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20 Part I / Basic Principles
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Chapter 1 / Thrombosis in Acute Coronary Syndromes 21
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22 Part I / Basic Principles
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Chapter 2 / Platelet Adhesion 23
23
From: Contemporary Cardiology: Platelet Glycoprotein IIb/IIIa
Inhibitors in Cardiovascular Disease, 2nd Edition
Edited by: A. M. Lincoff © Humana Press Inc., Totowa, NJ
2 Platelet Adhesion
CONTENTS
INTRODUCTION
SUBSTRATE PROTEINS FOR PLATELET ADHESION
PLATELET ADHESION RECEPTORS
ACKNOWLEDGMENTS
REFERENCES
Edward F. Plow, PhD
INTRODUCTION
Platelets are born from megakaryocytes and are bred to adhere. As such, these anuclear
particles represent one of the most highly specialized cells within the body. This func-
tional dedication to adhesion is essential in order to prevent excessive bleeding from sites
of vascular injury. The rapidity with which platelets seal an injured vessel is a remarkable
testament to their adhesive specialization and is essential to the maintenance and/or
restoration of vascular integrity. As with many physiologic systems, an overly exuberant
cellular response can have major pathophysiologic consequences. In the case of platelet
adhesion, the out-of-control response can be devastating, thrombosis, leading to life-threat-
ening or debilitating pathologies.
Platelet attachment and spreading reactions, together referred to as platelet adhesion,
are essential for the hemostatic function of platelets. Platelet adhesion depends primarily
on the interaction of these cells with components of the subendothelial matrix that become
exposed as a consequence of injury to the endothelial cell lining of the vessel wall. Engage-
ment of these substrates is mediated by an array of adhesion receptors that are displayed
on the cell surface. Platelet-platelet interaction (platelet aggregation) is essential for forma-
tion of a platelet-rich thrombus and thereby regulates the thrombotic properties of plate-
lets. Platelet aggregation depends primarily on the engagement of the plasma proteins,
fibrinogen and von Willebrand factor (vWF), by the cell surface glycoprotein GPIIb/IIIa
(integrin α
IIb
β
3
). Hence, at a theoretical level, it appears possible to block the thrombotic
function of platelets without severely compromising their hemostatic function by block-
ade of GPIIb/IIIa. Direct support for this supposition can be derived from the clinical obser-
vation that patients with Glanzmann’s thrombasthenia, who lack functional GPIIb/IIIa,
exhibit only episodic bleeding events. This set of observations provided a rationale and
an impetus for the development of GPIIb/IIIa antagonists.
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24 Part I / Basic Principles
From one perspective, this volume can be viewed as a testimonial to the principle that
the adhesion and aggregation functions of platelets are sufficiently separable that GPIIb/
IIIa can be targeted for antithrombotic therapy. This chapter focuses on the mechanisms
underlying platelet adhesion. The major receptor systems and their ligands will be dis-
cussed briefly. GPIIb/IIIa and its role in platelet aggregation are considered in Chapter 3.
Unfortunately, the adhesive and aggregation functions of platelets are not entirely sepa-
rable, and a bleeding risk appears to be intrinsic to total blockade of GPIIb/IIIa, particu-
larly in the context of an invasive procedure. This overlap between risk and efficacy may
reflect the requirement for some platelet aggregation for effective hemostasis, or to the
role of GPIIb/IIIa not only in platelet aggregation but also in platelet adhesion. At one
level, it has been the severity of the bleeding risk relative to the antithrombotic efficacy
that has shaped the development of GPIIb/IIIa antagonism.
