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Antithrombotic Drug Therapy in Cardiovascular Disease
THROMBOXANE A2 RECEPTORS
Thromboxane A2 (TXA2) is one of the bioactive metabolites of arachadonic acid. Cyclooxygenase
converts arachadonic acid into prostaglandin endoperoxide H2, which is then further metabolized to
TXA2 by thromboxane synthase. TXA2 is released from stimulated platelets and is a potent inducer
of platelet aggregation as well as vasoconstriction and bronchoconstriction
(96,97). TXA2 exerts its
influence on platelet aggregation by interacting with TXA2 receptors on the platelet surface. There
are two subtypes of TXA2 receptors, alpha and beta, but its effects in platelets are mediated primarily
by the alpha isoform. Thromboxane receptors are seven transmembrane G-protein-coupled receptors
as are a number of the agonist receptors on platelets. The transmembrane and extracellular regions of
thromboxane receptors are involved in ligand recognition. In addition, separate studies have provided
evidence that thromboxane receptors can couple to at least four separate G protein families
(98).
Consequently, thromboxane receptors can trigger a broad range of cellular responses. In platelets,
the TXA2 receptor can couple to Ga and Ga
13
. Ga
13
is phosphorylated through a PKA-dependent
mechanism
(98), linking TXA2 receptor signaling to cAMP levels. The long and well-established
effectiveness of aspirin as an antiplatelet agent raised the hope that intervention at other sites within
the arachondonic acid metabolic pathway or interference with the TXA2 receptor would provide new
antithrombotic drugs. TXA2 receptor antagonists were developed throughout the 1980s and 1990s. In
preclinical testing in animal models some of these antagonists were found to be protective in models
of thromboembolic disease (99). However, such TXA2 receptor antagonists have not yet resulted in
the development of effective antiplatelet drugs.
PLATELET AGGREGATION AND ITS RECEPTORS
aIIbb3
Deficiencies of integrin aIIbb3, either in humans or in mice, result in a failure of platelets to aggre-
gate in in
vitro assays, an absence of thrombus formation in murine models of arterial thrombosis, and
in an episodic bleeding syndrome in humans, Glanzmann’s thrombasthenia. The contribution of
aIIbb3 to platelet aggregation depends on its capacity to serve as a receptor for adhesive proteins.
Occupancy of the integrin by either fibrinogen or vWF supports platelet aggregation
(100–102). The
ligand repertoire of aIIbb3 includes still other protein ligands, such as fibronectin and vitronectin,
which can support aIIbb3-mediated platelet adhesion or modulate platelet aggregation but do not
themselves have multimeric structures appropriate for bridging platelets together
(103).
The essential role of aIIbb3 in thrombosis and hemostasis served as an impetus for extensive
analysis of its structure and function. Dedicated reviews of the structure aIIbb3 and of integrins in
general have been published
(100–102,104,105), and only a brief overview is provided here. aIIbb3
is a typical integrin composed of a noncovalent complex of its aIIb and b3 subunits. The complete
amino acid sequences of both subunits and the organization of their genes were determined in the
1980s (106–108). Each subunit can be divided into three regions: a large extracellular region, a single
membrane spanning region, and a short cytoplasmic tail. Contacts between subunits occur between
all three regions. The large extracellular regions consists of a series of domains that were initially
identified in the crystal structure of the aIIbb3 sister receptor, aVb3, which shares the same b3 subu-
nit (109,110). Today, several crystal structures of portions of the extracellular domain of aIIbb3,
including complexes with small molecule agonists and antagonists, have been determined (111). Low
molecular weight ligands nestle into a groove formed by domains contributed by each subunit: the
b-propeller from the aIIb subunit and the A-domain from the b3 subunit. In the b3A-domain, a metal
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ion bound in a MIDAS motif is critical to function. This metal ion is coordinated by an aspartic acid
residue present in the ligand
(112). The ability of ligands and antagonists to distinguish between
aIIbb3 and aVb3 depends upon the “reach” of the bound ligand from the MIDAS cation into the
b-propeller of the a subunit
(111). Hence, some ligands and antagonists can bind to aIIbb3, to aVb3
or to both integrins.
Critical to the function of aIIbb3 on platelets is its ability to undergo activation. On circulating,
unactivated platelets, aIIbb3 exists in a resting state where it exhibits low affinity for its natural
ligands. Hence, platelets can circulate in blood, a rich source of fibrinogen as well as other aIIbb3 ligands,
without occupancy of the receptor and spontaneous aggregation. When a platelet encounters an
appropriate agonist, intracellular signaling events are triggered which lead to conversion of aIIbb3
from its resting to an activated state in which its affinity for ligand increases to the point where recep-
tor occupancy, by fibrinogen and/or other ligands, occurs efficiently and leads to platelet aggregation
(113–115). Some of the agonists/receptors which induce aIIbb3 activation have been discussed earlier
and are being actively investigated as antithrombotic drug targets. Other natural agonists can trigger
aIIbb3 activation, particularly in combinations with low or subthreshold doses of ADP (115).
Included in this category are epinephrine and serotonin, with platelets expressing the G-protein-
coupled receptors that also recognize these agonists.
While the agonist receptors on platelets and the intracellular signaling processes that they elicit are
complex, the pathways converge to generate a common inside-out signal that changes the cytoplasmic
tails and initiates activation of aIIbb3 (116). As currently envisioned, the cytoplasmic tails of the aIIb
and b3 subunits interact with each other through several electrostatic and hydrophobic bonds between
their membrane proximal helices. This intersubunit clasp maintains the integrin in a resting state. The
signaling events from the agonist receptors converge and disrupt the clasp
(117,118). This change is
sensed in the transmembrane helices of the two subunits, which also are likely to be clasped, and
which then undergo an unclasping or a change in registry of their interaction to propagate the inside-
out activating signal across the membrane
(119,120). In the extracellular region, conformational
changes which alter the relationship between the b-propeller and the b3A domain prepare the ligand-
binding site to accept macromolecular ligands. A component of this conformational rearrangement
may be an unbending of the integrin to bring its ligand-binding site away from the membrane, permit-
ting easier access of large ligands and, once bound, for the ligands to bridge to another platelet
(121,122).
The contribution of aIIbb3 to thrombus formation does not end with its role in binding ligands.
Occupancy of aIIbb3 and other integrins results in their clustering. The consequence of such clustering
is to create adhesive patches where platelet cohesive properties are enriched; to recruit, activate, and
occupy other aIIbb3 molecules within the cluster; and to generate outside-in signals (105,123).
Certain signals can be generated across a single occupied aIIbb3 but most of the downstream signaling
events in platelets require clustering of occupied integrins for transmission. From the standpoint of
thrombus formation, such outside-in signals stabilize the thrombus, and interventions, either genetic
or pharmacologic that inhibit outside-in signaling, reduce the stability of platelet aggregates in
vitro
and destabilize thrombi in vivo (e.g., (124)).
Modulators of Platelet Aggregation
An emerging theme of platelet research has been the role of proteins other than aIIbb3 that modu-
late platelet aggregation. Antagonism of these molecules may blunt but not necessarily totally suppress
platelet aggregation. In principle, such partial antagonism of the functional response may limit platelet
participation in thrombosis while sparing their hemostatic activity. Thus, interference with the function
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Antithrombotic Drug Therapy in Cardiovascular Disease
of these molecules would suppress thrombosis but would not lead to the bleeding complications associated
with direct aIIbb3 antagonists. Included in this group of targets are membrane proteins as well as the
soluble ligands that bind to them. One of the first molecules displaying these properties was the
growth arrest-specific gene 6, Gas-6. Gas-6 is present in and released from platelet a-granules upon
platelet stimulation and aggregation (125). It is a 75-kDa protein which, like several coagulation pro-
teins, contains g-carboxylated glutamic acids. Gas-6 is a ligand for Axl, Sky, and Mer. These are all
transmembrane tyrosine kinases, and all are present on the platelet surface
(126). When Gas-6 binds
to these receptors, their kinase activities become activated. A downstream target of such activation is
phosphorylation of the two tyrosine residues in the cytoplasmic tail of the integrin b3 subunit, which
regulate outside-in signaling from the occupied integrin. As a result, thrombus growth and stability is
dependent upon Gas-6 and its receptors (125). Platelets from mice deficient in Gas-6 show an initial
aggregation response to various agonists but the aggregates are unstable. In vivo, the Gas-6
−/−
mice
are resistant to thrombosis but show normal bleeding times
(127). Mice in which the two tyrosine
residues in the b3 cytoplasmic tail have been the mutated to phenylalanines, the so-called DiY mice,
exhibit a similar phenotype
(124). Thus, outside-in signaling may provide multiple targets for dampen-
ing thrombosis while sustaining sufficient hemostatic function to avoid bleeding. Several other lig-
ands released from platelets and bound to platelet surface receptors (e.g., CD40L binding to CD40),
or membrane proteins involved in homotypic (e.g., PECAM-1 or JAM A and C) or heterotypic (e.g.,
Eph kinases and ephrins) interactions between platelets have been shown to modulate platelet aggre-
gation. Modulation of platelet response by such molecules has been the subject of recent reviews
(128,129). For several of these, deficiencies in mice show their potential to modify thrombosis
in
vivo.
Another category of platelet proteins that regulate platelet aggregation are ones that affect integrin
activation. Talin is a large (220 kDa) and abundant cytoskeletal protein within platelets as well as
many other cell types. A substantial body of evidence identifies talin as the final activator of aIIbb3
and other integrins (130,131). This function depends on binding of talin to the b3 cytoplasmic tail
and dissociating the cytoplasmic tail clasp
(117,132). Among the evidence that supports this role of
talin is the failure of platelets rendered deficient in talin or bearing a mutant aIIbb3 which does not
bind talin, to aggregate (132). Hence, talin and more specifically, the talin-b3 interaction, becomes
a potential target for antithrombotic drugs. Recently, our understanding of aIIbb3 activation has
become more complex. Members of the kindlin family of actin-associated proteins have been shown
to bind to the b3 cytoplasmic tail but at a different site than talin
(29,133,134). Platelets from mice
deficient in kindlin-3, one of the three kindlin family members, also fail to aggregate
(29). Hence,
kindlins also are candidate targets for suppression of platelet responses and thrombosis. Talin and kindlins
are widely distributed. Inhibitors of talin and kindlins that could be selectively delivered into platelets
or do not have deleterious effects in other cells would need to be developed to exploit these targets.
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From: Contemporary Cardiology: Antithrombotic Drug Therapy in Cardiovascular Disease
Edited by: A.T. Askari and A.M. Lincoff (eds.), DOI 10.1007/978-1-60327-235-3_2
© Humana Press, a part of Springer Science+Business Media, LLC 2010
2 The Role of Coagulation in Arterial
and Venous Thrombosis
Kandice Kottke-Marchant
Co n t e n t s
Introduction
Clinical Manifestations of Arterial and Venous Thrombosis
Coagulation Physiology
Regulation of Coagulation
Coagulation Dysregulation in Arterial and Venous Thrombosis
Summary
References
Ab s t r A c t The coagulation cascade is integral to the hemostatic process and serves to limit the amount of
blood loss during trauma. However, derangements in this process can result in venous thrombosis and con-
tribute to the development of arterial atherothrombotic disease. Indeed, in arterial thrombosis, the effects
of thrombin may extend far beyond coagulation activation and play an important role in activation of a
wide variety of cells and the inflammatory processes. Venous thrombosis and arterial thrombotic diseases
have traditionally been thought of as separate processes; however, they share many similarities in patho-
physiology and risk factors. The activation of the coagulation cascade underlies both arterial and venous
thrombosis, and biological triggers, such as inflammation, prothrombotic microparticles, and endothelial
activation provide a plausible link between the two.
Key words:
Coagulation cascade; Mechanism; Pathophysiology; Platelets; Thrombosis
INTRODUCTION
Hemostasis and thrombosis are related processes involving the coagulation system, platelets, endothe-
lial cells, and the vascular wall. Indeed, Rudolf VIrchow, in the 1800s, postulated a triad of causes for
thrombin formation: changes in the composition of blood, alterations in the vessel wall, and disruption of
blood flow (1). Physiological hemostasis occurs as a protective response to vascular injury; exposure of
blood components to subendothelial proteins stimulates activation of platelets and production of the key
coagulation enzyme thrombin leading to formation of a fibrin meshwork (2,3). This process prevents
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excessive bleeding or exsanguination after vascular injury or trauma. On the other hand, thrombosis can
be considered “hemostasis in the wrong place and at the wrong time”
(4), with dysregulation of one or
more elements of the hemostatic system contributing to formation of a platelet-fibrin thrombus that often
occludes blood vessels, leading to pathologic complications
(5). If the thrombus occurs in the arterial
system, distal ischemia can lead to acute coronary syndromes, myocardial infarctions, stroke, and peripheral
extremity necrosis. Thrombus formation in the venous system, typically deep venous thrombosis, leads to
local tissue congestion and decreased venous blood return, but dislodged thromboemboli may result in
the potentially devastating complications of pulmonary infarction or, paradoxically, stroke.
CLINICAL MANIFESTATIONS OF ARTERIAL AND VENOUS THROMBOSIS
Thrombosis in the arterial system typically occurs superimposed on vascular abnormalities that are
the result of other diseases, such as atherosclerosis or vasculitis
(6,7). Arterial atherosclerotic plaques
are well known to liberate prothrombotic and inflammatory substances, such as oxidized lipids
(8),
and express procoagulant molecules such as tissue factor
(9). While coagulation activation is undoubt-
edly involved in arterial thrombosis, platelet activation plays a crucial role, as described in Chap. 1,
and antiplatelet drugs are the predominant pharmacologic therapy for this class of disorders (10).
Arterial thrombosis in this setting, leading to vascular occlusion, can result in vascular-bed-specific
pathologies. Coronary artery occlusion results in acute coronary syndromes or myocardial infarction,
while cerebrovascular occlusion results in thrombotic stroke, and occlusion of peripheral arteries
leads to peripheral arterial disease and gangrene. Paradoxical embolism of a venous thrombus through
a patent foramen ovale may also lead to cerebrovascular thrombosis (11).
In contrast to arterial thrombosis, venous thrombotic disorders are not usually associated with
underlying vascular pathologies. Instead, venous thrombosis is associated with venous stasis or con-
genital dysregulation of coagulation proteins or natural anticoagulants
(12). Deep vein thrombi, most
often involving the veins of the lower extremities, may dislodge and result in embolism to the pulmo-
nary vasculature, with segmental pulmonary infarction and an attendant high morbidity rate (13).
Prolonged atrial fibrillation is often complicated by thrombosis of the atrial appendages. While multi-
factorial, stasis is a contributing factor in thrombosis observed with atrial fibrillation, the role of the
coagulation system is highlighted by success with targeted anticoagulant therapies
(14,15).
While a direct clinical link between arterial and venous thrombosis has not been demonstrated,
atherosclerosis and venous thromboembolic disease share similar risk factors, such as age, obesity,
diabetes mellitus, and the metabolic syndrome
(16). The activation of the coagulation cascade under-
lies both arterial and venous thrombosis, and biological triggers, such as inflammation, prothrombotic
microparticles, and endothelial activation provide a plausible link between the two. Some disorders,
such as the antiphospholipid antibody syndrome (APS) and heparin-induced thrombocytopenia (HIT),
have clear clinical associations with both arterial and venous thrombosis (17). The thrombosis in HIT,
for example, is thought to be multifactorial, with platelet activation, endothelial activation, and
thrombin generation all contributing to the pathophysiology (18). In the following sections, the physi-
ology of coagulation and its regulation will be described, followed by a discussion of coagulation-
associated risk factors for both arterial and venous thrombosis.
COAGULATION PHYSIOLOGY
The coagulation system, together with endothelial cells and platelets, is responsible for maintaining
blood in a fluid state, but, when activated, rapidly results in development of a fibrin clot by conversion
of the plasma protein, fibrinogen, to the polymer fibrin by the key enzyme, thrombin (2,3) (See Fig. 1).
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