COLMAN

Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice
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Editors:
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Colman, Robert W.; Clowes, Alexander W.;
Goldhaber, Samuel Z.; Marder, Victor J.; George, James N.

Title:
Hemostasis and Thrombosis: Basic Principles and
Clinical Practice, 5th Edition

Copyright 2006 Lippincott Williams & Wilkins
> Tabl e of C ontents > P art I - B asi c Pri nci pl es of Hem ostasi s and
Throm bosi s > Secti on B - Fi bri nol ysi s and i ts Regul ati on > C hapter 19 Pl as m i nogen Acti vator I nhi bi tor-1
Chapter 19
Plasminogen Activator Inhibitor-1
Manuel Yepes
David J. Loskutoff
Daniel A. Lawrence
Plasminogen activators (PAs) are specific serine proteinases that
activate the proenzyme plasminogen, by cleavage of a single
Arg-Val peptide bond, to the broad-specificity enzyme plasmin.
Plasminogen activation provides an important source of localized
proteolytic activity during a number of physiologic and pathologic
processes such as fibrinolysis, ovulation, cell migration, epithelial
differentiation, vascular disease, and cancer (1,2,3). Therefore,
accurate regulation of PAs constitutes a critical feature of many
physiologic and pathologic events. Two PAs are found in mammals:
tissue-type PA (tPA) and urokinase-type PA (uPA) (4). The
regulation of PAs is a complex process that involves regulation of
gene expression by factors such as hormones, growth factors, and
cytokines (1,4), as well as regulation of enzyme activity through
interactions with fibrin (5) or with specific receptors for uPA (6),
tPA (7), and plasminogen (8). PA activity is also regulated by
specific inhibitors termed plasminogen activator inhibitors (PAIs)
(9). However, of the five different PAIs [i.e., PAI-1 (10); PAI-2
(11); PAI-3, also called the activated protein C inhibitor (APCI)
(12); protease nexin-1 (13); and neuroserpin (14)], only PAI-1,
which is the most kinetically efficient, appears to play a significant
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role in regulating PA activity in blood and most tissues. The

exception to this may be in the central nervous system (CNS),
where neuroserpin is an important regulator of tPA activity
(15,16). This chapter focuses on the biochemical, genetic,
physiologic, and pathologic properties of PAI-1. The basic
properties of PAI-1 are analyzed in the first part, whereas the
second half examines the evidence linking PAI-1 to the
pathogenesis of human disease.
BASIC PROPERTIES OF PLASMINOGEN

ACTIVATOR INHIBITOR-1
Protein Structure and Function
Formerly known as the endothelial cell PAI (10,17,18,19), the
fast-acting inhibitor of tPA in plasma (20), and the PAI
(21,22,23), PAI-1 is a single-chain glycoprotein having an M r of
approximately 50,000 and was first identified in the year 1983
(10,24,25). PAI-1 has three potential N-linked glycosylation sites
and contains between 15% and 20% carbohydrate (19,23). It
belongs to the serine proteinase inhibitors superfamily (serpins),
which is a gene family that includes many of the proteinase
inhibitors found in blood, as well as other proteins with unrelated
or unknown functions (26) (see Chapter 13). The serpins share a
tertiary structure (27) and have evolved from a common ancestor
(28,29). They act as suicide inhibitors [i.e., they react only once
with their target protease to form sodium dodecyl sulfate
(SDS)stable complexes (30,31,32,33)]. The interaction between
the serpin and its target protease occurs at an amino acid residue
located in the reactive center loop (RCL) of the serpin, which is
known as a bait residue. This bait residue is thought to mimic
the normal substrate of the enzyme and to associate by its
side-chain atoms with the specificity crevice (S1 site) of the
enzyme (34,35,36,37). The bait amino acid is called the P1
residue, with the amino acids toward the amino-terminal side of
the scissile RCL bond being labeled in the order P1, P2, P3, and so
on, and the amino acids on the carboxyl side being labeled P1,
P2, P3, and so on (38). Upon cleavage of the bait amino acid by
a target proteinase, there is a large conformational change in the
serpin that involves the rapid insertion of the RCL into the major
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structural element of the serpin, the -sheet A (33,39,40,41). This

results in a tight docking of the enzyme to the serpin surface and
in a large increase in the structural stability of the serpin. This
leads to distortion of the enzyme structure, including the active
site, which traps the proteinase in an acylenzyme complex with
the serpin (42,43,44).
The PAI-1 complementary deoxyribonucleic acid (cDNA) encodes a
protein of 402 amino acids that includes a typical secretion signal
sequence (23,45,46,47). Mature human PAI-1 is composed of two
variants in approximately equal proportions (i.e., 381 and 379
amino acids), which likely arise from alternative cleavage of the
secretion signal sequence and generate proteins with overlapping
amino-terminal sequences of Ser-Ala-Val-His-His and
Val-His-His-Pro-Pro (45,48). The latter sequence does not contain
cysteines because the single cysteine residue present in the signal
peptide is removed during membrane translocation (46,47). This
property facilitates the efficient expression and isolation of
recombinant PAI-1 from Escherichia coli (48,49,50,51,52), which,
in contrast to PAI-1 purified from mammalian cell culture, is
predominantly produced in the active form (48).
Reactive Center Loop

The P1 bait amino acid of PAI-1 (Arg346) is contained within the
RCL near the carboxyl terminus of the molecule and serves as a
pseudosubstrate for the target serine proteinase (33,37). Either
arginine or lysine at P1 is essential for PAI-1 to function as an
effective inhibitor of uPA (36,37), and the residues surrounding P1
can modulate PAI-1 inhibitory activity by up to two orders of
magnitude and can also alter the targetprotease specificity
(42,53,54). In the case of tPA other P1 residues are tolerated, and
this is most likely due to tighter exosite interactions between
PAI-1 and tPA (53,55,56,57).
Plasminogen Activator Inhibitor-1

Conformations
Native PAI-1 exists in at least two distinct conformations: an
active form that is produced by cells and is secreted and an
inactive
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or latent form that accumulates in cell culture medium over time
(58,59,60) (see Fig. 19-1). In blood and tissues, most of the PAI-1
is in the active form; however, in platelets both the active and the
latent PAI-1 are found (21). In the active form of PAI-1, the RCL is
part of an exposed loop on the surface of the molecule (61). Upon
reaction with a proteinase, this RCL cleaves and integrates into the
center of its own -sheet A (33,61). In the latent form, the RCL is
intact, but instead of being exposed, the entire amino-terminal
side of the RCL is inserted as the central strand into the -sheet A
(60). This insertion accounts for the increased stability of latent
PAI as well as for its lack of inhibitory activity (58,62,63). The
active form spontaneously converts to the latent form with a
half-life of about 1 to 2 hours at 37C at neutral or slightly
alkaline pH (48,59,64,65,66). The latent form can also be
converted into the active form by treatment with denaturants or
negatively charged phospholipids, or can be converted very slowly
in the presence of the protein vitronectin (58,66,67). This
spontaneous reversible interconversion between the active and
latent structures is unique for PAI-1 and distinguishes it from other
serpins.
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FIGURE 19-1. Schematic illustration of the three

