formation. Prior to vascular injury, platelets are restrained from
activation by inhibitory factors that include PGI2 and NO released from endothelial cells, the presence
of CD39 on the surface of endothelial cells, and the inability of normal plasma VWF to bind
spontaneously to the platelet surface. The development of the platelet plug can be initiated by the
exposure of collagen and VWF in the vessel wall, and by the local generation of thrombin, a process that
occurs more rapidly on the surface of activated platelets. Rolling platelets adhere and spread on the
collagen matrix, forming a monolayer of activated platelets that can act as a surface for subsequent
recruitment of platelets by thrombin, ADP, and TxA2. During the perpetuation stage, close contacts
between platelets promote the growth and stabilization of the hemostatic plug, in part through
contact-dependent signaling mechanisms.
A
flow, the presence of inhibitors of thrombin, and the short
half-life of the platelet-derived agonist, thromboxane A2
(TxA2).
Given all of these barriers, platelet activation should
ideally occur only after substantial injuries. If this were
always the case, there would be little need for antiplatelet
agents in clinical settings. Formation of the platelet plug in
response to vascular injury can be thought of as occurring
in three stages: initiation, extension, and perpetuation
(Fig 1). Initiation begins with the tethering, rolling, and
arrest of moving platelets on collagen and their subsequent activation to form a platelet monolayer. Large VWF
multimers are essential to this process, particularly under
high shear conditions in the arterial circulation, but thrombin can also help to initiate platelet activation. Extension
refers to the recruitment of additional platelets through
the local accumulation of thrombin, ADP and TxA2.
Perpetuation refers to the events that stabilize the platelet
plug until wound healing can occur, some of which involve
molecules on the platelet surface that are capable of
generating intracellular signals only after platelets have
come into sustained contact with each other. The net
result is the formation of a fibrin-anchored platelet plug, a
structure in which platelet/platelet interactions are supported by the binding of fibrinogen and fibrin to the
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integrin ␣IIb␤3 (also known as glycoprotein [GP] IIbIIIa) and by VWF bound to GP Ib and ␣IIb␤3 (Fig 2).
Initiation
The arrest and eventual activation of moving platelets
by collagen plus VWF requires several receptors on the
platelet surface, including those that can bind directly to
collagen (GP VI and the integrin ␣2␤1) and those that bind
to collagen indirectly via VWF (the GP Ib/IX/V complex
and the integrin ␣IIb␤3). The presence of binding sites for
collagen and VWF on GP VI, GP Ib, ␣2␤1, and ␣IIb␤3
allows platelets to stop their forward movement in the
arterial circulation long enough to become activated and
fully adherent. This can happen rapidly, but only selectively. Videomicroscopy of blood vessels following focal
injuries shows that most platelets move by the site of injury
too quickly to stop.7 In a manner that is very much
reminiscent of how leukocytes escape from the circulation,
a small proportion of platelets rolling along the vessel wall
is able to react initially to injury and form the nidus for a
platelet plug. This requires platelets to both adhere to
collagen and be activated by it. The binding of collagen to
GP VI on the platelet surface causes the clustering of GP
VI and its associated ␥-chain within the plane of the
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B
Figure 2. Anatomy of a platelet plug: an enlarged view of the
assembled platelet plug, highlighting platelet/platelet interactions
mediated by the binding of fibrinogen, fibrin, and VWF to
activated GP IIb-IIIa (␣IIb␤3) and the binding of VWF to
GP Ib␣.
platelet plasma membrane, which leads to the phosphorylation of the ␥-chain by tyrosine kinases in the Src family,
creating a tandem phosphotyrosine motif that is recognized by the tyrosine kinase, Syk, and the activation of
phospholipase C␥2 (Fig 3).8 PLC␥2 hydrolyzes PI-4,5-P2
to produce 1,4,5-IP3 and diacylglycerol, raising the cytosolic-free Ca2⫹ concentration within the adherent platelets by discharging Ca2⫹ stores from within the dense
tubular system and activating protein kinase C. Clustering
of a cell-surface receptor (GP VI in this case) followed by
signaling through tyrosine kinases is a theme that occurs
again during the perpetuation phase of platelet plug
formation.
