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50 Part I / Basic Principles
Table 1
Agonist Receptor Platelet signaling
Adenosine P2Y
1
(P2T
PLC
) Gq-coupled, activation of PLC
β
diphosphate (ADP) P2Y
12
(P2T
AC
) Gi-coupled, inhibition of adenylate cyclase
P2X1 Ligand-gated ion channel, increase in
intracellular Ca
2+
levels
Thrombin PAR 1 Gi-coupled, inhibition of adenylate cyclase,
activation of non-receptor tyrosine kinases,
G protein
PAR 4 Coupled receptor kinases, PLC
β
, PI3K,
K
+
channel
Gq, activation of PLC
β
G
12/13
-coupled, activation of Rho proteins
Collagen α
2
β
1
Tyrosine phosphorylation and activation of
Syk, phosphorylation of PLC
γ2
PLC GP VI Associated with Fcγ, activation linked to Syk
Epinephrine α
2
Gz-coupled, inhibition of adenylate cyclase,
activation of PLC
Serotonin 5-HT receptor Gq-coupled
Thromboxane (TXA
2
)TPα Gq and G
12/13
-coupled, activation of PLCβ
vWF GPIb/IX/V Association with 14-3-3 εƒ, calmodulin
complex Coupled with Fc receptor γ-chain, association
with tyrosine kinases (Fyn, Lyn), activation
of Syk, PI3K
Immobilized fibrinogen GPIIb/IIIa Activation of tyrosine kinases (e.g., Src, and
Syk, activation of PLC, PI3K, SHIP
sCD40L GPIIb/IIIa Required for aggregate stability
Leptin Leptin receptor Aggregate stability cofactor
Gas6 Axl, Sky Aggregate stability cofactor
Eph kinase/ephrin Ephrin/Eph kinase Aggregate stability cofactors
The second level of redundancy is the signal transduction pathways from agonist recep-
tor stimulation through GPIIb/IIIa activation, as different receptors use overlapping
signal transduction pathways. For example, many of the agonist receptors on platelets are
heptahelical receptors (e.g., PAR1, thromboxane receptor, ADP receptor), which as a class
are known to be coupled to G proteins, a heterotrimeric family of signaling molecules
consisting of α-, β-, and γ-subunits that signal by dissociating into Gα (a GTPase) and
β-γ-dimers, both of which can propagate signals (98). Support for the direct involvement
of Gαq in GPIIb/IIIa inside-out signaling is provided by observations showing that
platelets from mice made deficient in this α-subunit have defective platelet aggregation
in response to ADP and thrombin (99). Gαq-deficient mice also have defective throm-
bosis in response to collagen and have prolonged bleeding times. Interestingly, Gabbeta
et al. (100) found that the platelets from a patient with defective GPIIb/IIIa activation in
response to thrombin or ADP were partially deficient in Gαq. Recently, platelets
from
Gα2
–/–
mice have also been found to be defective
for ADP-dependent inhibition of ade-
nylyl cyclase, integrin activation,
and aggregation (101).
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Chapter 3 / GPIIb/IIIa in Platelet Aggregation 51
Receptors for collagen or for vWf activate different intracellular signaling pathways
that ultimately result in GPIIb/IIIa activation. GPVI is normally bound to another mem-
brane protein termed Fcγ (102). Binding of collagen to GPVI causes phosphorylation of
the two tyrosine residues in the ITAM domain on the cytoplasmic face of Fcγ. Phosphory-
lated Fcγ then recruits Syk to the membrane so that it can be phosphorylated, activating its
tyrosine kinase so that it, in turn, can phosphorylate downstream effectors (e.g., phospho-
lipase Cγ) to propagate the signal through to GPIIb/IIIa activation. Platelets from mice
in which either the Syk or the Fcγ genes are disrupted are now known to have defective
collagen-induced signaling (103). More recently, a direct association of Fyn and Lyn
with the proline-rich domain of GPVI via their SH3 domain has been shown to regulate
intracellular signaling (104). GPIbα, the receptor for activated vWf, is also coupled to
the Fc receptor γ-chain, Fyn and Lyn (105).