conformations of plasminogen activator inhibitor-1 (PAI-1). The
major -sheet (-sheet A) is highlighted in light gray, and the
reactive center loop (RCL) is in dark gray. A: The active
conformation of a stable mutant of PAI-1 (61). B: The latent
conformation of PAI-1 (60). C: The cleaved conformation of
PAI-1 (287). In both the latent and cleaved forms of PAI-1, the
RCL is inserted into -sheet A to form a new -sheet (strand
4A) (see Color Fig. 19-1). (From Sharp AM, Stein PE, Pannu
NS, et al. The active conformation of plasminogen activator
inhibitor 1, a target for drugs to control fibrinolysis and cell
adhesion. Structure Fold Des 1999;7:111118, with
permission.)
Other inactive forms of PAI-1 have also been identified. The first
form results from the oxidation of one or more critical methionine
residues within active PAI-1 (68,69). This form differs from latent
PAI-1 in that it can be partially reactivated by treatment with an
enzyme that specifically reduces oxidized methionine residues
(68). Oxidative inactivation of PAI-1 may be an additional
mechanism for the regulation of the PA system. Oxygen radicals
produced locally by neutrophils or other cells could inactivate
PAI-1 and thereby facilitate the generation of plasmin activity at
sites of infections or in areas of tissue remodeling (70). A fourth
conformational form of PAI-1 has also been identified. This is a
noninhibitory substrate form that can be induced by the addition of
SDS and can be converted back to the active or latent forms by
treatment with 4 M guanidine HCl (71,72). The significance of this
form and whether it exists in vivo is not known. PAI-1 can also
exist in two different cleaved forms. As noted previously, PAI-1
that is complexed with a proteinase is cleaved at the site, and
PAI-1 can also be found not in complex with a proteinase but with
its RCL cleaved. This can arise from dissociation of the PAI-1PA
complex or from the cleavage of the RCL by a nontarget proteinase
at a site other than the (63,73). None of these forms of PAI-1 is
able to inhibit proteinase activity; however, they may interact with
other nonproteinase substrates.
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Biochemical Properties
Interaction with Tissue-Type Plasminogen
Activator, Urokinase-Type Plasminogen
Activator, and Plasmin
Inhibition of PAs by PAI-1 occurs in a rapid and stoichiometric
manner, resulting in the formation of a covalent bond between the
two molecules. Several studies indicate that PAI-1 is cleaved
during this reaction and that the amino-terminal end of the RCL
inserts as an antiparallel strand into -sheet A (74,75). The
inhibitor is consumed in the process, giving rise to the discussed
term suicide inhibitor.
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Kinetic studies demonstrate that PAI-1 inhibits the naturally
occurring single-chain form of tPA with a second-order rate
constant of approximately 10 6 M - 1 S -1 , a value that is at least
1,000 times higher than those for the interactions of PAI-2, PAI-3,
and protease nexin-1 with single-chain tPA (9,42). Moreover,
approximately 70% of the total tPA in carefully collected normal
human plasma is detected in complex with PAI-1, suggesting that
the inhibition of tPA by PAI-1 is a normal, ongoing process. PAI-1
is also an important uPA inhibitor because the second-order rate
constant for its interaction with uPA is also at least two orders of
magnitude higher than that of other PAIs (9,42,76). PAI-1 can also
efficiently and directly inhibit plasmin (64,77). Therefore, PAI-1 is
the chief regulator of plasmin generation in vivo, and, as such, it
appears to play an important role in both fibrinolytic and
thrombotic diseases (3,78,79,80).
Interaction with Vitronectin and Members of

the Low Density Lipoprotein Receptor Family
(LDL-Rs)
Most of the active PAI-1 in blood circulates in the form of a
complex with the glycoprotein vitronectin. The binding site of
vitronectin to PAI-1 has been identified in a region centered
around residues 101 to 123 of the three-dimensional structure
(81,82,83,84), whereas the binding site to members of the LDL-R
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family has been less well characterized and appears to be located

in a region associated with helix D containing residues Arg76 and
Lys69 (85,86). Vitronectin is present in plasma and in the
extracellular matrix (87,88), mainly at sites of injury or
remodeling. Vitronectin is also specifically incorporated into fibrin
clots (89). Vitronectin may be considered a cofactor for PAI-1
because it stabilizes PAI-1 in its active conformation, thereby
increasing its biologic half-life. In turn, PAI-1 converts vitronectin
from its native, plasma form, which does not support cell adhesion,
to an activated form that is able to bind ligands such as integrins
(90). Vitronectin also increases the inhibitory efficiency of PAI-1
for thrombin approximately 300-fold, making it a more efficient
inhibitor of thrombin than antithrombin III in the absence of
heparin (91,92) (see Fig. 19-2).
Upon complex formation with a proteinase, the conformational
change in PAI-1 associated with RCL insertion results in a loss in
high affinity binding to vitronectin but in a gain in high affinity
binding to the clearance receptors of the (LDL-R) family (85,93).
This is a result of a conformational change in PAI-1 that disrupts
the vitronectin binding site, exposing, at the same time, a cryptic
receptor binding site that is revealed only when PAI-1 is in an
active conformation complex with a proteinase (54,61,85). This
results in an approximately 1,000,000-fold shift in the relative
affinity of PAI-1 from vitronectin to a member of the LDL-R family,
which leads to a rapid clearance of PAI-1 by internalization through
the LDL-R family members (85).
The uPA receptor (uPAR) (6) is a glycosyl phosphatidyl inositol
(GPI)anchored protein that localizes uPA on the cell surface,
frequently at the invading edge of cells (94). Receptor occupancy
has been shown to activate intracellular signaling pathways. Like
PAI-1, uPAR also binds to vitronectin with high affinity as well as
to various integrins (95,96,97,98), and the binding of uPAR to
vitronectin is inhibited by PAI-1. The fact that uPAR and integrins
also may bind to vitronectin raises the possibility that, as
discussed later in this chapter, PAI-1 could regulate cell adhesion
and migration.
Binding to Heparin and Fibrin
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PAI-1 also binds to heparin with high affinity (48,99). This binding
does not affect the interaction of PAI-1 with uPA or tPA but, in
contrast, enhances the interaction of PAI-1 with thrombin
(91,100). The heparin binding domain on PAI-1 has been mapped
to a region homologous to the heparin binding domain of
antithrombin III on and around helix D (101). Critical residues
appear to include lysines 65, 69, 80, and 88, and arginine 76. It
has also been reported that PAI-1 binds to fibrin in vitro with a K d
of 3.8 M, and, while bound, remains capable of inhibiting uPA and
tPA (102,103,104,105). However, more recent data suggest that
most of the PAI-1 localized to fibrin clots is vitronectin dependent
(106,107).
Structure and Regulation of the