SUBSTRATE PROTEINS FOR PLATELET ADHESION
The Subendothelial Matrix
In addition to serving as a physical barrier, endothelial cells synthesize and elaborate
components, notably prostaglandin I
2
(PGI
2
), nitric oxide (NO), and enzyme(s) that degrade
ADP (1,2), which prevent platelet activation and adhesion. Also present on endothelial
cell surfaces are potent anticoagulant molecules, such as thrombomodulin and the tissue
factor pathway inhibitor (TFPI), which influence thrombin specificity or production and
hence the availability of this potent platelet agonist (3). Heparin sulfate and other pro-
teoglycans on the endothelial cell surface combine with antithrombin III to neutralize
thrombin and other coagulation proteases, which directly or indirectly affect the avail-
ability of thrombin. When the endothelium is disrupted, these platelet-suppressive activ-
ities are abolished, and components of the subendothelium that support platelet adhesion
and activation become exposed. As discussed shortly, a variety of subendothelial cell
constituents serve as substrates for platelet attachment and spreading. Furthermore, the
matrix is a mutable surface. It can be remodeled as a consequence of its interaction with
plasma proteins and/or by proteolysis, and these alterations influence its capacity to sup-
port platelet adhesion. Another relevant consideration is that, since the phenotypic proper-
ties of endothelial cells from different blood vessels varies, the composition of the matrix
they deposit will vary as well. Accordingly, certain proteins may play a dominant role in
supporting platelet adhesion in certain blood vessels. Moreover, even at the same anato-
mic location, the composition of the atherosclerotic plaque and the nature of the fissure or
injury to the endothelium will expose different substratum. Thus, certain plaques, even
when ruptured may have greater or lesser tendencies to support platelet adhesion and throm-
bus formation.
Some of major subendothelial matrix proteins that support platelet attachment and/or
spreading reactions are listed in Table 1. From the extent of this list, it is clear that plate-
lets can adhere to a variety of substrates once the endothelium has been disrupted. Not
all the adhesive proteins listed in Table 1 support the same spectrum of platelet responses.
For example, under some conditions, platelets attach to but do not spread on laminin (4),
whereas vWF and fibronectin appear to support both cell attachment and spreading (5–7).
Certain collagen types not only support the attachment and spreading of platelets but also
evoke platelet secretion (8). In turn, the secretory response, which can be induced by a vari-
ety of platelet agonists, directly influences the adhesive properties of platelets by altering
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Chapter 2 / Platelet Adhesion 25
their surface. Specifically, as a consequence of the secretory response, certain adhesion
receptors become expressed (e.g., P-selectin [9]), and the density and/or distribution of
others becomes altered (e.g., GPIIb/IIIa [10], GPIb/IX [11]). Of the multiple forms of
collagen, types I, III, IV, and VI are regarded as being particularly important in support-
ing platelet adhesion (12,13).
Although endothelial disruption is the primary stimulus for platelet adhesion, platelets
can interact with activated endothelial cells (14) and neutrophils, which in turn can inter-
act with matrix or endothelial adherent platelets (15,16). There is a body of literature sug-
gesting that such cell–cell interactions may play an important role in thrombus formation
(15,16). Furthermore, circulating platelet–leukocyte complexes in the blood may be
diagnostic of ongoing thrombotic events (17–19). Although not interacting directly with
platelets, erythrocytes also exert a marked effect on adhesion of platelet to the vessel wall
(20). Thus, the subendothelial matrix, and perhaps even intact endothelium, provide mul-
tiple substrates and mechanisms to support platelet adhesion, and other vascular cells can
modulate these interactions.
Nonmatrix Sources of Matrix Proteins
In addition to the subendothelial matrix, proteins that support platelet adhesion can be
derived from two other biologically relevant sources: blood, which comes in contact with
the injured vessel; and platelet α-granules, which secrete their content of adhesive pro-
teins when stimulated by agonists. The sources of key platelet adhesive proteins are listed
in Table 2. Some of the platelet adhesive proteins (e.g., vWF and fibronectin) reside in
Table 1
Major Subendothelial Proteins That Support Platelet Adhesion
Matrix constituent Comment
Collagens Large family of proteins that can support platelet adhesion aggregation
and secretion
von Willebrand factor Large multimeric protein critical for the hemostatic function of
platelets
Fibronectins Dimeric or multimeric proteins that support attachment and spreading
of platelets
Thrombospondins Trimeric or higher multimeric proteins exhibiting both adhesive and
antiadhesive properties for platelets
Laminins Proteins supporting platelet adhesion
Table 2
Origins of Platelet Adhesive Proteins
Adhesive protein Matrix Plasma Platelets
von Willebrand factor + + +
Fibronectin + + +
Thrombospondin-1 + − +
Vitronectin * + +
Fibrinogen * + +
Laminin + −−
Collagen + −−
*Constituent of provisional matrices.