The cytoplasmic domains of GPIbα and GPV in the GPIb/IX/V complex also bind pro-
tein 14-3-3ζ (94,106,107), an adapter protein that binds Raf-1 and PKC, both serine/thre-
onine kinases known to couple receptors to signaling pathways, and to phosphatidyl inositol
3-kinase (108). Shear conditions in which the thrombotic process happens also directly
influenced signaling events leading to GPIIb/IIIa activation. If, under static conditions,
binding of GPIbα to immobilized vWF induced a PI 3-kinase-independent intracellular
calcium release (109), under conditions of high shear rates, the same kinase is indispensable
for the calcium release and promotion of platelet adhesion to immobilized vWF (95). Inter-
estingly, GPIbα also seems to be able to mediate two different types of signaling path-
ways leading to GPIIb/IIIa activation, whether thrombin or vWF is the agonist (110). These
examples indicate that the intracellular signaling pathways within platelets for GPIIb/IIIa
activation can have diverse origins.
As of this writing it is unknown how signal transduction pathways initiated by the vari-
ous agonist receptors are coupled to GPIIb/IIIa activation. However, key players between
agonist receptors and GPIIb/IIIa have been identified. Two are phospholipase C (PLC)
and protein kinase C (PKC). PLC exists in several isoforms and functions to catalyze the
hydrolysis of phosphatidylinositol phosphates to produce inositol phosphate (IP
3
), which
induces the release of Ca
2+
from intracellular stores, and diacylglycerol, which markedly
enhances the catalytic activity of PKC (111). Phosphatidyl inositol phosphate hydrolysis
can be mediated by PLCβ, which is activated by Gαq, or by PLCγ, which can be activated
by kinases such as Syk. Support for the role of IP3, Ca
2+
, and diacylglycerol is derived
from studies showing that elevation of the cytoplasmic Ca
2+
concentration with the cal-
cium ionophore A23187 also induces GPIIb/IIIa activation (112) and that the GPIIb/IIIa
activation induced by certain platelet agonists can be diminished by PKC inhibitors (113).
Another key player between agonist receptors and GPIIb/IIIa activation is phospho-
lipase A
2
(PLA
2
), which catalyzes the release of arachidonic acid from the 2-position of
phospholipids. Arachidonate is metabolized to thromboxane A
2
(TXA
2
), the final reaction
being catalyzed by cyclooxygenase. TXA
2
acts on the TXA
2
receptor, a heptahelical recep-
tor, to propagate the signaling response. The effectiveness of aspirin, a cyclooxygenase
inhibitor, in blocking GPIIb/IIIa activation, particularly at low agonist concentrations,
demonstrates the importance of this pathway to GPIIb/IIIa signaling (114). However, the
ability of high agonist concentrations to overcome the effects of aspirin inhibition, even
when cyclooxygenase is completely inhibited, indicates the redundancy of the signal trans-
duction mechanisms for GPIIb/IIIa activation. Less well understood are the proximal events
at GPIIb/IIIa that result in its activation. Because chemical modifications of GPIIb/IIIa
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52 Part I / Basic Principles
have not been observed during inside-out GPIIb/IIIa signaling, it has been assumed that
signaling induces either the association or dissociation of a regulatory protein(s) to the short
cytoplasmic domains of GPIIb/IIIa. Several intracellular proteins have been identified
that bind to peptides corresponding to the cytoplamic domains of GPIIb/IIIa including
β3-endonexin (to GPIIIa) (115), CIB (to GPIIb (116,117), and several cytoskeletal proteins
(see below). Of these proteins, the binding of CIB seems most relevant as this protein has
been shown to activate GPIIb/IIIa directly via an interaction with the cytoplasmic domain
of αIIb (118). This conclusion, however, remains controversial (119) and requires further
investigation. Three transmembrane proteins have also been shown to bind GPIIb/IIIa,
CD9, a member of the tetraspanin class of proteins (120,121), CD47, integrin-associated
protein, which may assist in thrombospondin-induced GPIIb/IIIa activation (122), CD98,
a potential GPIIb/IIIa modulator (123), and CD151 (124).
Fibrinogen Binding on Unstimulated Platelets
Although GPIIb/IIIa must become activated to bind soluble fibrinogen, this is not the
case for fibrinogen immobilized on surfaces (e.g., vessel walls or plastic) or for fibrinogen
bound to GPIIb/IIIa. In both instances, binding apparently alters the conformation of fibrin-
ogen so that the γ-chain sequence becomes capable of binding GPIIb/IIIa on unstimulated
platelets (9,38,125,126). Importantly, fibrinogen in this instance becomes a platelet ago-
nist, as binding of GPIIb/IIIa by this mechanism initiates an outside-in signal transduc-
tion reaction through GPIIb/IIIa, resulting in platelet stimulation (see below for discussion
of mechanism).