Plasminogen Activator Inhibitor-1 Gene
The human gene for PAI-1 is located on chromosome 7q21.3-22
(108). It is 12.3 kb in length, composed of 9 exons and 8 introns,
and is similar in structure to that of protease nexin I and
neuroserpin (109,110,111,112,113,114,115). The promoter
contains a typical TATA box but no CAAT sequence. Segments of
PAI-1 promoter containing as little as 187 bp of upstream
sequence have shown to direct transcription in several mammalian
cell types (111,116,117). Comparison of the rat and human PAI-1
promoter sequences shows a striking region of conservation in
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the proximal promoter (from the TATA box to -90) and a distal
sequence of -510 to -753 of the rat sequence. Changes in plasma
PAI-1 levels can be correlated with variations in the structure of
the PAI-1 gene. To date, three polymorphic variations in the gene
have been reported: a single nucleotide insertion per deletion
(4G/5G) polymorphism in the promoter region, an allelic variation
at a (C-A) n dinucleotide repeat polymorphism in intron 3, and a
HindIII restriction fragment length polymorphism (RFLP) due to a
base change at the 3 flanking region of the gene (118). However,
no link between PAI-1 polymorphism and human disease has been
consistently demonstrated. Relative to the HindIII RFLP
polymorphism, three genotypes1/1, 1/2, and 2/2have been
described, on the basis of the presence (2 allele) or the absence
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(1 allele) of the HindIII polymorphic site. Interestingly, genotype

1/1 exhibits higher plasma PAI-1 activity than genotype 2/2 (119).
Two sizes of PAI-1 messenger ribonucleic acid (mRNA) are
observed in human cells, approximately 3 kb and 2 kb in length,
respectively. This difference has been shown to be because of
alternative polyadenylation, with both mRNAs encoding the same
protein, but with an additional 1 kb of 3 untranslated region
present in the larger message (109,110,111,112). The additional
3 untranslated region in the larger message contains a 75-base
pair AT-rich sequence that has been postulated to play a role in
PAI-1 gene regulation by a posttranscriptional mechanism
(47,120,121,122,123).
FIGURE 19-2. Plasminogen activator inhibitor-1 (PAI-1)

association with vitronectin and low density lipoprotein
receptors (LDL-Rs) is conformationally controlled. Surface
plasmin resonance analysis of PAI-1 or PAI-1uPA complexes
binding to either vitronectin or the LDL-R family member LRP.
The solid lines in each panel are PAI-1 only and the dashed
lines in each panel are the PAI-1uPA covalent complex. (From
Natalia Gorlatova and Daniel A. Lawrence.)
Although PAI-1 is present at low concentrations in plasma, its

relatively short half-life in blood (i.e., 10 minutes) suggests a high
biosynthetic rate (124). Moreover, its concentration rapidly
increases in response to a variety of agents or changes in
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physiologic state, indicating that the amount of PAI-1 in plasma is

subject to dynamic regulation. For example, the concentration of
plasma PAI-1 increases dramatically during endotoxemia (125).
Endotoxin [lipopolysaccharide (LPS)] induces PAI-1 mRNA in
virtually all tissues of the mouse (126), suggesting that plasma
PAI-1 may originate from multiple tissues during sepsis. In situ
hybridization analysis reveals that endotoxin induces PAI-1 mRNA
in endothelial cells at all levels of the vasculature, including larger
arteries, veins, and capillaries. PAI-1 gene expression is also
induced in hepatocytes and in adipocytes (127,128).
Many of the effects of endotoxin are mediated through the release
of cytokines from inflammatory cells [e.g., tumor necrosis factor-
(TNF-) and interleukin-1 (IL-1)]. Indeed, a variety of studies
have shown that most of the same tissues of the mouse that
produce PAI-1 in response to endotoxin (i.e., liver, heart, and
lung) also produce it in response to TNF- (126) Therefore,
endotoxin and TNF- upregulate PAI-1 expression primarily in
endothelial cells in most mouse tissues and induces it in vitro in
various bovine and human endothelial cells. Growth modulators
such as transforming growth factor- (TGF-) also induce plasma
PAI-1 antigen and tissue PAI-1 mRNA in several animal models
(126). In murine models, TGF- induces PAI-1 mRNA in vascular
and nonvascular smooth muscle cells, in adipocytes, and in cells in
the myocardium and kidney (126). Although TGF- induces PAI-1
in cultured bovine endothelial cells (129), it does not appear to
induce it in murine endothelium in vivo. Whether this apparent
inconsistency reflects differences between in vitro and in vivo
systems or between species remains to be determined.
Several studies have identified DNA sequence elements in the first
1.3 kb of the promoter/5-upstream flanking region of the PAI-1
gene that mediate cytokine responsiveness in transfected cells
(130). However, little is known about the role of these sequences
in PAI-1 promoter function in vivo. PAI-1 mRNA levels are
determined by both transcriptional and posttranscriptional
mechanisms. Factors that have been shown to increase the rate of
PAI-1 mRNA transcription in different cell types are
glucocorticoids, TNF- insulin, and IL-1 (130). In HepG2 cells,
IGF-1 (insulinlike growth factor-1) and insulin are also able to
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induce PAI-1 synthesis by increasing mRNA stability (131).

Moreover, it has been suggested that PAI-1 gene regulation is of
even greater complexity because it also could depend on genetic
polymorphisms.
Expression of PAI-1 has been observed in a wide range of cell
types including adipocytes, fibrosarcoma, hepatoma, and ovarian
and endothelial cells. However, studies in transgenic mice carrying
the PAI-1 promoter linked to an indicator gene suggest that PAI-1
expression may be much more limited in vivo (126). Although the
hepatoma cells produce large amounts of PAI-1 in vitro and have
been used as a model to study its regulation, it is known that
hepatocytes do not normally synthesize PAI-1 in vivo (132,133).
Nevertheless, these hepatocytes and the endothelial cells can be
induced by endotoxin to produce PAI-1 in vivo (133). These
observations raise important concerns about the validity of in vitro
tissue culture as a model for the regulation of fibrinolysis in vivo.
PLASMINOGEN ACTIVATOR INHIBITOR-1

IN PATHOLOGIC CONDITIONS
In healthy individuals, PAI-1 is expressed primarily in
megakaryocytes, smooth muscle cells, and adipocytes (128,
130,134,135). However, as noted in the preceding text, its
expression can be rapidly and markedly induced in many cell types
by stress, or injury, or by growth factors and cytokines
(133,136,137). The last part of this chapter analyzes the link
between PAI-1 and different pathologic events such as cancer,
obesity, atherosclerosis, and vascular, pulmonary, and renal
diseases (see Fig. 19-3).
Cancer
The existence of a link between fibrinolysis and tumor growth was
first suggested by the observations that patients with malignant
tumors have increased fibrinolytic activity and that tumor tissues
can degrade fibrin clots (1,138,139,140). Therefore, it was quite
unexpected when high PAI-1 levels were found to be strongly
correlated with a poor prognosis in patients with many different
neoplasias, including gastric and breast carcinomas, as well as with
brain, ovarian, and lung tumors and with metastatic lesions from
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renal cell carcinoma, melanoma,