Extension
The extension phase of platelet plug formation occurs
when activated platelets accumulate on top of the initial
monolayer of platelets bound to collagen (Fig 1). Key to
the extension phase is the presence on the platelet surface
of receptors that can respond rapidly to soluble agonists,
including thrombin, ADP, and TxA2. The local accumulation of these agonists allows circulating platelets to be
recruited into the growing hemostatic plug even when
they cannot arrest on collagen. Extension of the platelet
plug requires the activation of ␣IIb␤3 through what is
commonly called “inside-out” signaling, promoting the
formation of stable platelet/platelet contacts mediated by
bridges comprised of fibrinogen and VWF. The receptors
involved in these events are typically members of the
superfamily of GPCRs (Fig 3), which are membrane
proteins with an extracellular N-terminus, an intracellular
C-terminus, and seven transmembrane domains. Agonists
bind to the surface-accessible domains of GPCRs, causing
a conformational change that activates G proteins associated with the intracellular surface of the receptor.9 G
proteins interact with the cytoplasmic domains of the
receptor with specificity determined in part by the sequence of these domains and in part by the sequence of
the ␣ subunit of the G protein. G proteins are heterotri20S
mers comprised of a single ␣, ␤, and ␥ subunit. The ␣
subunit contains a guanine nucleotide-binding site that is
occupied by guanosine diphosphate (GDP) in the off state
and GTP in the on state. Activation of the receptor causes
exchange of guanosine triphosphate (GTP) for GDP, after
which partial dissociation of the GTP-bound G␣ from
G␤␥ exposes effector interaction sites on both. Fatty
acylation of G␣ and prenylation of G␤␥ cause them to
remain associated with the plasma membrane until hydrolysis of the GTP bound to G␣ allows the original heterotrimer to reform.
Mammalian G proteins fall into four families that are
typically referred to by the designation of the ␣ subunit.
Human platelets express at least one member of the Gs
family and four members of the Gi family (Gi1, Gi2, Gi3,
and Gz), which respectively stimulate and inhibit cAMP
formation by adenylyl cyclase, among other functions. In
addition, platelets express one or more members of the Gq
family, which stimulate ␤ isoforms of phospholipase C,
and two members of the G12 family (G12 and G13), which
help to regulate the platelet actin cytoskeleton.10 –14 Based
on evidence from knockout and reconstitution studies, the
abundance of G protein types in platelets appears to be
necessary to support the actions of multiple dissimilar
platelet agonists.15
The GPCRs that respond to platelet agonists differ in
their potency and their preferences for intracellular effector pathways. Some, such as the receptors for thrombin
(PAR-1 and PAR-4), TxA2 (TP), and ADP (P2Y12), cause
phosphoinositide hydrolysis and raise the cytosolic Ca2⫹
concentration by activating Gq (Fig 3).12 Others, such as
the P2Y12 receptor for ADP and the ␣2A-adrenergic
receptor for epinephrine, are coupled by Gi2 or Gz to the
inhibition of adenylyl cyclase and to the activation of PI
3-kinase and the Ras family member, Rap1.15–18 Optimal
platelet activation via GPCRs is thought to require activation of both a Gq-coupled receptor and a Gi-coupled
receptor.19 The ability of the Gi family members in
platelets to inhibit cAMP formation by adenylyl cyclase is
most relevant when PGI2 secreted by endothelial cells has
inhibited platelet activation. In the absence of PGI2, other
Gi effectors are more relevant.15 The essential role of the
Gi2-coupled P2Y12 receptor for ADP is suggested by the
phenotypes of the Gi2 and P2Y12 knockout mice and by
the proven utility of the P2Y12 antagonists ticlopidine and
clopidogrel as antiplatelet agents.15,17,20 –22 Thrombin and
TxA2 receptors can also cause the rearrangement of the
actin cytoskeleton that underlies platelet shape change by
coupling to guanine nucleotide exchange factors for Rho
via G12 and G13.10
Perpetuation
The third phase of platelet plug formation, perpetuation, occurs at a point when direct interactions between
platelets are of sufficient duration to make contact-dependent signaling feasible. Ultimately, these late events are
thought to stabilize the platelet plug, helping to prevent
premature disaggregation and regulating retraction of the
clot. A number of recent events have helped to define
some of the signaling events that are involved. The
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Figure 3. Overview of platelet activation. Most platelet agonists activate platelets via G proteincoupled receptors on the platelet surface. Critical responses include Gq-mediated activation of
phospholipase C␤ isoforms to allow an increase in cytosolic Ca2⫹, activation of phospholipase A2 and
protein kinase C, and G12-mediated activation of Rho family members to support rearrangement of the
platelet cytoskeleton (shape change). The increase in cytosolic Ca2⫹ is initially caused by the
IP3-triggered release of Ca2⫹ from within the dense tubular system of the platelet, which in turn
triggers Ca2⫹ influx across the platelet plasma membrane. Activated PGI2 receptors (not shown)
stimulate adenylyl cyclase, raising platelet cAMP levels and causing a generalized inhibition of platelet
responses to agonists. Gi family members support the suppression of adenylyl cyclase by platelet
agonists and also couple receptors to other critical effector pathways. TP ⫽ thromboxane receptor.
best-described of these events23 involves outside-in signaling through integrins. Other examples include the binding
of Eph kinases to ephrins,24 and the binding of CD40
ligand (CD40L) to ␣IIb␤3 and perhaps CD40.25 Each of
these depends on one or more molecules expressed on the
platelet surface, but the details differ in essential ways.
Eph kinases and ephrins can engage with each other
whenever platelets are in sufficiently close contact for a
long enough time. Interactions with ␣IIb␤3 require insideout signaling before the integrin can bind to its ligands,
particularly soluble ligands. CD40L is not expressed on
the surface of resting platelets, but appears there after
platelets have been activated. Once on the surface,
CD40L can bind to activated ␣IIb␤3 and (perhaps) to
CD40. Evidence exists for a role for each of these
contact-depending signaling mechanisms, but their relative contributions are still being studied.
Thrombin Receptor Structure
and Function
For years after thrombin was shown to activate platelets, little was known about how this might be accomplished. A variety of approaches established that the
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proteolytic activity of thrombin was required, and biochemical studies showed that G proteins are activated by
thrombin, but there was no precedent for G protein
activation by a protease. Binding studies26,27,48 identified
high-affinity interactions with several sites on the platelet
surface, including GP Ib␣, but efforts to show that any of
these constituted a receptor in the signaling sense were
not entirely successful. Substrates for thrombin were
identified on the platelet surface, including GP V. However, cleavage of GP V did not appear to be required for
platelet activation by thrombin. Before discussing the
receptors that have been identified, it is worth briefly
considering criteria that proved useful for establishing a
protein as a true signaling receptor for thrombin. Such
criteria included the following: (1) demonstrating the
presence of the candidate receptor on the surface of
resting platelets, (2) showing that it is a substrate for
thrombin or closely associated with a substrate for thrombin, (3) demonstrating a link to intracellular signaling
cascades, (4) showing that expression of the candidate
receptor could render a cell that was otherwise unresponsive to thrombin capable of responding, and (5) showing
that blocking, dismantling, or otherwise removing the
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candidate receptor would reduce platelet responses to
thrombin. So far, PAR family members are the only
receptors that meet all of these criteria. However, it
remains possible that other proteins on the platelet surface
also play a role, either by initiating signaling themselves or
by facilitating the activation of PARs.
Four members of the PAR family have been identified
to date. Three (PAR-1, PAR-3, and PAR-4) are thrombin
receptors. The fourth, PAR-2, is activated by serine proteases other than thrombin. All four of the PAR family
members have a structure similar to other GPCRs, including an exposed N-terminus (Fig 4). Studies1 on PAR-1
established the paradigm that applies (with certain exceptions) to the other three family members. In each case,
receptor activation begins when thrombin cleaves the
N-terminus of the receptor, exposing a new N-terminus
that serves as a tethered ligand. Given sufficient opportunity, proteases other than thrombin can also activate
PAR-1 or render the receptor unresponsive to thrombin
by cleaving the N-terminus in the “wrong” place. The
binding site for the tethered ligand has been mapped to
the extracellular loops of the receptor. Because the ligand
is not free to diffuse away, it presents a highly effective
local concentration at the receptor. As is the case for other
GPCRs, contact between PAR ligands and the receptor is
thought to initiate signaling because of an induced conformational change in the receptor that is transmitted
across the plane of the plasma membrane to promote
exchange of GTP for GDP on associated G proteins. In the
case of PAR family members, the activation paradigm that
was initially established for PAR-1 includes the ability to
respond to peptides based on the sequence of the tethered
ligand. The one exception to the rule is PAR-3, for which
no activating peptide agonist has been identified.
What about the other three members of the PAR
family? PAR-2 is expressed by a number of tissues,
including endothelial cells, but not by platelets. PAR-2 can
be cleaved and activated by trypsin and tryptase, but not
by thrombin.28,29 It can also be activated by the tissue
factor/VIIa complex and factor Xa, which may be particularly relevant for endothelial cells.30 –32 PAR-3 was identified after a gene ablation study33 showed that platelets
from mice lacking PAR-1 were still responsive to thrombin. PAR-3 is a major regulator of thrombin responses in
rodent platelets,34 but little else is known about it. When
overexpressed, human PAR-3 can respond to thrombin.
However, on murine platelets PAR-3 serves solely to
facilitate cleavage of PAR-4 by thrombin.35 The fourth
family member, PAR-4, was identified by database
searches using conserved domains of the other three
family members.36,37 PAR-4 is expressed on human and
mouse platelets and accounts for the continued ability of
platelets from PAR-3 knockout mice to respond to thrombin.36,37 Simultaneous inhibition of human PAR-1 and
PAR-4 with blocking antibodies or a small-molecule antagonist completely abolishes platelet responses to thrombin,38 as does deletion of the gene encoding PAR-4 in
mice.39
Thus, the four PAR family members have some features
in common, but also have differences. Of the three that
can be activated by thrombin, two (PAR-1 and PAR-3)
have similar dose/response curves. The third, PAR-4,
requires 10- to 100-fold higher concentrations of thrombin, apparently because it lacks the hirudin-like sequences
that can interact with the anion-binding exosite and
facilitate receptor cleavage of thrombin.35–37,40 This distinction is important for understanding the role of PAR-4
in human and mouse platelets.
Platelet Activation by Thrombin
Figure 4. Structure and features of PAR-1. Cleavage of PAR-1
by thrombin between arginine 41 and serine 42 exposes a new
N-terminus that serves as a tethered ligand. Activation of PAR-1
is followed by a rapid burst of signaling before the receptor is
desensitized and, in some cases, cleared from the cell surface.49
22S
Putting all of this together, current evidence suggests
that thrombin activates human platelets by cleaving and
activating PAR-1 and PAR-4 (Fig 5). In turn, these
receptors activate Gq, G12, and perhaps Gi family members, leading to the activation of PLC␤, PI 3-kinase, and
the monomeric G proteins, Rho, Rac, and Rap1, and also
causing an increase in the cytosolic Ca2⫹ concentration
and inhibiting cAMP formation. This process is supported
by released ADP and TxA2, which bind in turn to their
own GPCRs on the platelet surface (Figs 3, 4). Cleavage of
human PAR-4 requires a higher concentration of thrombin than does cleavage of PAR-1, and it is likely that
PAR-1 is the predominant signaling receptor at low
thrombin concentrations, but PAR-4 activation may be
more sustained.41,42 Mouse platelets provide an interesting
contrast to human platelets. Where human platelets express two functional PAR family members (PAR-1 and
PAR-4), mouse platelets express PAR-3 and PAR-4, but
signaling appears to be mediated entirely by PAR-4, with
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Figure 5. Receptor-mediated platelet activation by thrombin. Platelet responses to thrombin are
mediated largely by members of the PAR family. Human platelets express PAR-1 and PAR-4, which
collectively are coupled to Gq-, G12-, and Gi-mediated effector pathways. Secretion of ADP acts as a
further activator of Gi-mediated pathways via the receptor, P2Y12. Cleavage of PAR-1 by thrombin
appears to be facilitated by the binding of thrombin to GP Ib␣ in the GP Ib/IX/V complex.
PAR-3 serving solely to facilitate the cleavage of PAR-4 at
low thrombin concentrations.35,39
One issue that remains unresolved is the contribution of
other thrombin receptors on platelets, particularly the
members of the GP Ib/IX/V complex. GP Ib is a heterodimer comprised of an ␣ and a ␤ subunit, which are
disulfide linked to each other. The heterodimer forms a
complex with GP IX and GP V that serves as both a
binding site for VWF and an anchor for the platelet
cytoskeleton.43 A high-affinity binding site for thrombin
located at approximately residues 268 –287 on GP Ib␣ is
thought to interact with domains other than the active
site.44 Deletion of the extracellular domain of GP Ib␣ or
blockade of the thrombin-binding site decreases platelet
responses to thrombin.45– 48 In theory, the binding of
thrombin to GP Ib␣ could facilitate the cleavage of a PAR
family member on human platelets, much as the binding
of thrombin to PAR-3 is thought to facilitate cleavage of
PAR-4 on mouse platelets (Fig 5).48 Thus, although it is
very clear that PAR-1 and PAR-4 provide the primary
response elements for thrombin on human platelets, it
remains possible that interactions with one or more members of the GP Ib/IX/V complex may facilitate cleavage/
activation of PAR-1 or otherwise regulate platelet activation by thrombin.
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Conclusion
In summary, there is no simple answer to the question
“how does thrombin activate human platelets?” At least
two GPCRs, several heterotrimeric G proteins, and a long
list of intracellular signaling molecules are involved. Other
surface molecules, including GP Ib␣, may play an accessory role. The identification of PAR-1 and the recognition
of its novel mechanism of action suggested that it might be
possible to develop small-molecule antagonists of platelet
activation by thrombin. A number of efforts to do so have
been launched over the past 10 years. As might be
expected (at least in hindsight), such efforts have met with
qualified success. Antagonists for PAR-1 have been identified and tested in vitro and in animal trials. Despite the
competitive advantage of having a tethered ligand, it has
proved possible to block PAR-1 activation by agonist
peptides and, in some cases, thrombin as well. One
problem is that platelets also express functional PAR-4 and
full blockade of thrombin responsiveness requires inhibition of both receptors. One might imagine administration
of a combination of PAR-1 and PAR-4 antagonists.
Whether such a combination will be more useful as
antiplatelet therapy than aspirin or an ADP receptor
antagonist awaits demonstration. Orally active inhibitors of
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thrombin are currently under investigation as potential
substitutes for warfarin in patients needing long-term
anticoagulation. Whether they offer an advantage based in
part on their ability to block platelet activation via PAR-1
and PAR-4 remains to be seen.
20
21
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CHEST / 124 / 3 / SEPTEMBER, 2003 SUPPLEMENT
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25S
Thrombin and Platelet Activation*
Lawrence F. Brass
Chest 2003;124; 18S-25S
DOI 10.1378/chest.124.3_suppl.18S
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