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26 Part I / Basic Principles
all three locations. Fibrinogen and vitronectin are present in platelets and plasma but are
not synthesized by endothelial cells. However, they are components of the provisional
matrix formed at sites of vascular injury and are, therefore, particularly relevant sub-
strates for platelet adhesion. It should be noted that the molecular forms of the adhesive
proteins derived from these different sources are not identical. For example, the degree
of vWF multimerization differs for the plasma and platelet forms (21), and the splicing
variants of fibronectin differ depending on their sites of synthesis (22). As still yet another
variable, a number of these adhesive proteins interact with one another. For example, vWF,
fibronectin, and thrombospondin-1 (TSP-1) all bind to collagen (23–25). These inter-
actions may indirectly bridge platelets to a matrix protein or may modulate the adhesive
properties of the matrix. Although TSP-1 can be synthesized by many cell types in vitro,
the platelet appears to be its primary source in vivo. Members of the thrombospondin
gene family, including TSP-1, have been identified as risk factors for premature athero-
sclerosis (26).
Shear and Platelet Adhesion
Shear rate developed by flowing blood varies with vessel caliber. Local turbulence
may develop as blood flows across the irregularities of an atherosclerotic plaque or a
developing thrombus (27). Variations in the fluid dynamics of blood greatly influence
platelet adhesion (28–30). Over the past two decades, more and more studies of platelet
adhesion have been conducted under flow conditions to help ensure the biologic rele-
vance of the analyses. Shear is particularly important in defining the contribution of vWF
to platelet adhesion. In in vitro experiments, a role of vWF in platelet adhesion is readily
demonstrable at high but not at low shear or under static conditions (6,31). By contrast,
a role for fibronectin in supporting platelet adhesion can be demonstrated both high and
low shear (32). Nevertheless, patients with von Willebrand disease have a major bleeding
syndrome, which indicates the importance of vWF and shear in supporting platelet func-
tion. The development of vWF-deficient mice has attested to the importance of vWF in
supporting platelet adhesion and thrombus formation (33), although evidence for still other
mechanisms of platelet thrombus formation exists in mice that lack not only vWF but also
fibrinogen (34). Shear may exert its influence on platelet/vWF adhesion by affecting the
conformation of the adhesive protein or its platelet receptor, GPIb, or both (30,35,36).
Also coming into play in the complex interrelationship between shear and vWF is the multi-
meric structure of vWF; the higher multimers of vWF are hemostatically more active (37).
PLATELET ADHESION RECEPTORS
The individual adhesive proteins discussed above interact with platelets by serving as
ligands for specific cell-surface receptors. The major platelet adhesion receptors are listed
in Table 3. Several nomenclature systems have been used to identify the same membrane
proteins of the platelet. The original nomenclature was based on electrophoretic mobil-
ity, giving rise to GPI, -II, -III, and so on, with GPI having the highest molecular weight.
As gel separation and protein detection systems became more sophisticated, several pro-
teins were discerned with similar electrophoretic mobilities (e.g., GPI became GPIa,
GPIb, and GPIc). Several of the membrane proteins exist on the platelet surface as non-
covalent complexes; thus, GPIb/IX, GPIc/IIa, and GPIIb/IIIa should be viewed as single-
membrane proteins with multiple subunits. Despite these ambiguities, the nomenclature
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Chapter 2 / Platelet Adhesion 27
based on electrophoretic properties remains the most widely used. Other nomenclature
systems have arisen from the fact that several platelet membrane proteins are present and
have been assigned different names on other cell types. This is the basis for the very late
antigen (VLA), which is now rarely used, and leukocyte differentiation (CD) designations,
which receive sporadic usage in the platelet literature. Some receptors are also identified
on the basis of their function (e.g., the vitronectin and the fibronectin receptors). Such
functional designations have major limitations since multiple membrane proteins can
function as receptors for the same ligand (e.g., vitronectin [α
IIb
β
3
and α
V
β
3
], fibronectin
[α
IIb
β
3
and α
5
β
1
], and several platelet membrane proteins can function as collagen recep-
tors [see the Platelet Collagen Receptors section]). Beyond creating a nomenclature com-
plexity (nightmare), the functional redundancy of the platelet receptors enables the cell
to establish multiple contacts with a single matrix constituent. In turn, a single ligand may
initiate several distinct functional responses by engaging multiple receptors.
Platelet Integrins
Many of the adhesive protein receptors on platelets, including GPIIb/IIIa, are integrins.
The integrins are a broadly distributed family of heterodimeric cell surface molecules
that share certain structural, immunochemical, and functional properties (38–44). A pro-
totypic integrin structure is illustrated in Fig. 1A. The β-subunits, of which eight are known
currently, are highly homologous to one another, exhibiting extensive (35–45%) identity
at the primary amino acid sequence level. The α-subunits, of which 15 are presently known,
are also similar to one another but exhibit less extensive sequence identity. The α-sub-
units are synthesized as single-chain polypeptides, but some, such as GPIIb (α
IIb
), are pro-
teolytically processed to a two-chain, disulfide-linked form (45–48). Each α-subunit has
a short cytoplasmic tail, a single transmembrane segment, and a large extracellular por-
tion, which contains several cation binding sites. Each β-subunit is the product of a sepa-
rate gene and combines in a noncovalent complex with an α-subunit to form an adhesive
protein receptor. Each β-subunit also spans the membrane once and typically has a short
cytoplasmic tail.
Table 3
Major Platelet Adhesion Receptors
Receptor Common alternative designations Major adhesive ligands
GPIa/IIa α
2
β
1
, VLA-2 Collagen
GPIb/IX/V vWF
GPIc/IIa α
5
β
1
, VLA-5, fibronectin receptor Fibronectin
GPIIb/IIIa α
IIb
β
3
Fibrinogen, vWF,
fibronectin, vitronectin,
thrombospondin
GPIV GPIIIb, CD36 Collagen, thrombospondin
GPVI Collagen
α
6
β
1
Laminin
α
V
β
3
Vitronectin receptor Vitronectin, fibrinogen,
fibronectin, vWf
thrombospondin, osteopontin
Abbreviations: GP, glycoprotein; vWF, von Willebrand factor; VLA, very late antigen.
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28 Part I / Basic Principles
The extracellular domain is formed as a complex between the α- and β-subunits, and
high-affinity binding of adhesive proteins depends on contributions from both subunits.
The crystal structure of the extracellular domain of integrin α
V
β
3
has recently been reported
(49). Each subunit is composed of a series of structural motifs, which are strung together
into stalks that can extend at some distance from the cell membrane. A single β-subunit
can combine with several α-subunits, with each complex having distinct functional prop-
erties. The cytoplasmic tails of the subunits also appear to interact with each other (50,51)
and link to the cytoskeleton and to signaling pathways. These intracellular interactions
determine the roles of integrins in cell adhesion, cell spreading, and signal transduction.
Key to the regulation of the ligand binding function of many integrins is their activation
state. Stimulation of cells by a variety of agonists can generate intracellular signals that
are transmitted from within the cell to the extracellular domain of integrins, enhancing
their affinity for ligands (inside-out signaling). Similarly, occupancy of integrins can gen-
erate signals that are transmitted across the membrane and initiate intracellular responses
(outside-in signaling). Such bidirectional signaling, as it relates to GPIIb/IIIa function,
is discussed separately in Chapter 3 and reviewed elsewhere (43,52). Other integrins
that are present on platelets can undergo activation on other cell types and on platelets
(53–56), but the importance of such activation to the hemostatic function of platelets is
uncertain.
Fig. 1. (A) Schematic depiction of the structure of a prototypic integrin adhesion receptor.
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Chapter 2 / Platelet Adhesion 29
Platelets express two major β-subunits, β
1
and β
3
(β
2
has been reported to be present
at low levels) and five α-subunits (Table 4). Of the integrins expressed on blood cells,
GPIIb/IIIa (α
IIb
β
3
) is the most narrowly distributed and is restricted predominantly to
platelets/megakaryocytes. GPIIb/IIIa may contribute to the association of platelets with
Fig. 1. (B) Schematic depiction of the GPIb/IX complex on the platelet surface. (Adapted from ref. 30)
Table 4
Platelet Integrins
Integrin Ligands
α
2
β
1
Collagen
α
5
β
1
Fibronectin
α
IIb
β
3
Fibrinogen, fibronectin, vitronectin, von Willebrand factor,
thrombospondin, factor XIII, prothrombin
α
V
β
3
Vitronectin, fibrinogen, von Willebrand factor, thrombospondin,
fibronectin, prothrombin, osteopontin
α
6
β
1
Laminin
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