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and colorectal cancer (141,142,143,144,145,146). Analysis of
human colon carcinomas has demonstrated the presence of PAI-1
mRNA in vascular endothelial cells, in the stroma surrounding the
invasive tumor, in granulation tissue, and in some capillaries
within the tumor (147). Strong staining for PAI-1 antigen has also
been observed in proliferative vessels in intracranial tumors such
as high-grade gliomas and metastatic tumors, as well as in blood
vessels near the necrotic center of tumors (147,148). In these
studies, PAI-1 was localized to the vascular basement membrane
and perivascular connective tissue, whereas endothelial cells
themselves showed only weak reactivity. In contrast, an
immunochemical study on Lewis lung carcinoma transplanted in
mice showed PAI-1 protein in the tumor cells themselves (149).
Like PAI-1, uPA and its receptor, uPAR, have also been correlated
with a poor prognosis in cancer (150,151). In this section of the
chapter, the role of PAI-1 in cancer has been addressed from two
perspectives: cell migration and tumor angiogenesis.
FIGURE 19-3. Plasminogen activator inhibitor-1 (PAI-1)
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association with normal and pathologic physiology.
Cell Migration
Although uPA has been associated with physiologic events that
involve cell migration such as wound healing, vascular remodeling
(152,153,154), neural cell migration (155), and monocyte
invasiveness (156), its precise role in these processes is not clear.
Increased uPA levels have been reported in many transformed cells
including myeloid leukemic cells (157), hepatomas (158), gliomas
(159), and carcinomas (160), and inhibitors of uPA have been
shown to reduce cell migration and metastasis (161,162,163,164).
The long-established explanation of these data was that the main
role of uPA was to activate plasmin, which directly, and indirectly
through the activation of matrix metalloproteases (MMPs),
degrades the basement membrane and clears a path for cells to
migrate (165,166) (see Fig. 19-4A). However, by the 1990s, this
matrix barrier model of proteases and cell migration started to
be challenged. For example, it was shown that uPA and plasmin as
well as other proteases could produce a limited proteolysis that
preserved the architecture of the basement membrane but exposed
cryptic sites within the matrix that could enhance cell adhesion and
or migration (167,168,169,170,171). Plasmin could also promote
cell proliferation and migration through the activation of growth
factors such as TGF- (172,173) or through the release of
sequestered growth factors from the matrix such as fibroblast
growth factor (FGF) and vascular endothelial growth factor (VEGF)
(174,175,176,177). More recent studies have expanded on these
observations and have begun to suggest that the role of uPA and,
in particular, PAI-1 in cell migration may be far more subtle and
elegant than previously thought and may involve interactions on
the cell surface with a number of receptors including uPAR,
integrins, and members of the LDL-R family.
Cell surface uPA and uPAR were originally shown to localize at the
focal contacts (178,179) and at the leading edge of migrating cells
(180). As already noted, uPAR not only serves as a receptor for
uPA but also binds to vitronectin in the matrix, and, through this
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binding, can mediate cell adhesion directly (181). uPAR can also
associate either directly or indirectly with integrins, and this
association can affect both cell signaling and migration
(95,96,97,98,182,183). In vitro studies have demonstrated that
both PAI-1 and uPAR bind to the somatomedin B-domain of
vitronectin, although uPAR binds with significantly lower affinity
(184,185). Therefore, PAI-1 competes with uPAR for binding to
vitronectin and may inhibit uPAR-mediated cell attachment
(185,186,187). PAI-1, uPA, and uPAR can also modulate
integrin-mediated cell adhesion (167,168,188). For example, the
integrin v 3 binding site on vitronectin overlaps the PAI-1 binding
site, resulting in a potential competition between v 3 and PAI-1
for vitronectin binding (167,184). Therefore, in smooth muscle
cells where vitronectin promotes cell migration, the inhibition of
v 3 binding to vitronectin by the addition of PAI-1 results in an
inhibition of cell migration (167). Exogenous PAI-1 also inhibits the
migration of stimulated endothelial, human amnion WISH and
epidermal carcinoma cells, as well as the invasion of human
monocytes (167,168,189,190). However, the effects of PAI-1 on
cell adhesion and migration are fully reversible by PAs because, as
discussed at the beginning of this chapter, upon complex formation
with a protease, PAI-1 undergoes a conformational change that
alters the PAI-1 vitronectin binding site and renders the PAPAI-1
complex unable to bind to vitronectin. Therefore, the relative
concentrations of uPA (or tPA) and active PAI-1 at the cell matrix
interface are able to regulate cell-matrix interactions mediated
through vitronectin.
In addition to blocking cell-matrix interactions and cell migration,
PAI-1 can also promote cell migration under some conditions. In
the studies discussed in the preceding text, high concentrations of
exogenous PAI-1 were used to block migration. However, recent
work by Palmieri et al. has established that the stable expression
of PAI-1 can actually stimulate adhesion and migration on several
different matrix proteins (191). And although this activity required
the inhibition of uPA, it did not specifically require PAI-1, because
another uPA inhibitor, PAI-3, could also stimulate this activity. The
inhibition of uPA by either PAI-1 or PAI-3 also increased the
surface expression of several integrin subunits. Czekay et al.
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demonstrated that the binding of uPA to uPAR promotes the

association of the uPAuPAR complex with the integrins v 3 or
v 3 and showed that by adding PAI-1 to this complex, they could
induce the cells to detach from the matrix by stimulating
endocytosis of the integrinuPARuPAPAI-1 complex by a member
of the LDL-R family (188). The endocytosed integrins could then
recycle back to the cell surface, where, upon activation, they could
reengage the matrix. These studies have lead to the hypothesis
that the inhibition of uPA by PAI-1 could promote the type of
cycled attachmentdetachmentreattachment required for cell
migration (166,188). Importantly, this activity appears to be
independent of the matrix composition, suggesting that it may
represent a general mechanism in which inhibition of uPA promotes
cell migration (Fig. 19-4B). This model is consistent with much of
the literature examining the roles of uPA and PAI-1 in cell
migration. It provides a compelling explanation as to why both uPA
and its inhibitor PAI-1 are strongly correlated with a poor
prognosis in cancer patients because both molecules are needed
for the maximal enhancement of cell migration. In this context, it
is also interesting to note that PAI-1 differs from other serpins in
that it is a trace protein in plasma and tissues, with a relatively
short half-life (approximately 10 minutes). Moreover, it is an
immediate early gene and has been shown to accumulate at focal
points of adhesion (192). Finally, its biosynthesis is stimulated
rapidly by a variety of inflammatory mediators, growth factors, and
hormones (130,136). The short half-life and ability to be rapidly
and dramatically unregulated are the expected properties of
molecules with the potential to rapidly initiate or terminate biologic
processes (i.e., molecular switches) (80,185). Therefore, by
regulating the production rate or activity of PAI-1, cells may be
able to control their adhesiveness and movement.
Finally, PAI-1 can also regulate cell motility by effecting
uPA-dependent cell signaling by phosphorylation of the
extracellular signal-regulated kinase (ERK). This signaling requires
uPA binding to uPAR and requires endocytosis of the
PAI-1uPAuPAR complex by the VLDLR and recycling of uPAR back
to the cell surface (193). The effect of PAI-1 inhibition of
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uPA and endocytosis of the complex is unclear: It was shown to

inhibit uPA-induced chemotaxis in one study (194), whereas in
another report, PAI-1uPA complexes increased cell migration and
proliferation in a process that required association with the LDL-R
family and was dependent upon uPAR recycling (193).
FIGURE 19-4. The evolution of our understanding of the role