There are potentially three important physiologic consequences of this reaction. First,
it provides a mechanism for platelet aggregation whereby fibrinogen is the primary ago-
nist and the bound fibrinogen is responsible for recruiting and activating platelets by out-
side-in GPIIb/IIIa signaling. Second, fibrinogen bound to GPIIb/IIIa on surfaces is also
capable of binding Mac-1 to mediate the arrest and adhesion of neutrophils (39). Third, fibrin-
ogen is one of the proteins bound to artificial surfaces, including extracorporeal devices
such as in-dwelling catheters. The immobilization of fibrinogen on these surfaces, leading
to the generation of an active conformation of fibrinogen capable of activating GPIIb/IIIa,
may also account for the thrombotic potential of artificial surfaces when exposed to plasma
proteins (9), and may explain the thrombocytopenia associated with bypass surgery. This
idea is supported by observations showing that GPIIb/IIIa antagonists reduce platelet loss
in animal models of cardiopulmonary bypass (127).
OUTSIDE-IN GPIIb/IIIa SIGNAL TRANSDUCTION
The binding of adhesive ligands (e.g., fibrinogen, vWF) to GPIIb/IIIa, together with the
resultant clustering of the integrin following adhesion or aggregation, initiates outside-in
signaling events leading to further platelet activation. These activation events are required
for maximal platelet responses when platelets are activated with ADP or suboptimal amounts
of other platelet agonists. Such responses include secretion of the contents of α-granules
and dense bodies when platelets are activated with ADP or suboptimal amounts of other
agonists as well as activation of the remaining GPIIb/IIIa molecules.
Although outside-in signals are required for maximal platelet aggregation, adhesion, clot
retraction, and secretion, in and of themselves they are insufficient to induce these responses:
low concentrations of other agonists (e.g., ADP) are required as costimuli. However,
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Chapter 3 / GPIIb/IIIa in Platelet Aggregation 53
since low concentrations of other platelet agonists are normally present at sites of vascu-
lar injury such as would occur with plaque rupture or following percutaneous interven-
tion, it can be anticipated that platelet activation via outside-in GPIIb/IIIa signaling occurs
in these settings and that GPIIb/IIIa antagonists will have effects on platelet functions like
secretion, GPIIb/IIIa activation, and enhancement of coagulation that are normally attrib-
uted to the classical platelet agonists such as ADP, thrombin, and collagen. In support of
this conclusion, Carroll et al. (128) showed that the GPIIb/IIIa antagonist Ro 44-9883
inhibited α-granule secretion when platelet aggregation was induced by ADP or low doses
of TRAP or collagen. Studies claiming that GPIIb/IIIa antagonists do not affect these
reactions (129) were apparently performed under conditions in which aggregation and out-
side-in GPIIb/IIIa signaling were not involved. The requirement for low levels of other
agonists may also allow for a further level of regulation of platelet aggregation by con-
fluent endothelium, which expresses CD39, an ecto-ADPase that acts by metabolizing
the ADP released by platelets (130), and thrombomodulin (131), which neutralizes throm-
bin. A lack of these two activities at sites of disrupted endothelium may facilitate platelet
activation through outside-in GPIIb/IIIa signaling. However, the antithrombotic pheno-
type displayed by the CD39-deficient mice demonstrates the complexity of pro- and anti-
thrombotic activities present at sites of vascular injury (132).
The mechanism by which GPIIb/IIIa propagates outside-in signaling is of extreme
importance in understanding GPIIb/IIIa function. Analysis of mutations of GPIIb/IIIa
revealed that the extracellular adhesive protein binding pocket and the cytoplasmic
domains were crucial for outside-in signaling events. Previous work in our laboratory has
identified a postaggregation phosphorylation of the cytoplasmic domain of GPIIIa that
appears to act as scaffolds for the recruitment of signaling proteins and propagation of
the outside-in signal, and as anchor sites for the cytoskeletal proteins myosin (133–136).
The clear demonstration of a physiologic role for the GPIIIa tyrosine phosphorylation
signaling pathway following engagement of GPIIb/IIIa came from the generation of trans-
genic mice (diYF) in which both tyrosines in the cytoplasmic domain of GPIIIa were replaced
by phenylalanines. Inside-out signaling is not affected in diYF mice, as demonstrated by
the normal binding of FITC-fibrinogen upon platelet activation, whereas outside-in sig-
naling is impaired, which in turn affects both thrombosis and hemostasis. Indeed, whereas
the velocity of platelet aggregation induced by weak agonists is normal in diYF mice, the
platelet aggregates spontaneously disaggregate. This defect in aggregation causes the diYF
mice to generate unstable hemostatic plugs and to retract clots poorly (5,134,135).
The downstream signaling events induced by outside-in GPIIb/IIIa signaling include
protein tyrosine phosphorylation of numerous cytoplasmic proteins, as well as reorgani-
zation of the cytoskeletal/contractile apparatus within the platelet. Several of the proteins
phosphorylated are tyrosine kinases, including multiple Src family members (e.g., Src,
Lyn, Fyn), Syk, and FAK (137,138). The effects of tyrosine kinase inhibitors have high-
lighted the importance of tyrosine phosphorylation in platelet signaling, as a wide range
of tyrosine kinase inhibitors including genistein, erbstatin, the tyrphostins B42 and B46,
and piceatannol have been found to inhibit platelet aggregation and secretion (83,139–
141). Both Syk and Src become phosphorylated and activated within seconds of fibrino-
gen binding to GPIIb/IIIa and translocate to the cytoskeleton upon aggregation. Upon
fibrinogen binding, Csk, which is a negative regulator of Src under nonstimulated con-
ditions, dissociates from GPIIb-IIIa, which allows the Src activation loop to be phospho-
rylated (142).
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54 Part I / Basic Principles
Chemical crosslinking studies have demonstrated that Src is physically associated with
GPIIb/IIIa within platelets as is another Src family member, Lyn (143). The lack of any
defect in platelet function in platelets from mice in which the gene for Src has been dis-
rupted is probably owing to the redundancy between members of the Src family of tyrosine
kinases (144). Indeed, normal aggregation in response to γ-thrombin was observed with
Src- and Fyn-deficient platelets, whereas Lyn deficiency reduced the aggregation response
(145). In contrast to the phosphorylation of Syk and Src, the FAK tyrosine kinase only
becomes phosphorylated and activated upon full spreading or aggregation of platelets and
requires agonist costimulation in addition to integrin ligation (146). FAK autophosphor-
ylation at tyrosine 397 leads to its association with Src, which can phosphorylate other
tyrosine residues within the protein to provide docking sites for other phosphotyrosine
binding proteins including GRB2 (147). Thus, the common theme emerging from the study
of outside-in GPIIb/IIIa signaling involves the tyrosine phosphorylation of a series of pro-
teins, including the phosphorylation and activation of tyrosine kinases, followed by the
assembly of signaling complexes.
Other second messenger pathways have also been implicated in outside-in GPIIb/IIIa sig-
nal transduction events, one of which is the hydrolysis of phosphoinositides. As described
earlier, platelet activation leads to activation of phospholipases, leading to the production
of inositol 1,4,5-trisphosphate. This polyphosphoinositide can be phosphorylated on the
D3 position, subsequent to platelet activation, by at least two types of phosphoinositide
3-kinases (p85/PI3K and PI3Kγ) (148). D3 phosphoinositides have been implicated in
actin assembly and filopodial extension in the platelet (149). The activation of p85/PI3K
and its translocation to the cytoskeleton are dependent on GPIIb/IIIa outside-in signaling
and platelet aggregation (150). Treatment of platelets with the PI3K inhibitor wortmannin
results in the reversal of TRAP-induced platelet aggregation and affects aggregation
induced by the LIBS6 antibody, although platelet secretion remains unaffected. That wort-
mannin also inhibited the maintenance of GPIIb/IIIa in its high-affinity conformation
suggested that PI3K may be involved in the formation of stable platelet aggregates, possi-
bly by sustaining GPIIb/IIIa in an active state (151). This hypothesis was confirmed by the
defect in thrombus formation observed in the PI 3-kinase-deficient mice (152). Another
protein involved in this signaling pathway that is also phosphorylated and induced to trans-
locate to the cytoskeleton upon GPIIb/IIIa-mediated platelet aggregation is the inositol
5-phosphatase, SHIP. This 145-kDa protein can dephosphorylate inositol 1,3,4,5-tetra-
kisphosphate and phosphatidyl inositol 3,4,5-trisphosphate, leading to the further produc-
tion of D3 phosphoinositides (153).
The calcium-activated protease calpain also becomes activated during platelet aggre-
gation, possibly indicating that this protein is another second messenger in GPIIb/IIIa
signaling (154–156). Indeed, recent studies in the calpain knockout mouse showed that
calpain deficiency causes the same aggregation and clot retraction defect as occurred in
the diYF mouse (157). A direct link between GPIIb/IIIa and calpain was established by
observations showing that calpain-deficient platelets fail to tyrosine phosphorylate the
cytoplasmic domain of GPIIIa (157) and by observations showing that the cytoplasmic
domain of GPIIIa is a calpain substrate and that calpain-mediated hydrolysis of GPIIIa
during platelet aggregation leads to inhibition of fibrin clot retraction (156). However,
since calpain cleaves several proteins in addition to GPIIIa, it is not clear which hydro-
lytic event is responsible for this effect (158). The small GTPases including Ras, Rho, Rac,
and CDC42 constitute a further class of second messengers that appear to be involved in
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Chapter 3 / GPIIb/IIIa in Platelet Aggregation 55
GPIIb/IIIa-mediated signaling downstream of calpain. The Rho family (Rho, Rac, CDC42)
are important in GPIIb/IIIa-mediated actin assembly (159,160). The role that Ras plays
in platelet integrin signaling remains to be established. Although in many cell types Ras is
involved in activation of the MAP kinase cascade, it is not clear that this is its role in plate-
let biology. Indeed, Ras may even act to regulate Rho negatively, since the Ras-associated
protein GAP can associate with p190 Rho-GAP, leading to the inactivation of Rho (161).
GPIIb/IIIa Cytoskeletal Interactions
Many of the signaling events described above involve the translocation of proteins to
the cytoskeleton, leading to reorganization of the cytoskeleton/contractile apparatus so
that it can consolidate a platelet aggregate and retract a clot, processes important to throm-
bus stability. The binding of fibrinogen and subsequent platelet aggregation result in an
increased association of GPIIb/IIIa with cytoplasmic actin filaments and the formation of
focal contact-like structures (162,163). Important issues concern (1) how outside-in signal-
ing induces GPIIb/IIIa association with the cytoskeleton and (2) the identity of the cytoskel-
etal protein used for integrin attachment. It is estimated that approx 30% of GPIIb/IIIa
becomes associated with the cytoskeleton following platelet aggregation. GPIIIa tyrosine
phosphorylation is involved in this interaction, since tyrosine-phosphorylated GPIIIa
is retained preferentially with the cytoskeletal fraction from aggregated platelets (164).
Phosphorylated GPIIIa appears to bind to platelet myosin, as phosphorylated GPIIIa cyto-
plasmic domain peptides but not unphosphorylated peptides have been found to interact
selectively with the heavy chain of platelet myosin (165). This suggests a mechanism for
regulation of the interaction of GPIIb/IIIa with the platelet cytoskeleton: the cytoplasmic
domain of GPIIIa becomes tyrosine phosphorylated upon integrin ligation and platelet
aggregation. In addition to the phospho-GPIIIa interacting with signaling proteins (described
previously), it also translocates to the cytoskeleton, where the phosphotyrosine residues
may direct binding to the contractile protein myosin. GPIIb/IIIa on adherent platelets aligns
with cytoplasmic actin filaments (166) and, with time, redistributes to the abluminal sur-
face, causing the platelet to become refractory for the recruitment of additional platelets
(167). The resultant loss, on the luminal surface, of accessible, activated GPIIb/IIIa may
serve to limit the recruitment of platelets to a growing thrombus (167).
Clot retraction is GPIIb/IIIa-dependent: the integrin serves as the transmembrane bridge
between extracellular fibrin and the cytoplasmic contractile machinery. The direct bind-
ing of GPIIb/IIIa with several purified cytoskeletal proteins in addition to myosin has been
demonstrated, e.g., the binding of purified GPIIb and GPIIIa to talin (168), the binding
of GPIIb/IIIa incorporated into phospholipid vesicles with α-actinin (169), and the bind-
ing of the GPIIIa cytoplasmic domain, in a yeast two-hybrid screen, to skelemin (170). An
unsolved problem is the identification of the cytoskeletal interactions that are responsible
for the clot retraction process. In this regard, it is of interest that mutations in the cytoplas-
mic domains of GPIIb/IIIa that affect integrin signaling can also inhibit clot retraction
(171), including those mutations involving GPIIIa tyrosine residues (as described previ-
ously) (164,172). Since tyrosine mutations would prevent GPIIIa phosphorylation, it is
possible that one way such mutations can exert their effect on clot retraction is by pre-
venting the association of phosphotyrosine-GPIIIa with myosin. In support of this idea,
tyrosine kinase inhibitors can regulate the cytoskeletal attatchment of GPIIb/IIIa and can
inhibit the clot retraction process (173). Furthermore, diYF-deficient mice also presented
a defect in fibrin clot retraction (134).
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56 Part I / Basic Principles
GPIIb/IIIa POLYMORPHISMS
AS A CARDIOVASCULAR RISK FACTOR
Our initial understanding of GPIIb/IIIa structure-function relationships came from the
study of patients with Glanzmann’s thrombasthenia. This hereditary disease, which
results in a life-long bleeding diathesis, is caused by a lack of GPIIb/IIIa function. More
than 38 mutations in GPIIb and 27 in GPIIIa have been characterized at the genetic level
(174). The identified mutations cause either (1) defective biosynthesis or (2) normal or
nearly normal expression of a defective protein owing to a loss of function mutation.
Mutations that lead to defective expression of GPIIb/IIIa include those that produce defec-
tive proteins caused by early terminations, deletions, or frameshifts, those that generate
a GPIIb/IIIa complex that is sensitive to dissociation [e.g., an Arg214Trp point mutation
in GP IIIa (175)], and those affecting mRNA stability [e.g., Arg53Stop in GPIIb (176)]
or RNA processing [e.g., an extensive 3-kb insertion in GPIIIa (177)]. Loss of function
mutations include those that affect the ligand binding and Ca
2+
binding sites in GPIIb/IIIa
and also those that affect GPIIb/IIIa signaling. Some of these latter mutations highlight the
importance of the cytoplasmic domains in GPIIb/IIIa function. For example, a patient with
a single point mutation in the cytoplasmic domain of GPIIIa where serine 752 is replaced
by a proline (S752P) had platelets that expressed nearly normal amounts of GPIIb/IIIa but
that failed to aggregate, to spread, or to undergo FAK phosphorylation in response to fibrin-
ogen binding (178). This mutation helped validate the CHO cell expression system as a
model for studying GPIIb/IIIa. When transfected into CHO cells with GPIIb, GPIIIa pro-
tein bearing S752P also showed a loss of GPIIb/IIIa function with impaired integrin sig-
naling (171).
In addition to the mutations arising in GPIIb/IIIa that lead to Glanzmann’s thrombasthe-
nia, there are a number of naturally occuring allotypic variants of GPIIb/IIIa. One of these
is the Pl
A
alloantigen system, which has frequently been implicated in syndromes of immune-
mediated platelet destruction. The Pl
A1
and Pl
A2
isoforms arise through a Leu33Pro amino
acid polymorphism in GPIIIa (179), a polymorphism that has generated considerable inter-
est since it has been postulated that the Pl
A2/2
genotype is a possible risk factor for coronary
thrombosis. A correlation between the Pl
A2/2
genotype (found in approx 2% of the Caucas-
ian population) and acute coronary thrombosis was observed in one study (71 patients,
68 controls) (180). This correlation was strongest in patients who had had coronary events
at a relatively young age (<60 yr). However, although a number of studies support this
putative association between the Pl
A2/2
genotype and an increased chance of coronary
disease (181,182), it remains controversial since other investigators have failed to observe
such an association (183–185). Although it remains to be established whether the Pl
A2/2
polymorphism is indeed a risk factor indicator for coronary disease, recent studies do sug-
gest that platelet glycoprotein polymorphisms may provide a whole new array of thrombo-
tic risk factors (186). Indeed, it is of particular interest that Greenland Inuits, a population
with a low incidence of thrombotic disease, have a lower frequency of the Pl
A2
allele than
does the general Caucasian population (187). An increased aggregability in response to
ADP and epinephrine stimulation might be associated with Pl
A2
-bearing platelets (188).
One study noted that the Pl
A2
polymorphism of β3 enhances outside-in signaling (189);
even more interestingly, two recent studies highlight decreased levels of inhibition achieved
by abxicimab in platelets with the Pl
A2
polymorphism and enhanced thrombin generation
associated with the Pl
A2
polymorphism (190,191). A higher risk of a primary event and
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Chapter 3 / GPIIb/IIIa in Platelet Aggregation 57
myocardial infarction in Pl(
A2
) carriers compared with noncarriers was observed in patients
receiving orbofiban, suggesting that an analysis of the polymorphism might be required
upon the use of oral GPIIb/IIIa antagonists (192).
GPIIb/IIIa IN INFLAMMATION
Although the role of GPIIb/IIIa in platelet aggregation is well established, recent find-
ings suggest that GPIIb/IIIa is also involved in vascular inflammation via its interaction
with CD40 ligand (CD40L, CD154, gp39), a transmembrane protein in platelets (Fig. 3).
CD40L is a member of the TNF-α family of proteins, which was originally identified in
T-lymphocytes (193), where it functions in the immune response by binding to its recep-
tor on B-cells, CD40, to mediate a variety of responses including antibody class switch-
ing (194). CD40L and its receptor, CD40, have in addition been identified on cells within
the vasculature including endothelial cells, smooth muscle cells, monocytes, and macro-
phages where it has been implicated in various inflammatory responses (194), including
expression of inflammatory adhesion receptors [e.g., E-selectin, VCAM-1, and ICAM-1
(195)], expression of tissue factor (196), and the release of chemokines (e.g., MCP-1, IL-6,
and IL-8]. Studies utilizing blocking monoclonal antibodies and gene targeting have now
determined that the CD40L/CD40 system has roles in atherosclerotic lesion progression
(197), establishing CD40L as a major mediator of vascular inflammation.
Fig. 3. Model showing the interelations existing among GPIIb/IIIa, thrombosis, inflammation,
coagulation, and restenosis. Soluble CD40L (sCD40L) is released from platelet-rich thrombi and con-
tributes to various steps in atherosclerotic lesion progression: (1) inflammation—sCD40L induces the
production and release of proinflammatory cytokines from vascular cells and matrix metalloproteinases
from resident cells in the atheroma; (2) thrombosis—sCD40L stabilizes platelet-rich thrombi; and (3)
restenosis—sCD40L inhibits the reendothelialization of the injured vessel, potentially leading to the
activation and proliferation of smooth muscle cells.
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58 Part I / Basic Principles
The pioneering studies of Henn and associates have shown not only that CD40L also
exists in platelets but that platelets contain most of the circulating protein (198–200).
CD40L is cryptic in unstimulated platelets, but platelet agonists cause this protein to
become rapidly exposed on the platelet surface, where it is subsequently cleaved, gener-
ating a soluble hydrolytic fragment termed sCD40L. Essentially all the sCD40L gen-
erated during the clotting of whole blood is derived from platelets (198,200). Although
the CD40L expressed on activated platelets is proinflammatory, sCD40L appears to be
involved in both thrombosis and inflammation. The prothrombotic activity of sCD40L
was demonstrated in mice harboring a CD40L gene deletion. These mice have a throm-
bosis defect, but infusion of recombinant sCD40L normalizes this deficiency (6), demon-
strating the prothrombotic activity of this protein. Other studies have shown that sCD40L
is also proinflammatory, capable of inducing chemokine production in peripheral blood
mononuclear cells (201,202), PGE
2
production in lung fibroblasts (203), adhesive protein
expression in endothelial cells (195), and proliferation of B-lymphocytes (204); yet other
studies have failed to observe these activities (200). Thus, sCD40L has the potential of
mediating several events within the vasculature. Indeed, elevated levels of sCD40L have
been documented in several thrombotic and inflammatory conditions including acute cor-
onary syndromes (205), peripheral arterial occlusive disease (206), and systemic lupus ery-
thematosus (207). Furthermore, elevated levels of sCD40L are associated with an increased
cardiovascular risk in women (208).
GPIIb/IIIa is linked to CD40L function in two ways. A primary role for GPIIb/IIIa
in CD40L function is demonstrated by observations showing that GPIIb/IIIa antagonists
inhibit the release of sCD40L from the surface of activated platelets (209). Thus, the
ligand binding activity of GPIIb/IIIa is involved in the release of this proinflammatory
protein. An additional role for GPIIb/IIIa is to serve as a receptor for the released sCD40L.
Direct binding measurements have shown that sCD40L binds to purified GPIIb/IIIa and
to activated platelets in a GPIIb/IIIa-dependent manner. sCD40L is involved in platelet
function, as it can serve as a substrate allowing for platelet spreading, it is also required for
platelet aggregation under conditions of high shear.
In addition to the linkage of GPIIb/IIIa to CD40L, there are additional ways that GPIIb/
IIIa can be linked to vascular inflammation. One example is by modulating the level of
several soluble inflammatory markers released from activated platelets. Platelet α-gran-
ules contain several proinflammatory proteins in addition to CD40L, e.g., TGF-β, PF4,
RANTES, and P-selectin. GPIIb/IIIa is indirectly involved in the activities of each of these
proteins, as their release following activation of platelets by weak agonists such as ADP is
promoted by the outside-in signaling through GPIIb/IIIa induced by platelet aggregation.
In another example, fibrinogen bound to GPIIb/IIIa on the surface of activated platelets
is capable of also binding to the β2-integrin Mac-1 and recruiting neutrophils into a grow-
ing thrombus (39). In yet another example, the coagulation factors generated by GPIIb/IIIa
signaling, including factor Xa and thrombin, are also proinflammatory (210).
One study has tested the hypothesis that GPIIb/IIIa antagonists are antiinflammatory.
Increased levels of inflammatory markers including C-reactive protein, IL-6, and TNF-α
normally accompany percutaneous coronary revascularization (211): elevations of these
markers were found to be prevented by performing the procedure in the presence of abcixi-
mab (212). Although the authors of this manuscript suggested that the crossreactivity of
abciximab with α
M
β
2
might explain the antiinflammatory effects of this drug, two consid-
erations argue against this conclusion. First, abciximab has low affinity for α
M
β
2
and would
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Chapter 3 / GPIIb/IIIa in Platelet Aggregation 59
have very low receptor occupancy (<5%) on this integrin during the clinical administration
of abciximab. Second, a recent study has shown that Integrilin, which does not crossreact
with α
M
β
2
, also significantly reduced C-reactive protein concentration after angioplasty
(213). Third, as outlined above, there are several mechanisms by which GPIIb/IIIa antag-
onism can be antiinflammatory. Thus, GPIIb/IIIa antagonists can be antiinflammatory
by a GPIIb/IIIa mechanism. Additional studies are required to determine whether the clin-
ical benefits of GPIIb/IIIa antagonists are derived not only from their direct inhibition
of platelet aggregation, but also from their inhibition of inflammation by their blockade
of the release of CD40L and other inflammatory mediators from platelets.
ADDITIONAL FUNCTIONS FOR GPIIb/IIIa
It is well known that the presence of platelets in blood shortens the time required for
clot formation. Several studies (214–216) have shown that GPIIb/IIIa antagonists reduced
tissue factor-induced thrombin formation, indicating that clot formation and GPIIb/IIIa
may be linked. This finding raises the interesting possibility that GPIIb/IIIa antagonists
may decrease thrombin formation at sites of vascular injury. In support of this suggestion,
ex vivo samples have prolonged clot formation time when they were obtained from patients
receiving GPIIb/IIIa antagonists (217). Although unstimulated platelets have poor clot-
promoting activity, activated, adherent platelets assemble prothrombinase and factor Xase
on their surface to greatly enhance the rate of thrombin generation (218). Since aggregation
induces platelet activation through outside-in GPIIb/IIIa signaling, one possibility is that
GPIIb/IIIa antagonists reduce thrombin formation by their ability to reduce aggregation-
induced platelet stimulation. An alternative hypothesis is that prothrombin binding to
GPIIb/IIIa on unstimulated platelets may enhance the rate of prothrombin activation (219,
220). On the other hand, it is also likely that GPIIb/IIIa antagonists prolong blood clotting
by inhibiting outside-in signaling through GPIIb/IIIa. Either explanation could account
for the long clotting times in the platelet-rich plasma from patients with Glanzmann’s thromb-
asthenia (221).
An additional way that GPIIb/IIIa may affect coagulation is by prevention of α-granule
secretion. α-Granules contain several proteins involved in blood coagulation including
CD40L P-selectin, and factor V. CD40L and P-selectin are both expressed on the surface of
activated platelets and subsequently cleaved (199). CD40 ligand has been shown to upreg-
ulate the expression of tissue factor in macrophages (222), and soluble P-selectin has been
shown to be procoagulant (223). These data suggest a third mechanism by which GPIIb/
IIIa antagonists will reduce the ability of ruptured lesions to synthesize thrombin, i.e.,
by prevention of outside-in signaling through GPIIb/IIIa and the subsequent α-granule
secretion.
Platelets provide perhaps the most abundant source of platelet-derived growth factor
(PDGF), a growth factor implicated in atherosclerosis and restenosis (224). PDGF is released
with other α-granule proteins during platelet stimulation, and it has been speculated that
sufficient PDGF is released during thrombosis to affect the vessel wall (225). Tests of this
idea using GPIIb/IIIa antagonists in the setting of angioplasty have proved negative in that
the large, randomized clinical trials have not shown that the use of these drugs prevents
the need for revascularization (226–228). The exception was the EPIC trial using abcixi-
mab, which did show a decreased need for revascularization, a result attributed to the
α
v
β
3
crossreactivity of abciximab (229). However, since subsequent trials using this drug
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