of plasminogen activator inhibitor-1 (PAI-1) in cell migration.
A: Before the multiple actions of urokinase PA (uPA),
urokinase-type plasminogen activator receptor (uPAR), and
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PAIs were fully understood, the simplest model for the function
of uPA in cell migration was based solely on its ability to
activate plasmin. Plasmin in turn degraded extracellular matrix
(ECM) proteins, which allowed the cell to escape its matrix
barrier and migrate. B: A model of the potential role of PAI-1
in cell migration that assumes that the cell surface receptors
uPAR, integrins, and low density lipoprotein receptor (LDL-R)
family, such as the very low density lipoprotein receptor
(VLDLR), are expressed by the cell. The integrin may or may
not be attached to the matrix and connected to the
cytoskeleton, and uPA may be expressed by the same cell or by
a nearby cell. Binding of the secreted uPA to uPAR may
enhance the association of uPAR with an integrin, which may
also promote adhesion. If there is also PAI in the surrounding
milieu, it can then bind to the uPA present in the ternary
complex. This PAI would most likely be PAI-1 because its
expression is stimulated by conditions associated with cell
migration and because it will specifically localize to the matrix
through binding to vitronectin. The binding of PAI-1 to uPA
induces the conformational change in PAI-1 that simultaneously
reduces its affinity for the matrix while enhancing its affinity
for the clearance receptor (Fig. 19-2). This association also
promotes integrin disengagement from the matrix, and very
likely from the cytoskeleton as well, and binding to the
clearance receptor (VLDLR). Whether the integrin first
disengages from the matrix and then binds to the clearance
receptor or whether it is the association of the quaternary
complex with the clearance receptor that induces the integrin
to disengage is not yet clear. Regardless of the exact sequence
of events, the quaternary complex is then endocytosed, and, in
the endosome, the PAIuPA complex separates from the three
receptors and is targeted to the lysosome for degradation. The
LDL-R, uPAR, and the integrin are then recycled back to the
cell surface where the process can be repeated. This cycled
attachmentdetachmentreattachment of integrins is necessary
for cell migration. This figure does not indicate the many
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potential intracellular signaling events that each one of these

interactions could generate, but each one of these receptor
interactions could signal the cell. The downstream
consequences of these events undoubtedly also modulate cell
adhesion and migration through other pathways (see Color Fig.
19-4). (From Stefansson S, Lawrence DA. Old dogs and new
tricks, proteases, inhibitors, and cell migration. Sci STKE
2003;e24, with permission.)
Tumor Angiogenesis
Angiogenesis is an important factor for tumor growth and
metastasis. It has been proposed that extracellular matrix
remodeling is necessary to allow invasion of the newly forming
blood vessels, which would explain why uPA and its receptor
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are elevated in several types of cancers. However, it is the spatial
relationship between uPA and PAI-1 that seems to be critical for
the tumorigenic response. Immunohistochemical staining and in
situ hybridization of aortic explants and cocultures of endothelial
cells and fibroblasts have shown uPA to be predominantly located
in the sprouting endothelium, whereas PAI-1 expression is strong
in the population of stromal fibroblasts that are directly in contact
with the migrating endothelial cells (195). These findings have also
been described in breast carcinomas, where PAI-1 expression is
found primarily in fibroblasts, and this expression is associated
with an increased incidence of tumor invasion (196,197).
The effects of PAI-1 on tumor growth and angiogenesis in ectopic
or transplant tumor models are variable and are sometimes
opposed (77,198,199,200,201,202,203,204,205,206). A potential
explanation for these differences is that PAI-1, in vivo, normally
plays a regulatory role to either enhance or reduce cell migration
and/or angiogenesis during wound healing. This system then may
also play a role in tumor growth, and the potential importance of
this regulation is likely to be very context specific. For example, in
transplant tumor models, small differences in the initial conditions
of either the tumor or the recipient of the transplant may have
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very large effects on early events such as the number of

transplanted cells that survive or the adhesion and engraftment of
the tissue, and it may be these very early events that are largely
determining the final outcome of the experiments. Nevertheless,
the potential for PAI-1 to regulate angiogenesis has been shown in
several nontumor models, where PAI-1 has been found to be a
potent regulator of angiogenesis (204). The inhibitory effect of
PAI-1 on angiogenesis in this model can be explained by the ability
of PAI-1 to inhibit both integrin access to vitronectin and
proteinase activity (77).
A dual role for PAI-1 in tumor growth and angiogenesis has been
demonstrated by the finding that physiologic levels of PAI-1
promote the growth of M21 human melanoma tumors in nude mice,
whereas pharmacologic levels of PAI-1 are inhibitory (204). This
pattern of growth is mirrored in the extent of angiogenesis, with
the treatment of tumors with low doses of PAI-1 increasing the
density of vessel branching, whereas substantially fewer branches
are observed upon treatment with higher doses (80,204). This
dose-response curve has also been observed in Matrigel implants
in mice (204) (see Fig. 19-5) and in an ex vivo aortic ring explant
assay (205). Therefore, PAI-1 concentrations at or near to the
normal physiologic range appear to promote angiogenesis, whereas
pharmacologic levels of PAI-1 appear to inhibit angiogenesis.
These data yield a classic bell-shaped curve for the effects of
PAI-1 on angiogenesis, consistent with its potential role as a
regulator of angiogenesis in vivo.
Clinical Implications
As noted previously, high tumor levels of PAI-1 are one of the
most informative markers of poor prognosis in several types of
cancers (141,142,143,144,145,146). This would suggest that,
despite the various results obtained in different in vivo andin vitro
models of tumor growth and angiogenesis, PAI-1 might be used as
a prognostic marker in patients with cancer, as well as a predictive
factor for therapy response. Moreover, these results also suggest
that therapeutic modulation of PAI-1 levels might be a potential
target for anticancer treatment.
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Plasminogen Activator Inhibitor-1 in

Vascular Disease
In addition to tumor growth and angiogenesis, PAI-1 is also
important in vascular disease. In this section, the role of PAI-1 in
vascular disease is addressed from four different perspectives:
vascular fibrinolysis, vascular wound healing, atherosclerosis, and
cardiovascular and cerebrovascular disease.
Vascular Fibrinolysis
The coagulation cascade is a stepwise activation and amplification
of plasma proteinases resulting in the conversion of prothrombin to
thrombin, with the generation of the fibrin clot. The coagulation
cascade also leads to platelet activation
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by a proteinase-activated receptor (207). Activated platelets then
release their granular content into the clot, which includes PAI-1
and vitronectin. The dissolution of the fibrin clot is primarily
mediated by tPA, which is synthesized and stored within
endothelial cells. tPA binds fibrin and is incorporated into the clot
along with its substrate plasminogen, which also binds fibrin. PAI-1
released from platelets is also localized within the thrombus, where
it is associated with vitronectin (107). In humans most of the
PAI-1 in platelets is latent (21,208,209,210). However, there is
enough active PAI-1 in platelets to ensure that the initial fibrin
matrix is not prematurely lysed by tPA activation of plasminogen
(211). PAI-1 deficiency in humans results in a hyperfibrinolytic
state (212), and complete absence of PAI-1 results in
posttraumatic bleeding without other manifestations
(212,213,214).
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FIGURE 19-5. Quantitative analysis of angiogenesis in Matrigel

implants. Wild-type, plasminogen activator inhibitor
(PAI)-1deficient or PAI-1transgenic C57/B6J mice were
injected subcutaneously with Matrigel alone or with Matrigel
containing fibroblast growth factor (FGF)-2 and heparin at a
final concentration of 250 ng per mL of FGF-2 and 0.0025 U per
mL of heparin. Wild-type mice were also injected with
FGF-2/heparin and with increasing concentrations of PAI-1.
After 5 to 7 days, the Matrigel implant was harvested and
solubilized in 0.1% Triton X-100 and then centrifuged to
remove particulates. The concentration of hemoglobin in the
supernatant was then determined directly by measuring the
absorbance at 405 nm and by comparing to a standard curve
for purified hemoglobin. A: Hemoglobin content from implants
containing FGF-2 in wild-type, PAI-1deficient (*, P<, 0.05), or
PAI-1transgenic mice (*, P<, 0.05), B: Hemoglobin content in
implants containing FGF-2 with increasing concentrations of
fully active PAI-1. In both panels, n4 for each condition, and
the SE (standard error) are shown. As a control for
background, the hemoglobin levels from implants without
FGF-2 was subtracted form each sample. (From McMahon GA,
Petitclerc E, Stefansson S, et al. Plasminogen activator
inhibitor-1 regulates tumor growth and angiogenesis. J Biol
Chem 2001;276:3396433968, with permission.)
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Vascular Wound Healing and Restenosis

Abnormal expression of the fibrinolytic system may also promote
the development of luminal stenosis resulting from arterial
neointima formation after vascular injury. As with tumor growth
and angiogenesis, studies analyzing the role of PAI-1 in neointima
formation and restenosis are variable and are frequently
contradictory. A role for PAI-1 in vascular restenosis was first
suggested by studies where an electrical injury to the femoral
artery was induced in mice genetically deficient in tPA, uPA, or
PAI-1 (215,216). These studies demonstrated that in this model,
neointima formation was primarily the result of smooth muscle cell
migration and proliferation, which, compared to wild-type animals,
was enhanced in the PAI-1 -/ - , reduced in uPA - /- and unchanged in
tPA -/ - mice. These results were similar to those obtained in in vitro
cell migration assays, showing that PAI-1 can inhibit smooth
muscle cell migration by obscuring the cell adhesion site to
vitronectin (167,217). However, work by other groups using
different vascular injury models has yielded opposite conclusions
about the role of PAI-1 in neointima formation. In studies using
either copper-induced (218,219) oxidative vascular injury
(220,221) or carotid ligation (221), PAI-1 was found to inhibit
neointima formation. Consistent with these reports, PAI-1
overexpression by adenovirus transgene transduction was found to
increase vascular stenosis after balloon injury in a rat (222).
The binding of PAI-1 to vitronectin has also been shown to alter
smooth muscle cell migration in different experimental models
(167). Studies with vitronectin - / - mice (221) have shown that
vitronectin deficiency results in a smaller neointima formation
following vascular injury. Likewise, there is no difference between
the smooth muscle cell proliferation in the vitronectin-null mice
compared to wild-type mice, suggesting that vitronectin could
promote neointima formation by enhancing smooth muscle cell
migration. These results raise the possibility that, because
vitronectin can bind simultaneously to fibrin and PAI-1, it can also
promote the inhibition of fibrinolysis by targeting PAI-1 to the
fibrin surface. Therefore, vitronectin may support neointima
formation both by stabilizing fibrin in a PAI-1-dependent manner
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and by enhancing the interaction between the provisional matrix

and the smooth muscle cells through integrins.
Atherosclerosis
Although most patients with generalized arterial atherosclerosis
exhibit normal plasma fibrinolytic profiles, the local fibrinolytic
balance in these patients may be severely disturbed. Intravascular
or mural thrombosis is a frequent histologic feature of
atherosclerotic lesions and appears to play a role in the intimal
thickening and fibrosis characteristic of advanced lesions (223). To
determine whether localized alterations in fibrinolytic activity could
influence this process, PAI-1 mRNA expression was evaluated in
segments of severely diseased and relatively normal human
arteries obtained from patients undergoing reconstructive surgery
for aortic occlusive or aneurysmal disease (224) (see Fig. 19-6).
Compared with normal or mildly affected arteries, PAI-1 mRNA
levels in severely atherosclerotic vessels that were analyzed by
Northern blot were considerably increased. In most cases, the level
of PAI-1 gene expression correlated with the degree of
atherosclerosis. Analysis by in situ hybridization demonstrated an
abundance of PAI-1 mRNA-positive cells within the thickened
intima of atherosclerotic arteries, mainly around the base of the
plaque, and in cells scattered within the necrotic material. In
contrast to these results, PAI-1 mRNA was detected primarily
within the luminal endothelial cells of normal-appearing aortic
tissues (224). These observations have been confirmed and
extended (225) in studies showing that both endothelial cells and
smooth muscle cells of apparently normal arteries were positive for
PAI-1 antigen and mRNA. In advanced atherosclerotic lesions,
increased expression of PAI-1 was seen in the smooth muscle cells
within the fibrous cap of the necrotic core. In addition, large
quantities of PAI-1 were present in the extracellular matrix in close
association with elastic lamina and collagen bundles.
PAI-1 has also been found to increase considerably during the
progression from normal vessels to fatty streaks to the developed
atherosclerotic plaque (226). Staining for PAI-1 is strongly
positive, particularly in the areas adjacent to the plaque, whereas
tPA shows the opposite trend, being lowest in lesions with plaque
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(226). Therefore, higher concentrations of PAI-1, together with

lower levels of tPA, are characteristic of advanced atheromatous
lesions and alterations in the balance of the fibrinolytic system
that favor its inhibition appear to be a component of the
atherosclerotic process and its common clinical complications.
The role of PAI-1 in the development of atherosclerotic lesions was
also studied in mice lacking either apolipoprotein E (apoE - /- ) or the
LDR-R -/ - These mice develop atheroscleroticlike lesions when fed a
high fat diet. Studies where apoE - / - or LDR-R -/ - mice were crossed
with either transgenic mice overexpressing PAI-1 or mice deficient
in PAI-1 have indicated that, contrary to observations made in
human autopsy specimens (226), PAI-1 expression levels have no
effect on the progression of the disease in the region of the aortic
arch (227). In contrast, a different study, where apoE -/ - mice and
animals deficient in both apoE and PAI-1 were compared, showed a
statistically significant protection from the development of
atherosclerosis at the carotid artery bifurcation in mice lacking
PAI-1 (228).
Cardiovascular and Cerebrovascular Disease

A role for PAI-1 in thrombotic events has been studied in animals
and humans. PAI-1transgenic mice develop age-dependent
coronary artery thrombosis (229), and mice exhibit accelerated
clot-lysis time (230). There is a statistically significant increase in
the PAI-1 expression in mice as a result of stress (231). The
importance of PAI-1 in human vascular disease is suggested by the
finding that human plasma concentrations of PAI-1 increase by
almost 10-fold at the site of injury when platelets are activated
(232). Likewise, a link between increased PAI-1 levels, decreased
fibrinolytic activity, and cardiovascular disease has been suggested
by several studies (233,234,235), and, as was discussed earlier,
increased PAI-1 levels constitute the link between obesity, insulin
resistance, and the resultant increase in the risk of cardiovascular
disease (135,236,237).
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A number of sequence variations in the promoter region of the
PAI-1 gene have been described. One of them, referred to as the
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4G/5G polymorphism, is the guanosine deletion/insertion 675 bp

upstream from start of transcription. Although some in vitro
studies have suggested that the 4G allele is associated with higher
PAI-1 activity compared to the 5G allele (238), no association
between PAI-1 genotype (4G/5G polymorphism) and arterial or
venous thrombosis has been found (239), except in patients with
combined protein S deficiency (240). Therefore, there is no reason
to include the study of PAI-1 4G/5G polymorphism as part of the
diagnostic process in patients with cerebrovascular or
cardiovascular disease. Nevertheless, some studies with human
subjects have suggested that patients with 4G/5G polymorphism
and insulin resistance syndrome or sepsis have an increased risk of
myocardial infarction (240) and vascular complications during
systemic infections (118).
FIGURE 19-6. Plasminogen activator inhibitor-1 (PAI-1) gene

expression in the abdominal aortic aneurysm wall. Tissue
samples were analyzed for the expression of PAI-1 mRNA
(messenger ribonucleic acid) by in situ hybridization. A:
Atherosclerotic aneurysm wall. The PAI-1 transcript is
expressed in cells (arrows) aligned at the base of the necrotic
atheroma core, 200, bright field. B: Atherosclerotic aneurysm
wall. Macrophagelike cells expressing PAI-l mRNA (arrows)
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within the inflammatory infiltrate, 400, epiluminescence. C:

Atherosclerotic aneurysm wall. PAI-l mRNA expression in
circumferentially arranged cells (arrows), which depict a
cross-section of ringlike structures that are assumed to be
small caliber capillaries (C) within an inflammatory infiltrate,
1,000 bright field. D: Normal aorta. PAI-1 mRNA signal is
detected in luminal endothelium and in a few subintimal cells,
400 epiluminescence (see Color Fig. 19-6). (From
Schneiderman J, Bordin GM, Engelberg I, et al. Expression of
fibrinolytic genes in atherosclerotic abdominal aortic aneurysm
wall. A possible mechanism for aneurysm expansion. J Clin
Invest 1995;96:639645, with permission.)
The role for PAI-1 in cerebral ischemia is less well characterized.

In animal models, PAI-1 mRNA substantially increases in the
ischemic area early after the onset of cerebral ischemia (241,242),
and inactivation of the PAI-1 gene results in a considerable
increase in the volume of the ischemic area following middle
cerebral artery occlusion (243). However, studies in nonhuman
primates have failed to show any link between PAI-1 levels and
neuronal death following cerebral ischemia (242). Likewise, as with
cardiovascular disease, no link has been demonstrated between
4G/5G polymorphism and increased risk of ischemic stroke (244).
Obesity
PAI-1 synthesis has been reported in murine adipocyte cell lines
(128,245,246,247,248), human adipose tissue explants
(236,245,249), and primary cultures of human adipocytes
(250,251) (see Fig. 19-7). There is a considerable correlation
between the amount of visceral fat and plasma levels of PAI-1 in
humans (236,252,253,254) and mice (255,256). Adipose tissue
PAI-1 mRNA levels are enhanced in obese individuals (128), and a
potential role for PAI-1 in obesity has been suggested by the
finding that genetically obese and diabetic (ob/ob) mice crossed
into a PAI-1-deficient background had considerably reduced body
weight and had improved metabolic profiles compared to lean mice
(237). Likewise, nutritionally induced obesity and insulin
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resistance were dramatically attenuated in wild-type mice lacking

PAI-1 (257). The improved adiposity and insulin resistance may be
related to the observation that PAI-1-deficient mice fed a high fat
diet had increased metabolic rates and total energy expenditure
compared to wild-type mice and that peroxisome
proliferator-activated receptor (PPAR) and adiponectin were
maintained (257). These observations suggest that PAI-1 has a
direct role in obesity and insulin resistance.
PAI-1 is dramatically upregulated in obesity, and it is now clear
that accelerated atherosclerosis, increased risk for fatal myocardial
infarction, hypertension, insulin resistance, and type 2 diabetes
also frequently accompany this disorder. The possibility that
adipose tissue itself may directly contribute to
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the elevated expression of PAI-1 in obesity has gained
considerable attention. Initial clues for such a hypothesis came
from the observation that the adipose tissue of mice contains
relatively high levels of PAI-1 mRNA (126). Moreover, clinical
studies demonstrated that weight loss due to surgical treatment,
diet, and so on, considerably reduced plasma PAI-1 levels in obese
humans (258). These findings were noteworthy because, in
obesity, the size and number of adipocytes and, therefore, the
amount of adipose tissue mass typically increase severalfold.
Therefore, in obesity, the PAI-1 biosynthetic capacity of adipose
tissue may approach or even exceed that of other tissues.
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FIGURE 19-7. Localization of plasminogen activator inhibitor-1

(PAI-1) messenger ribonucleic acid (mRNA) in the adipose
tissue of CB6 mice after lipopolysaccharide (LPS) or tumor
necrosis factor (TNF)- treatment. In situ hybridization of
paraffin sections showing vasculature from epididymal fat pads
of untreated mice (A) or from mice treated with LPS (B) or
with TNF-(C) for 3 hours. e, endothelial cells; a, adventitial
cells; s, cells within smooth muscle layers. In situ hybridization
on sections of epididymal fat padcontaining adipocytes and
microvascular endothelial cells from untreated mice (D) or
from mice treated with LPS (E) or TNF- (F) for 3 hours. Some
positive cells are indicated by arrowheads. Slides were exposed
for 8 weeks at 4C and were stained with hematoxylin and
eosin (see Color Fig. 19-7). Original magnification, 400.
(From Samad F, Yamamoto K, Loskutoff DJ. Distribution and
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regulation of plasminogen activator inhibitor-1 in murine

adipose tissue in vivo. Induction by tumor necrosis
factor-alpha and lipopolysaccharide. J Clin Invest
1996;97:3746, with permission.)
These initial observations have been extended considerably by

studies in genetically obese (ob/ob) mice (128,255). Plasma PAI-1
activity is approximately fivefold higher in these mice than in their
lean counterparts, and this elevation increases further with age
(255). Moreover, studies with genetically obese and diabetic mice
(ob/ob) lacking the PAI-1 gene (PAI-1 -/ - ) have demonstrated that
elevated PAI-1 is associated with hyperglycemia, hyperinsulinemia,
and insulin resistance syndrome (237).
A variety of observations implicate specific hormones or cytokines
in the increased expression of PAI-1 by adipose
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tissue in obesity. For example, the observations that TNF-, TNF-,
and insulin are elevated in obesity and induce PAI-1 in the plasma
and adipose tissue of lean mice (135) certainly suggest the
involvement of these mediators in the regulation of PAI-1 in
obesity. The observation that inhibiting TNF- in obese mice
considerably decreases plasma PAI-1 antigen and adipose tissue
PAI-1 mRNA (259) strongly supports this hypothesis. Triglycerides
and free fatty acids also may be involved because they stimulate
PAI-1 gene expression in adipocytes. These studies of PAI-1
suggest that these mediators may promote the increased risk for
cardiovascular disease in obesity or type 2 diabetes.
Renal Disease
Although PAI-1 is essentially undetectable in normal kidneys, PAI-1
mRNA and PAI-1 protein levels have been found to be increased in
several renal diseases (260,261,262,263). A role for PAI-1 has
been described in acute renal disorders such as proliferative
glomerulonephritis, renal vasculitis, thrombotic microangiopathy,
and membranous nephropathy, as well as in chronic progressive
renal disease.
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PAI-1 in Acute Renal Disease. PAI-1 mRNA has been detected in

human and animal models of proliferative glomerulonephritis,
particularly in those cases associated with fibrin deposition (264).
Moreover, it has been observed that inhibition of plasminogen
activators results in a considerable worsening in the severity of the
glomerular injury. PAI-1 reduces glomerular mesangial turnover by
inhibiting plasminogen activators, thereby decreasing plasmin
generation and plasmin-mediated matrix degradation. Because
fibrin accumulation is an important mediator of acute glomerular
injury, strategies designed to enhance fibrinolysis by blocking
PAI-1, or to prevent fibrin formation, might be therapeutic in
patients with glomerulosclerosis. Although the role of PAI-1 in
renal vasculitis is less clear, PAI-1 protein has been identified
together with fibrin deposits in renal biopsy specimens from
patients with focal necrotizing glomerulonephritis due to systemic
lupus erythematosus (265).
In thrombotic microangiopathy, which is a condition that typically
involves fibrin deposition in glomerular capillaries and
extraglomerular arterioles, PAI-1 plays an active role in the
generation of the fibrin thrombi that are formed in response to
glomerular endothelial cell injury. PAI-1 deposition has been
identified in the kidneys of patients with this disorder (266).
Likewise, elevated plasma PAI-1 levels have been associated with
disease outcome in several studies of patients with hemolytic
uremic syndrome (267). Finally, in membranous nephropathy,
PAI-1 transcripts have been found to be abundant (265), and PAI-1
protein colocalizes with vitronectin within the transmembranous
deposits (268).
PAI-1 in Progressive Chronic Renal Disease. In this disease,
there is a pathologic accumulation of extracellular matrix
(269,270). PAI-1 mediates several effects that may facilitate
matrix accumulation through the impairment of matrix turnover.
Likewise, TGF-, which is a critical mediator of renal fibrosis, is a
powerful inducer of PAI-1 expression (271), and several studies
have demonstrated a link between the
reninangiotensinaldosterone cascade, TGF-, and PAI-1 in the
pathophysiology of glomerulosclerosis (269). Angiotensin II
stimulates PAI-1 production (272), and the renoprotective effects
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of pharmacologic inhibition of angiotensin II are mediated, at least

in part, by a reduction in PAI-1 expression (273). Likewise, recent
studies using a rat model of mesangioproliferative nephritis, known
as antiThy-1 nephritis, demonstrated that treatment with a
dominant-negative human mutant PAI-1 that binds to matrix
vitronectin but does not inhibit plasminogen activators enhances
plasmin generation, increases matrix turnover, and decreases
matrix accumulation in experimental glomerulonephritis
(274,275,276).
Lung Disease
PAI-1 has been identified as a major deleterious mediator in many
acute and chronic inflammatory lung disorders. Adult respiratory
distress syndrome (ARDS) (277,278), idiopathic pulmonary fibrosis
(IPF) (279,280), sarcoidosis (281), hyperoxic lung injury (282),
and bronchopulmonary dysplasia (283) are all associated with
prominent intraalveolar fibrin deposition and development of
pulmonary fibrosis. Fibrin turnover is tightly regulated by the
concerted action of proteases and antiproteases, and inhibition of
plasmin-mediated proteolysis could account for fibrin accumulation
in lung alveoli. The fibrinolytic activity in bronchoalveolar lavage
(BAL) supernatant fluids obtained from patients with these
diseases has been found to be suppressed (282,283). Moreover,
PAI-1 levels in BAL fluids from patients with ARDS and IPF are
considerably elevated, and it was shown that this PAI-1
upregulation impairs the fibrinolytic capacity of the fluid
(277,278). Bleomycin administration has been used extensively to
study the pathogenesis of pulmonary fibrosis in a variety of animal
models (284). Bleomycin causes a pneumonitis that progresses to
fibrosis in a dose-dependent manner within 2 weeks, while
simultaneously suppressing the fibrinolytic activity of BAL fluid in a
pattern similar to that seen in human inflammatory lung disease
(284). In mice, it was demonstrated that PAI-1 is upregulated after
bleomycin administration and that the PAI-1 expression localizes to
areas of fibrin-rich fibroproliferative lesions (285).
Transgenic mice that either overexpress PAI-1 or are genetically
deficient in PAI-1 have been used to determine whether a
cause-and-effect relation exists between PAI-1 expression and the
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development of pulmonary fibrosis. The results of one study

demonstrated a strong relation between PAI-1 gene expression and
the degree of pulmonary fibrosis that followed bleomycin
administration (286). In another study, it was shown that the
lungs of mice exposed to hyperoxia overproduced PAI-1 and that
PAI-1 upregulation impaired fibrinolytic activity in the alveolar
compartment (282). It was found that mice genetically deficient in
PAI-1 did not develop intraalveolar fibrin deposits in response to
hyperoxia. These findings suggest that alterations in the
fibrinolytic environment during inflammatory injury influence the
subsequent development of pulmonary fibrosis and provide
evidence for the pathologic contribution of PAI-1 in this event.
In conclusion, there is a strong link between PAI-1 and human
disease. Currently, there are several ongoing clinical trials testing
the effects of the modification of human PAI-1 in different
diseases. Results of these studies will provide new and definitive
insight into the role of PAI-1 in the pathologic and physiologic
processes.
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Cancer Res 1985;44:139266.
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activator/plasmin system. J Clin Invest 1991;88:10671072.
3. Kohler HP, Grant PJ. Plasminogen-activator inhibitor type 1
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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

Colman, Robert W.; Clowes, Alexander W.;

Goldhaber, Samuel Z.; Marder, Victor J.; George, James N.

Hemostasis and Thrombosis: Basic Principles and

Clinical Practice, 5th Edition

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

role in regulating PA activity in blood and most tissues. The

BASIC PROPERTIES OF PLASMINOGEN

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

structural element of the serpin, the -sheet A (33,39,40,41). This

Reactive Center Loop

Plasminogen Activator Inhibitor-1

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

FIGURE 19-1. Schematic illustration of the three

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

Interaction with Vitronectin and Members of

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

family has been less well characterized and appears to be located

Binding to Heparin and Fibrin

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

Structure and Regulation of the

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

(1 allele) of the HindIII polymorphic site. Interestingly, genotype

FIGURE 19-2. Plasminogen activator inhibitor-1 (PAI-1)

Although PAI-1 is present at low concentrations in plasma, its

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

physiologic state, indicating that the amount of PAI-1 in plasma is

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

induce PAI-1 synthesis by increasing mRNA stability (131).

PLASMINOGEN ACTIVATOR INHIBITOR-1

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

renal cell carcinoma, melanoma,

FIGURE 19-3. Plasminogen activator inhibitor-1 (PAI-1)

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

association with normal and pathologic physiology.

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

demonstrated that the binding of uPA to uPAR promotes the

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

uPA and endocytosis of the complex is unclear: It was shown to

FIGURE 19-4. The evolution of our understanding of the role

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

potential intracellular signaling events that each one of these

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice