Plasminogen-Plasmin System: Editors

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 18 Pl as m i nogenP l asm i n System
Chapter 18
PlasminogenPlasmin System
Nuala A. Booth
Fedor Bachmann
Hemostasis requires mechanisms both to stop bleeding by
formation of a hemostatic plug and to limit the plug's development,
allowing reestablishment of normal blood flow. The latter function
is largely accomplished by localized activation of the
plasminogenplasmin enzyme system, also called the fibrinolytic
system. The system is finely tuned, as it has to be to accomplish
healing of a vascular lesion without compromising the early
stability of the hemostatic plug and to limit activity to the injured
area. There is a dynamic balance between coagulant and
fibrinolytic activities, each of which represents a further balance
between proteolytic and inhibitory proteins. Excessive local or
systemic fibrinolytic activity can result in bleeding, often occurring
after a delay, as the weakened plug is dissolved. Conversely, an
inadequate fibrinolytic response may retard lysis of a thrombus
and contribute to its extension.
Plasmin is the main fibrinolytic enzyme. It is a trypsinlike serine
protease that degrades fibrin, and it is generated by activation of
the zymogen, plasminogen. The central players dictating the
activity of the system are the plasminogen activators (PAs) and the
inhibitors of both the activation stage and of plasmin activity. The
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two major PAs that occur in the circulating blood, tissue-type

plasminogen activator (tPA) and urinary-type plasminogen
activator (uPA), also called urokinase or UK, are also serine
proteases. Inhibitors, most of which are of the serine protease
inhibitor (serpin) family, regulate the proteases. These are PA
inhibitors, plasminogen activator inhibitor 1 (PAI-1; see Chapter
19) and PAI-2, and 2 -antiplasmin the principal inhibitor of
plasmin. Receptors and binding proteins for plasminogen and the
PAs on endothelial cells (see Chapter 22), platelets and leucocytes
regulate local generation of plasmin. The best characterized of
these receptors is the urinary-type plasminogen activator receptor
(uPAR). Tables 18-1 and 18-2 list several of the properties of the
genes, messenger ribonucleic acids (mRNAs), and proteins of the
components of the fibrinolytic system, together with modulating
factors. These include factor XII and the contact phase of
coagulation, in which interactions with prekallikrein and kininogen
also promote plasminogen activation. They also include
apolipoprotein (a), cytokines, growth factors and hormones (see
Chapter 6), the PAI-1binding protein vitronectin, the
thrombin-activatable fibrinolytic inhibitor (TAFI) (see Chapter 20),
the low density lipoprotein receptorrelated protein (LRP, identical
to the 2 -macroglobulin receptor), the mannose receptor, annexin
II, and several other inhibitors that affect the physiology of
fibrinolysis (see Chapter 23).
Like the serine proteases of blood coagulation, fibrinolytic
proteases generally exist in a zymogen form, a single-chain form
of the molecule that is activated by cleavage of a single peptide
bond. All the active serine proteases cleave arginine and/or lysine
bonds and originated from a simple trypsinlike ancestor protease
(1,2). In the case of the fibrinolytic proteases, the products are all
in a two-chain form held together by one or more disulfide bonds.
An exception to this rule is tPA, which is an active protease in its
single-chain form; tPA can be considered to be at one end of the
scale of zymogenicity, with plasminogen as a totally inactive
example at the other (3).
The typical structure of these proteases (Fig. 18-1) is that they
have an N-terminal A chain containing several independently folded
modules or domains, which have particular binding properties, and
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a C-terminal B chain, which is the protease domain. The kringle

domainnamed after a Danish pastryis approximately 80
residues long and is held together by three internal disulfide
bonds. The finger domains are homologous to type I and type II
fibronectin regions. Type I finger domains are approximately 50
residues long, contain two internal disulfide bonds, and may play a
role in fibrin binding. The epidermal growth factor (EGF) domains,
which are homologous with a module in the EGF precursor, contain
53 residues and three internal disulfide bonds. In several proteins,
these domains bind to specific cell-surface receptors or binding
proteins. The apple domain has been found only in prekallikrein
and factor XI, in which it occurs as a quadruple repeat of 90
residues in the N-terminal portion of these molecules. Like the
kringle domains, it contains three internal disulfide bonds. The
protease domain contains the catalytic triad, Ser195, His57, and
Asp102 (chymotrypsin numbering), and the domain comprises
approximately 250 amino acid residues and four disulfide bonds.
The protease domain is well conserved, with particularly high
sequence identity in the vicinity of the three active site residues
and at the N-terminus.
The components of the fibrinolytic system are discussed next,
beginning with the central enzyme, plasmin, and its precursor,
plasminogen, then considering in turn plasminogen activators, the
different inhibitors of plasmin, and plasmin generation, with a final
summary on the balance of the system.
PLASMINOGEN
Human plasminogen (PLG) is the zymogen form of the main
fibrinolytic protease, plasmin (EC 3.4.21.7). PLG is a single-chain
glycoprotein of 92 kDa, consisting of 791 amino acid residues and
approximately 2% carbohydrate (Table 18-1). In the human adult,
the plasma concentration of PLG is approximately 200 mg per L,
which corresponds to M (Table 18-2). The half-life (t 1/ 2 ) of native
PLG, which has a glutamic acid residue at its N-terminus
(GluPLG), is approximately 2.2 days; the slightly degraded
LysPLG (see subsequent section, Activation of Plasminogen to
Plasmin) has a much shorter t 1/ 2 of 0.8 days (4). The principal
production site for PLG is the liver, but PLG is present in the
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extravascular space of most tissues, and some, such as

eosinophils, the kidney, and the cornea, may be capable of
synthesizing it (5,6,7,8).
FIGURE 18-1. Structural elements of some serine proteases.

A, apple domain (approximately 80 residues); AP, activation
peptide; E, epidermal growth factor domain (approximately 32
residues); F, fibronectin finger (approximately 40 residues); K,
kringle (approximately 90 residues); P, protease domain (B
chain) (approximately 250 residues); scuPA, single-chain
urokinase; tPA, tissue-type plasminogen activator.
TABLE 18-1 CHROMOSOME LOCATION AND GENE ORGANIZATIO

CONSTITUENTS OF THE PLASMINOGEN-PLASMIN SYSTEM
Factor
Symbol
Chromosome
Gene
mRNA
(kb)
(kb)
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Plasminogen
PLG
6q26-q27
52.5
2.9
tPA
PLAT
8p12-p11
32.7
2.7
uPA
PLAU
10q24
6.4
2.4
PAI-1
PLANH1
7q21.3-q22
12.2
2.4/3.2
PAI-2
PLANH2
18q22.1
16.5
1.9
Factor XII
F12
5q33-qter
12
2.6
Prekallikrein
KLK3
4q34-q35
22
2.4
HMW kininogen
KNG
3q27
27
3.2
Vitronectin
VTN
17q11
4.5
1.6
C1-inhibitor
C1NH
11q12-q13.1
17
1.8
2 -Antiplasmin
PLI
17p13
16
2.2
2 -Macroglobulin
A2M
12p13.3-p12.3
48
4.6
Histidine-rich
HRG
3q27
15.5
2.1
TNA
3p22-p21.3
12
0.9
APOAL2.1
6q26-27
glycoprotein
Tetranectin,
monomer
Apolipoprotein
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(a)
TAFI
CPB
13q14.11
48
1.8
uPAR
PLAUR
19q13.1-q13.2
23
1.4
Annexin II a
ANX2
15q21-q22
1.3
LRP ( 2 -MR)
LRP1
12q13-q13.3
92
15
-Enolase
ENO1
1pter-1p36.13
>18
1.7
Mannose-receptor
MRC1
10p13
5.1
mRNA, messenger ribonucleic acid; tPA, tissue-type plasminogen activ

uPA, urinary-type plasminogen activator; PAI-1, plasminogen activato
inhibitor 1; PAI-2, plasminogen activator inhibitor 2; HMW,
high-molecular-weight; C1-inhibitor, complement component 1 esteras
inhibitor; TAFI, thrombin-activatable fibrinolysis inhibitor; uPAR,
urinary-type plasminogen activator receptor; LRP, low density lipoprot
receptorrelated protein; MR, macroglobulin receptor.
a
Heavy chain of tetramer.
Variable, depending on the number of kringle 4 inserts.
TABLE 18-2 PROPERTIES OF PROTEIN CONSTITUENTS OF TH
Amino
Factor
Plasminogen
M r (kDa)
acid
residues
Concentration
(mg/L)
92
791
200
Mo
concent
2
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tPA
68
530
0.005
70
uPA
54
411
0.002
40
PAI-1
52
379
0.01
200
PAI-2
46/70
393
<0.005
<70
Factor XII
80
596
30
0.375
Prekallikrein
88
619
40
0.45
110
626
70
0.60
Vitronectin
78
459
350
4.5
C1-inhibitor
105
478
180
1.7
70
452
70
725
1,451
2,500
75
507
100
HMW kininogen
2 -Antiplasmin
2 -Macroglobulin
Histidine-rich
1.5
glycoprotein
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Tetranectin,
22.5
181
10
0.5
300800
<7
TAFI (pro-CpU)
60
401
uPAR
55
313
Annexin II b , f
38
338
LRP ( 2 -MR)
600
4,525
54
433
175
1,437
monomer b , c
Apolipoprotein(a) d
-Enolase
Mannose receptor
75
z, protease zymogen; tPA, tissue-type plasminogen activator; p, prote

activator; PAI-1, plasminogen activator inhibitor 1; i, inhibitor; PAI-2,
high-molecular-weight; c, cofactor; C1-inhibitor, complement compone
TAFI, thrombin-activatable fibrinolysis inhibitor; uPAR, urinary-type p
density lipoprotein receptorrelated protein; MR, macroglobulin recept
M r of mature proteins is listed.
a
Estimated value, calculated from difference of molecular weight deter
amino acids derived from complementary DNA.

b
c
M r of nonglycosylated form as listed in Swiss protein base ExPASy.
Heavy chain of homotrimer.
Many isoforms due to 13 up to 37 type 4 kringle motifs.
Activated form.
Heavy chain of heterotetramer (formed by two heavy and two 10-kDa
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Structural Organization
The PLG gene is 52.5 kb, the largest among the fibrinolytic
proteins. It is located on chromosome 6q26-q27 (9), close to two
genes for apolipoprotein (a) and for the PLG-related genes A and B
(10,11). Amino acid sequence analysis showed the structural
features of N-terminal activation peptide, five kringles, and the
protease domain (12). Subsequent isolation of mRNA, and later of
the gene, confirmed the original sequence, except for an additional
isoleucine residue at position 67 (13).
The 19 exons of the PLG gene are separated by 18 introns that
follow the general GT-AG rule found in other eukaryotic genes. The
first exon encodes the signal sequence, with two exons encoding
the activation peptide and each kringle; the remaining six exons
code for the intervening sequence and the protease domain (see
Fig. 18-2). Each of the five kringles consists of 78 to 80 residues,
and they are well-conserved between species (14). Functionally,
the kringles give PLG the ability to bind to exposed lysyl residues
in fibrin (15,16), -antiplasmin (17), tetranectin (18),
histidine-rich glycoprotein (19), collagen (20),
high-molecular-weight (HMW) and low-molecular-weight (LMW)
kininogen (21), thrombospondin (22), and cell surface receptors.
The affinity of the kringles for lysyl residues is also exploited to
isolate it from human plasma by affinity chromatography (23).
The kringle structure is shaped by a characteristic 16, 24, 35
disulfide bond pattern involving six conserved Cys residues in
positions 1, 22, 51, 63, 75, and 80 (kringle 5 numbering). A single
nonconserved Cys in position 4 of kringle 2 forms an interkringle
disulfide bond with Cys43 in kringle 3 (not shown on Fig. 18-2)
and thereby restricts the mobility of kringles 2 and 3. Solution
structures have been determined for kringle 1 (24), kringle 2 (25),
kringle 2 + 3 (26), and kringle 4 (27). Crystallographic structures
have been solved for kringle 1 (28,29), kringle 4 (30), and kringle
5 (31). The human PLG kringles have different affinities and
specificities for -amino acid ligands. The tightest binding site for
-aminocaproic acid (ACA) is provided by kringle 1 [dissociation
constant (K d ) of 9M] (32,33) followed by kringle 4 (32,34,35) and
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kringle 5 (34,35). Kringle 2 displays only a weak interaction with

ACA probably not of functional significance, whereas kringle 3
shows no interaction (36).
Experimentally, kringle 4 can be isolated from PLG by digestion
with elastase, which cleaves after small amino acids and at
relatively frequent ValVal sequences. Therefore, digestion with
elastase typically yields three major components: a fragment
containing kringles 1 through 3, isolated kringle 4, and kringle 5
attached to the protease portion of PLG (12). The latter fragment,
also called mini-PLG, can be activated to plasmin by PAs
(37,38,39). Although it has some affinity for fibrin (16,40), it does
not bind to lysineSepharose and therefore is separated from
kringles 13 and kringle 4, which do bind lysine (12,32). Kringle 1
can be obtained from kringles 13 by further digestion with other
enzymes, such as Staphylococcus aureus V8 protease (41).
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FIGURE 18-2. Sequence of the human plasminogen molecule.

Positions of the 18 introns are indicated by solid arrows at (for
type 1 and 2 introns) or in between (for type 0 introns)
residues. The 19-residue signal peptide (shown in a box with
negative numbers) is cleaved by signal peptidase at the
GlyGlu peptide bond. Conversion of plasminogen to plasmin
occurs after the peptide bond Arg561Val562 (shown by an
open, curved arrow) is cleaved by plasminogen activators. PAP
refers to the preactivation peptide generated by the action of
plasmin on Gluplasminogen; primary (but not sole) cleavage
site is Lys77Lys78 bond (shown by an open, straight arrow).
K1K5, kringles, located in the A chain; the B chain remains
attached to the A chain after cleavage of the 561562 bond via
the disulfide bonds Cys548Cys666 and Cys558Cys566.
Carbohydrate attachment sites (Asn289 and Thr346) are shown
by diamonds. (From Petersen TE, Martzen MR, Ichinose A, et
al. Characterization of the gene for human plasminogen, a key
proenzyme in the fibrinolytic system. J Biol Chem
1990;265:6104, with permission.)
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Crystallography has revealed that the kringle 4 structure is highly
stabilized by an internal hydrophobic core and an extensive
hydrogen bonding network. The lysine binding site (LBS) on the
surface of kringle 4 is an open, elongated, shallow trough, formed
by the hydrophobic residues Trp62, Phe64, and Trp72, surrounded
by the positively charged Lys35 and Arg71 on one side and, at a
distance of approximately 7 , the negatively charged side chains
of Asp55 and Asp57 (see Fig. 18-3) (30). This structure provides
for ideal docking of zwitterions such as lysine or ACA, whose
opposite charges also are approximately 7 apart. The interaction
between binding site and ligand is enhanced further by the close
van der Waals contacts between the methylene carbons of ligand
and the aromatic residues of the amino acids in the center of the
binding site (30). The binding sites in kringles 1 and 5 are
similarly constructed. In kringle 1, Arg34 takes the place of Lys35
in kringle 4 (30,42); in kringle 5, Leu71 is substituted for Arg71 in
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kringle 4 (31).
The catalytic domain of PLG (B chain) is made up of 230 residues
and is homologous with the trypsin family of serine proteases. In
the active site, it contains three amino acids: His603, Asp646, and
Ser741. Two groups of investigators have elucidated the crystal
structure of micro-PLG mutants. The main observations, a
deformed catalytic triad and a blocked S1 specificity pocket, are
similar in the two crystal structures (43,44). This unusual
structural feature has only been observed in the crystal structure
of factor D of the complement system, in contrast to the preformed
catalytic triad observed in other trypsinlike structures.
Activation of Plasminogen to Plasmin

All known PAs cleave the Arg561Val562 bond in PLG (see Fig.
18-4). The B chain of the resulting two-chain Gluplasmin
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molecule remains attached to the A chain by two disulfide bonds.
When these PAs are added to GluPLG in vitro, the generation of
trace amounts of plasmin results in cleavage of the N-terminus by
hydrolysis of one or several of the following bonds: Arg68Met69,
Lys77Lys78, or Lys78 Val79 (45,46). The final product obtained
is Lysplasmin (Fig. 18-4). Under physiologic conditions, the
plasmin-mediated conversion to LysPLG probably does not occur
in circulating blood (47) because free plasmin is inactivated rapidly
by 2 -antiplasmin. Studies with monoclonal antibodies that
recognize LysPLG but not GluPLG revealed no free LysPLG nor
bound Lysplasmin in normal human plasma and only minimal
increases of these forms in patients who received thrombolytic
therapy (48).
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FIGURE 18-3. Model of the interaction site of plasminogen

kringles 1 and 4 with -aminocaproic acid (depicted in heavy
lines), a lysine analog. A hydrophobic core on the surface of an
elongated shallow trough is surrounded by two clusters of
positively and negatively charged amino acids.
Several -aminocarboxylic acids, such as 6-aminohexanoic acid

(ACA, 6-AHA), p-aminomethyl benzoic acid (p-AMBA), and
trans-4-amino-methyl-cyclohexane-carboxylic acid (trans-AMCA,
also known as tranexamic acid), inhibit the functional activity of
plasmin in vitro and in vivo by binding to the LBS of
plasmin(ogen), thereby inhibiting the binding of plasmin(ogen) to
fibrin (49,50). These compounds are therefore termed
antifibrinolytic. It should be noted that their effects depend
strongly on the concentration used; at micromolar to low millimolar
concentrations, these compounds actually increase the activation
rate. They do this by inducing a conformational change, making
GluPLG adopt a structure similar to that of LysPLG, which is
more easily activated to plasmin. The change reflects the loss of
interaction of the N-terminal preactivation peptide with LBS on the
kringles (51,52). It causes a marked decrease in the Michaelis
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constant K M for activation with tPA or uPA, by approximately one

order of magnitude, down to the range of the physiologic
concentration of PLG in human plasma (53,54). In GluPLG, Lys50
binds to LBSs on kringles (51,52), making these LBS less available
for binding to C-terminal lysine in fibrin, PLG receptors, and other
proteins. Therefore LysPLG binds with higher affinity to fibrin
than does GluPLG.
Conformational differences between Glu and LysPLG have been
revealed by small-angle neutron scattering. GluPLG goes from
having a radius of gyration of 39 to 56 in the presence of ACA
(55). The closed form, in which the five kringle domains and the
catalytic domain interact to produce an ellipsoid shape, is less
activatable than the open form (see Fig. 18-5),
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whose increased flexibility facilitates the binding of PAs, leading to
approximately a 10-fold decrease of K M Furthermore, the open
form, taken up by PLG after binding to fibrin, enables the
proteolytic domain to sweep over a larger area and therefore be
more efficient in the digestion of fibrin than a closed form with
restricted mobility. Activatability of GluPLG by PAs is dependent
on other factors as well, such as the presence of anions, which in
the human plasma are mainly chloride ions, and of the divalent
cations Ca 2+ , Mg + and Mn + All these ions counteract the effect of
ACA and stabilize PLG in the Glu-form, which is poorly activatable
by PAs (54,56).
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FIGURE 18-4. Activation of native Gluplasminogen. In the

absence of plasmin inhibitors, tissue plasminogen activator
(tPA) or urinary plasminogen activator (uPA) converts
Gluplasminogen to Glu plasmin; the latter then converts
Gluplasmin(ogen) to Lysplasmin(ogen).
FIGURE 18-5. A sketch of the conformational change in
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plasminogen induced on occupation of a weak lysine binding

site by the ligand -aminocaproic acid. In the absence of
ligand, the domains in plasminogen interact to form a molecule
with the overall shape of a prolate ellipsoid. Binding of the
ligand converts this into an extended flexible structure in
which the interaction between the domains is abolished. The
closed and open forms are drawn to scale. (From Mangel WF,
Lin B, Ramakrishnan V. Characterization of an extremely large,
ligand-induced conformational change in plasminogen. Science
Many modulators increase or decrease the rate of PLG activation;

the most important of these is fibrin. PLG binds to fibrin, directing
the process of clot lysis to its target, binding that is competed by
-amino carboxylic acids (57). Identification of the binding sites
was achieved by examining the products of elastase digestion of
PLG. Kringle 13 and miniplasminogen bound to fibrin, but kringle
4 did not (15,16,58,59). Notably, there is no close correlation
between the affinities of kringles for fibrin and lysine/ACA (see
Table 18-3).
Partial degradation of fibrin by plasmin creates a positive feedback
mechanism, whereby native GluPLG is bound to newly created
C-terminal lysines on the fibrin molecules, particularly on the
C-terminal portion of the (60,61,62,63,64). A few studies
support the concept that PLG might bridge adjoining fibrin
molecules and that probably both kringle 5 and kringle 1 bind to
fibrin, albeit not at the same binding site (59,65,66). There is little
consensus on the values of the dissociation constants and number
of binding sites for the binding of PLG, particularly of GluPLG to
fibrin. Taking all reported figures together, it appears that an
average K D for the binding of GluPLG to native fibrin is
approximately 5 M and that of LysPLG, roughly one order of
magnitude lower. In the presence of partially digested fibrin, the
K d for the binding of GluPLG approaches that of LysPLG for
native fibrin (0.5 M) (63,64). This probably means that GluPLG,
after binding to fibrin, takes on the open configuration (Fig. 18-5)
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and functionally behaves like LysPLG.
TABLE 18-3 AFFINITIES OF PLASMINOGEN KRINGLES FOR

2 -AMINOCAPROIC ACID AND FOR NATIVE FIBRIN
Kringle
Affinity for
Affinity for native
number(s)
ACA
fibrin
13
High
Moderate
Moderate
Very low a
Low
High
A nuclear magnetic resonance imaging study has shown
that kringle 4 can bind to partially digested fibrin, which

has many C-terminal lysyl sites exposed (58). This binding
was abolished by -aminocaproic acid (ACA).
Recombinant microplasminogen, consisting of residues 542791,

can be activated to microplasmin by all four common PAs: tPA,
uPA, streptokinase (SK), and staphylokinase (SAK) (39).
Autocatalytically produced microplasmin consisting of the last 31
amino acid residues of the A chain and B chain exhibited a similar
affinity for fibrin as regular plasmin but a markedly lower catalytic
rate constant (k cat = 0.0076 per second vs. 0.064 per second for
plasmin) (38).
Cell Binding of Plasminogen

PLG binds to different cell types and a number of binding proteins
have been identified on human and other cells (67), -enolase (68)
and annexin II (69) being the most studied. All are abundant
proteins, and generally the binding occurs through Lys residues.
Interestingly, PLG binding to the prion protein PrPsc distinguishes
between the normal and misfolded conformers (70). The
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association of PLG with the various binding proteins is not always

of high affinity, and the fact that so many proteins can serve this
function precludes their being viewed strictly as PLG receptors. The
abundance of such binding sites does make it likely that this
phenomenon indeed potentiates local plasmin generation (71).
Angiostatins
Many primary tumors have been found to suppress the growth of
their remote metastases or of a second and different tumor. At
least in part, this effect is due to angiostatin, originally described
as a 38-kDa protein and identified as an internal proteolytic
fragment of PLG comprising kringles 14 (72). It appears that
tumor-mediated proteolysis is responsible for the generation of
angiostatin, through uPA-induced PLG activation and the opening
up the inter-kringle and other disulfide bonds by plasmin
reductases (73,74), which include known PLG-binding proteins that
act as plasmin reductases (67,75,76). Similarly, proteolytic
cleavage of PLG by macrophage elastase (MMP-12), or
stromelysin-1 (MMP-3), or a 24-kDa endopeptidase from a
Chrysobacterium species generated angiostatinlike fragments
(77,78).
The antiproliferative effect of PLG fragments is not restricted to
the originally described kringle 14 or 15 fragments. Recombinant
fragments containing kringles 13 (79,80,81), or even isolated
kringles 1, 2, 3, or 5, but not kringle 4, had antiproliferative
activity similar to the originally described larger fragments
(79,82,83). On the other hand, isolated kringle 4 was potent in
inhibiting endothelial cell migration (84). Endothelial cell
proliferation assays revealed that angiostatin induces apoptosis
(85) and also appears to have a role in the prevention of
atherosclerosis, in that it inhibits smooth muscle cell proliferation
and migration in vitro (86). In all of these assays, intact PLG was
inactive. The sum of these data suggests that various internal
fragments of PLG exert powerful biological effects and that
fragments of HMW kininogen (87) and of apolipoprotein (a) (88)
have similar effects.
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Variants of Plasminogen
The PLG molecule is subject to considerable variation in the normal
population. In addition to the limited proteolysis and activation
processes already discussed, the two important sources of
variation are glycosylation and genetic polymorphism.
Glycosylation
Human GluPLG occurs in two variants that differ in glycosylation.
PLG variant 1 is diglycosylated, with N-linked carbohydrate moiety
of the mannose type with 10 to 11 monosaccharide units on
Asn289, and an O-linked moiety of three to four residues on
Thr346 (89). PLG 2, which accounts for a little more than half of
the PLG molecules, contains only the carbohydrate group at Thr346
(90). Both of these forms exhibit, on isoelectric focusing,
additional heterogeneity with respect to their sialic acid content
(45,46,91,92). Therefore, each of the two variants produces six
additional isoforms with pIs between 6.1 and 6.6. All 12 molecular
forms are present in plasma from single donors (4).
In the absence of fibrin, PLG 1 appears to be more easily activated
to plasmin than is PLG 2, but this difference is not observed in the
presence of fibrin. Variant 1 changes its conformation more easily
in the presence of fibrin or tranexamic acid than does variant 2
(93). Variant 2 was cleared more rapidly than variant 1 and bound
five times faster to a deendothelialized vessel surface (94,95,96).
A novel glycosylation site containing a trisaccharide on Ser249 has
been described (97), and another may exist at Ser339 (98); a
further serine residue, Ser578, can be phosphorylated (99).
Polymorphism
Isoelectric focusing of neuraminidase-treated plasma (which
removes sialic acid) revealed several bands of activatable PLG,
representing the genetic polymorphism of PLG alleles. The
nomenclature, adopted in 1984 (100), is that the two most
commonly observed variants found in all races are called PLG A (A
for acidic pI) and PLG B (B for basic pI). Recently, the molecular
basis of these common polymorphisms was reported: PLG A have
Asp453, and PLG B have Asn453 (101). Variants with intermediate
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pI are designated as PLG M. Variants with more acidic pI than PLG

A receive, in addition to the letter A, a numerical suffix that
increases with decreasing pI of the variant. The variants with more
basic pI than PLG B receive a numerical suffix that increases with
increasing pI of the variant. Alleles are designated with an asterisk
(e.g., PLG*A3), and silent (null) PLG alleles are designated
PLG*Q0, with Q0 standing for quantitatively zero or nonexpressed.
The frequency of the alleles in 1,330 unrelated West Germans was
70.1% for PLG*A, 27.7% for PLG*B, 1.5% for PLG*A3, 0.6% for
PLG*R (R: the sum of all other rare PLG alleles), and 0.35% for
PLG*Q0 (100). In the Japanese population, the frequency of the
allele PLG*A is considerably higher (95%) than in the Western
population (102).
Since the first report in 1978 of a hereditary molecular abnormality
in PLG in a patient with recurrent thrombosis (103), several
hundred cases have been described with a deficiency of PLG
antigen and activity (type I), or a normal level of antigen but
reduced activity (type II, dysplasminogenemia). In most instances,
a thromboembolic event or the presence of ligneous conjunctivitis
has been the starting point for family studies. Only those cases in
which the molecular defect has been identified are listed in Table
18-4
(101,102,103,104,105,106,107,108,109,110,111,112,113,114);
updates can be found
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on the OMIM (Online Mendelian Inheritance in Man) Web site. The
sum of all data does not prove conclusively that the heterozygous
presence of one mutant PLG gene results in an increased incidence
of thromboembolism (115,116). In healthy subjects, the Ala601Thr
mutation is found in 2.2% and 2.9%, respectively, of the Japanese
and Chinese Han populations (107,112). Similarly, Lys19 to Glu
occurs relatively frequently in association with
hypoplasminogenemia (104,117), and there is controversy over
whether it should be regarded as a polymorphism (118).
Homozygous deficiencies, however, are clearly associated with
ligneous conjunctivitis, caused primarily by Arg216 to His or
Trp597 to stop mutations (106), and replacement therapy with PLG
is effective (108).
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TABLE 18-4 CONGENITAL PLASMINOGEN MUTATIONS
Residue
9
Mutation
Thr to
References
Other
names
Type
Clinica
manifestat
106
106
101
104
101
106
105
Ligneous
Asn
19
Lys to
Glu
128
Lys to
Pro
133
Cys to
stop
134
Arg to
Lys
212
Lys
deletion
216
Arg
toHis
355
Val to
conjunctivi
107
II
Nagoya
Thromboph
107
Nagoya-I
Thromboph
Phe
374
Val to
Phe
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453
Asp to
101
108
106
108
Thromboph
Asn
460
Glu to
stop
513
Arg to
His
572
Ser to
Pro
(secretion
defect)
597
Trp to
105,106
Cys
597
Trp to
conjunctivi
104
stop
601
Ala to
Ala to
Ligneous
conjunctivi
107,112
II
Thr
620
Ligneous
Tochigi,
Thromboph
Osaska II
107
Thr
Nagoya-II,
Thromboph
Tochigi,
Kagoshima
675
Ala to
109,111
112,113
II
Thromboph
Thr
676
Asp to
Asn
693
Gly to
Osaka-II,
None
III
101
II
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Arg
732
Gly to
114
II
Kanagawa-I
Arg
It is not yet clear why some mutations cause ligneous

conjunctivitis and why very low functional PLG levels can be
tolerated in some individuals, without thrombosis occurring (106).
PLG-deficient mice, depending on genetic background, also develop
ligneous conjunctivitis (119). These mice are predisposed to
severe thrombosis and deposit intravascular and extravascular
fibrin in many tissues (120,121). Growth is retarded, rectal
prolapse is frequent, and Plg - /- mice die prematurely, but
ovulation, embryonic development, and reproduction are normal
(122). Wound healing and tissue remodeling are impaired
(123,124), and response to an inflammatory stimulus is diminished
(125). After electric injury to the femoral artery, wound healing,
removal of necrotic debris, leukocyte infiltration, smooth muscle
cell immigration, and arterial neointima formation were greatly
delayed in Plg - /- mice (126), suggesting that PLG deficiency might
reduce the development of atherosclerosis. However, in Plg - /- mice
crossed with apolipoprotein Edeficient mice prone to develop early
atherosclerotic lesions, the spontaneous development of intimal
lesions was greatly accelerated (127). Plg - /- mice crossed with
fibrinogan
- /-
mice were restored to normality in many of these
cases, demonstrating the causative role of intravascular and

extravascular fibrin deposition (128,129).
Regulation of Expression
Plasma levels of PLG are generally rather stable, as discussed
further in the section, Balance of the System, but there is an
increase in the acute-phase response (130). Two sequence
elements of CTGGGA common to acute-phase reactant genes are at
position 76 to 81 and -553 to -558 (13). Three interleukin-6
(IL-6)responsive elements are at positions -830, -518 and +117
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in the 5-flanking region (131).

The PLG gene has a TATA-like sequence, TGTAA, at position -16
rather than a canonical TATA box. This sequence corresponds
exactly to the TATAA box of the homologous apo(a) gene, located
31 bp upstream from its transcription start site. Accordingly, the
TGTAA sequence is probably involved in the expression of the
TATA-less promoter in the PLG gene (131). Two main regions
conferring liver specificity of expression to the PLG gene were
found in the cis-acting element, AAAAATA, at -2194 to -2188 and
+48 to +61 (132). Several other putative regulatory transcription
elements are present in the 5-flanking region (reviewed in 131).
On the 3-flanking region, the location of the consensus
polyadenylation sequence AATAAA and of potential CAYTG signals
suggests the presence of three polyadenylation sites (13).
PLASMINOGEN ACTIVATORS
There are two physiologic PAs, tPA (tissue-type) and uPA
(urinary-type, UK), named after the original sources of purified
proteins. A further activation system, which is dependent on factor
XII, prekallikrein, and HMW kininogen, is known as the contact PA
pathway. There are also PAs from bacteria and the vampire bat,
which are included here because of their therapeutic use in
thrombolysis; they are also of mechanistic interest. The two human
proteases, tPA and uPA, are also implicated respectively in
neurobiology (133) and tumor biology (134). These aspects are not
considered further in this chapter, which focuses on PAs in
hemostasis.
TISSUE-TYPE PLASMINOGEN ACTIVATOR

tPA, a serine protease of 68 kDa (EC 3.4.21.68), also known as
vascular PA or extrinsic activator, is a glycoprotein of 527
residues. It exerts its effect primarily in the vascular system,
because it is produced and secreted by endothelial cells (135);
many other cells in culture also synthesize tPA. In normal plasma,
the antigen concentration of tPA is approximately 5 g perL, which
corresponds to approximately 70 pM (136). Most of the tPA is
present in a complex with its primary inhibitor, PAI-1 (137,138).
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The gene for tPA comprises 32.7 kb and is located on bands
p12-p11 on chromosome 8 (139), containing 14 exons (140) (Table
18-1). The mature protein exists in two forms of different length.
The N-terminus of Bowes melanoma cell tPA can be either glycine
or serine (141), but serine is nearly always the N-terminus of
recombinant tPA (142). In this chapter, the numbering is based on
the Ser-terminus of recombinant tPA because of the extensive
literature that exists on structure-function in this form of tPA. In
this numbering, glycine -3 probably represents the real N-terminus
of tPA.
The structure of tPA (Fig. 18-1) shows a finger and EGF domain,
and two kringle domains in the A chain, whereas the protease
domain is the B chain. The finger domain extends from residues 6
to 43 and is 34% identical to the first finger of bovine fibronectin.
It is involved in the binding of tPA to fibrin, as shown by mutants
lacking kringle domains, which still bound to fibrin (143).
Consistent with this, a degraded form of tPA that had lost the
N-terminal 12 kDa bound less well to fibrin than wild-type tPA
(144). The binding of the finger domain is independent of LBS, in
that it cannot be blocked by ACA (145). Structurally, the finger
domain is very similar to the seventh type 1 repeat of human
fibronectin (146).
The epidermal growth factor domain (residues 4492) is
structurally similar to other EGF structures, as shown by nuclear
magnetic resonance (NMR) (147). There is some evidence from
deletion mutagenesis that the EGF domain binds to the mannose
receptor and is involved in tPA clearance. Kringle 2 has affinity for
lysine, -amino acids such as ACA, and fibrin, whereas no
function has yet been ascribed to kringle 1 (143). The affinity for
the binding of ACA (a model for C-terminal lysine residues) and of
N-acetyllysine methyl ester (a model for intrachain lysine residues)
is approximately equal, suggesting that tPA does not prefer
C-terminal lysine residues (as PLG does) for binding. Intact tPA
and a variant consisting only of kringle 2 and the protease domains
were found to bind to the cyanogen bromide (CNBr) fibrinogen
fragment FCB-2, which also binds PLG and acts as a stimulator of
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tPA-catalyzed PLG activation. In both cases, binding was

completely inhibited by ACA, pointing to the involvement of LBS
in this interaction (143).
The structurefunction relations among kringle 2 and -amino
acids and lysine have been studied in detail, using
P.343
NMR (148), microcalorimetry (149), crystallography (150), and
site-directed mutagenesis (42,149,151). The crystal structure of
kringle 2 resembles that of PLG kringle 4; there are, however,
differences in the lysine-binding pocket. The core of kringle 2 is
formed by a hydrophobic cluster of three tryptophan residues in
positions 25, 63, and 74, surrounded by aromatic and hydrophobic
side chains that form, at the surface of the kringle, a hydrophobic
grove. Ligand binding appears to rely mostly on the integrity of
Trp63 and Trp74, and an aromatic residue at position 76, which is
normally Tyr (152). Mutation of the critical amino acids Lys33,
Asp55, Asp57, or Trp72 markedly diminished binding to
lysineSepharose, or interaction with ACA, or both (153).
The catalytic domain of tPA is typical of other serine proteases,
with the catalytic triad His322, Asp371, and Ser478. The 2.3-
crystal structure of the protease domain revealed strong structural
similarity with other trypsinlike serine proteases, thrombin in
particular (154). The active site cleft is shaped and narrowed by
four surface loops. The loop around Arg299 exhibits five additional
residues, Arg298-Arg-Ser-Pro-Gly302, compared with
chymotrypsin. It projects out of the molecular surface as a
-hairpin and is of fundamental importance for the interaction with
PAI-1 (3). The 60-loop around Arg327 is similar to but shorter than
the corresponding loop in thrombin. Further loops are found around
Ser381 and Gly465. The fully solvent-exposed hydrophobic region,
comprising amino acids 420 to 423 of tPA, which forms a surface
loop near one edge of the active site of tPA, constitutes an
important secondary site for the interaction of tPA with PLG in the
absence of fibrin.
tPA is secreted as a single-chain molecule (sctPA) but can easily be
converted to the two-chain form (tctPA) by plasmin, which cleaves
the Arg275Ile276 peptide bond. Surprisingly, the single-chain
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form is not a zymogen but a protease which, in the presence of

fibrin, is nearly as active as the two-chain form (155). The
nonzymogenicity of tPA has been explored in a series of
comparisons, with confirmatory mutagenesis studies (3,156). It
emerges that other zymogens have a triad Asp194, His40 and
Ser32 (chymotrypsin numbering), the His and Ser of which are
replaced in tPA by Phe305 and Ala292, respectively. The
constructed double mutant Phe305His and Ala292Ser was more
zymogenic (156).
Enzymatic Properties of Tissue-Type

Plasminogen Activator
tPA is unusually specific; its major substrate is PLG, in which it
cleaves the Arg561Val562 bond. It is an inefficient activator of
PLG in the absence of fibrin, but in its presence the activation of
PLG is greatly potentiated (157). Since Thorsen et al.'s original
observation that fibrin binds tPA but not urokinase (158), this
phenomenon has been much studied. Binding of sctPA and tPA is
roughly comparable, although the single-chain form may bind
slightly better (159). The K d for binding of tPA to fibrin clots in the
absence of PLG ranges from 140 nM to 400 nM (155,159,160). In
the presence of PLG, the affinity of tPA to fibrin increases
approximately 20-fold (K d 20 nM) (161). These observations are
explained by formation of a ternary complex comprising tPA, PLG,
and fibrin or binding of tPA to PLG, which, on binding to fibrin, has
taken on an open conformation. The isolated A chain of tPA was
indeed shown to bind to Glu- and mini-PLG with a K d of 100 nM
(162).
The kinetic parameters reported for the activation of PLG by tPA
show great variation. This is due to many potential experimental
variables, different tPA and PLG preparations, different substrate
concentrations, and, for studies using fibrin or fibrin derivatives,
the nature of the fibrin stimulator used. In the absence of fibrin,
K M values for the activation of GluPLG by tPA range from M to
slightly more than 100 M (155,163,164). In general, K M is three
to four times lower with tPA than with sctPA when the activation of
GluPLG is investigated in the absence of fibrin. This difference
disappears when fibrin is present and K M values typically are two
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orders of magnitude smaller, with only moderate increases of K c at .

Values for K M in the presence of fibrin range from 0.16M to 1.1
M PLG, and k c at values from 0.1 per second to 1.1 per second
(155,164). Several authors found nonlinear enzyme kinetics
(162,163,165), and there appear to be two phases in the activation
of GluPLG by tPA in the presence of fibrin, with an initial K M of
1.05 M and k c at 0.15 per second. Later in the process, as new
high-affinity binding sites for PLG and tPA are exposed in partially
digested fibrin (61,62,63,166,167), K M decreased to 0.07 M
whereas the k c at was unchanged (165). The important message
from these studies is that PLG is not activated by tPA except in the
presence of fibrin, because it is only in the presence of fibrin that
the K M for the reaction is consistent with the circulating
concentration of 2 M.
Distinct sites in the fibrin molecule have been identified as
accelerating PLG activation; notably they are not available for
binding in fibrinogen but become exposed in fibrin (168). The
D-region of fibrin binds both PLG and tPA in a region that
encompasses the sequence A148-160, and tPA alone, in a site
including 312-324. Both these associations are of low affinity and
in the circulation the A148-160 site would bind PLG, which is
present in much higher concentrations than tPA. Another binding in
the D-region encompasses 311-336 and 337-379, which are
linked by a disulfide bond (169). Antibodies have revealed that
there is a tPA binding site that includes 312-324 (170). Higher
affinity sites for binding tPA and PLG are in the C-terminus of the
chain, within the region A392-610 (171). The conformational
changes that occur on fibrinogen cleavage and fibrin assembly
(172) reveal new sites for binding of tPA and PLG, and for
enhancement of PLG activation.
Huge numbers of mutations have been engineered into tPA to
change its characteristics, with the aim of making it an even more
effective therapeutic agent. The most striking of these is the series
of mutations to make tPA resistant to PAI-1. Madison et al.
identified the importance of interactions between the positively
charged tPA sequence 298302 and the negatively charged PAI-1
sequence 350355 (173). Mutagenesis of Arg298, 299, and 304
resulted in a mutant that was inhibited 120,000 times less rapidly
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by PAI-1 than wild-type tPA (174). These observations led, in part,

to the development of the new tPA mutant tenecteplase (TNKtPA),
in which the sequence 296299 (Lys-His-Arg-Arg) is replaced by
four alanines (175). This variant also exhibits slower clearance, by
virtue of its changed glycosylation, and its enhancement of PLG
activation is more selective for fibrin above fibrinogen and the
fibrin fragment DDE (176).
Tissue-Type Plasminogen Activator

Receptors and Binding Proteins
A number of structurally unrelated components that bind tPA have
been described. Some have the potential to localize tPA on a
surface to which PLG also binds, and therefore to potentiate
activation. Many of the proteins in this group contain a C-terminal
lysyl group and therefore also are receptors for PLG, as already
discussed (69). This group includes 42-kDa annexin II (69,177),
45-kDa actin (178), heparan sulfate and chondroitin sulfatelike
proteoglycans (179), cytokeratin 8 and 18 (180), and tubulin
(181). Overexpression of the tPA receptor annexin II, as it occurs
in patients with promyelocytic leukemia exhibiting the t(15;17)
translocation in the
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leukemic cells, is associated with a hyperfibrinolytic state. The
abnormally high levels of annexin II bind PLG and tPA and
generate plasmin, with consumption of 2 -antiplasmin, leading to
bleeding (182). Interestingly, other studies show that such
patients also have abnormally high fibrinolytic activity that is
clearly due to uPA (183).
Several as yet poorly defined tPA receptors have been identified on
endothelial cells, and it is not clear yet whether some of these are
identical with each other (184). In addition to actin, human
umbilical vein endothelial cells (HUVEC) express two further
37-kDa and 45-kDa tPAbinding proteins (178), whereas another
study found a 20-kDa tPA binding protein, which was characterized
and purified (185). This protein did not interact with PLG, but
binding was inhibited by -amino acids, suggesting that the
lysine-binding site of tPA is involved, but the purified protein was
not recognized by antibodies to annexin II or -enolase. In the
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presence of this receptor, PLG activation by tPA was enhanced

90-fold. A receptor that interacts with the B chain of tPA and
potentiates PLG activation approximately 100-fold has been
described on vascular smooth muscle cells (186). It has now been
defined as type-II transmembrane protein p63 (CKAP4) (187).
Glycosylation of Tissue-Type Plasminogen

Activator
tPA has four potential glycosylation sites, of which three are
occupied in type I (Asn117, Asn184, and Asn448) and two in type
II tPA (Asn117 and Asn448), making it 3 kDa smaller (163).
Residue 117 is predominantly N-glycosylated with
oligomannose-type structures, whatever the source. Residues 184
and 448 are predominantly associated with complex-type
structures in recombinant tPA and in tPA isolated from fibroblast
cell lines, but with both complex- and oligomannose-type
structures when isolated from melanoma cells (188). Glycosylation
at residue 184 influences the biological properties of tPA. The type
II form has a higher affinity for lysine (and fibrin) and a higher
fibrinolytic activity (189). tPA, like uPA, has an O-linked fucose on
Thr61 in the EGF domain (190) and this affects binding to HepG2
cells and may be relevant to clearance (191).
Polymorphisms
The best-studied polymorphism is an Alu insertion/deletion
polymorphism. Most clinical studies including a large prospective
study (192), found no correlation with acute myocardial infarction
or stroke, but one casecontrol study found that homozygotes for
the insertion had twice as many cases of acute myocardial
infarction as homozygotes for the deletion (193). The homozygous
insertion polymorphism is also associated with a high rate of
release of tPA in response to stress (194). Further study of the
promoter identified additional polymorphisms, one of which
(-7351C> T) has been shown to be functional at the level of
transcription, with decreased release of tPA in individuals carrying
the T allele (195).
Synthesis, Release, and Clearance of
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Tissue-Type Plasminogen Activator

tPA is produced by many different cell types in culture, and its
synthesis can be upregulated by diverse agents. Endothelial cells
are a principal source and, in these cells, release rather than
synthesis is also important in controlling local tPA. Finally, tPA is
very rapidly cleared from the circulation. These three aspects are
now reviewed briefly.
Synthesis
tPA expression in cultured cells can be stimulated through multiple
intracellular signaling pathways. Vasoactive substances, such as
thrombin and histamine, increase tPA synthesis in HUVEC, acting
through their G-coupled receptors and the protein kinase C (PKC)
pathway (196,197,198). Steroid hormones (199,200) and retinoids
can increase synthesis of tPA (201,202). It is important to note
that not all endothelial cells synthesize tPA in vivo and that
expression is restricted to small vessels (203,204,205).
The tPA gene contains common transcription elements, with three
TATA boxes; a CAAT box is present at position -112 to -116. DNase
I protection analysis of the tPA gene promoter in human
endothelial- and phorbol-stimulated HeLa cells revealed several
protected regions. A cytotoxic factor/nephritic factor-1like
element is at -92 to -77; three Sp1 binding sites are at positions
-72 to -66, -48 to -39, and +60 to +74; a cyclic adenosine
monophosphateresponsive element CRE-like/tetradecanoyl
phorbolacetateresponsive element TRE-like element is between
-102 and -115; and three GC/GT boxes are between -43 and +68
(196,206,207). Several of these cis-acting elements have been
shown to be involved in constitutive and phorbol esterinduced
expression of tPA mRNA. Between -2,288 to -2,129 and -2,390 to
-2,289 are two further regulatory elements necessary for
constitutive expression of the tPA gene in Bowes melanoma cells
(208). Far upstream at -7.3 kb is located a functional retinoic acid
response element consisting of a direct repeat of the GGGTCA
motif spaced by five nucleotides (209). The region -7,145 to
-9,758 comprises a multihormone-responsive enhancer that is
activated by glucocorticoids, progesterone, androgens,
mineralocorticoids, and 1,25-dihydroxyvitamin D 3 but not by
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estrogens (199,200). A nuclear factor NF1 site, 600 bp upstream

from the transcription start site, acts as a repressor of tPA
expression and confers cell specificity to tPA expression (205).
Release
Acute release of tPA is established from early studies on venous
occlusion for 10 or 20 minutes, a procedure widely used in patients
(210,211,212). During venous stasis, at midway between systolic
and diastolic blood pressure, venous outflow from the occluded
segment decreases more than the (diminished) arterial inflow and
represents approximately one third of remaining arterial inflow
(210). Much of the locally produced tPA therefore remains in the
occluded limb and is not cleared. Young patients with a history of
recurrent venous thromboembolism exhibited an abnormal
response to venous occlusion and this could be attributed either to
subnormal tPA release or, more often, to elevated PAI-1
(137,211,212). In isolated cases with severe forms of von
Willebrand disease, a complete lack of response to venous stasis
and the infusion of 1-deamino-8-D-arginine vasopressin (DDAVP)
was found (213). These patients may have a functional or
structural defect in their endothelial cells. It should be noted,
however, that release of von Willebrand factor (VWF) and tPA
occur by different mechanisms (214). Lower PA activity is found in
leg veins than in proximal veins. It has been estimated that the
synthesis and release rate of tPA during forearm occlusion ranges
between 0.5 to 1.1 ng per mL of blood, but only 0.08 to 0.11 ng
per mL in an occluded leg (210). Increased hydrostatic pressure in
the leg veins may be responsible for the diminished production of
tPA. Applying a 20-minute venous occlusion test, patients who
were immobilized were found to release considerably more tPA
from leg veins after 12 to 33 days of recumbency than at the
beginning of immobilization (215). The low content of tPA in
human calf veins therefore may be one etiologic factor for
development of deep venous thrombosis.
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There is evidence that continuous release of tPA from the
vasculature can diminish a thrombotic response. The human tPA
gene was cloned into an adenoviral vector under the control of the
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Rous sarcoma virus (RSV) promoter and the construct was tested
in an in vivo model of arterial thrombosis. All animals developed
obstructive thrombosis except those treated with the viral vector;
this was actually more effective than tPA therapeutic regime (216).
This echoes two independent studies in the 1980s of patients who
had consistently raised tPA activity as the cause of a hereditary
lifelong hemorrhagic disorder (217,218). One of these (217) had a
total absence of fibrin deposits or thrombi, despite widespread
arterial disease; he died of cerebral bleeding. The underlying cause
was not a deficiency of PAI-1 or any other known inhibitor. No
further cases have been reported, despite an intensive search in
many laboratories.
The mechanism of release has been studied in cultured endothelial
cells, from which much of the tPA is released continuously, but
there is an additional regulated pathway, where tPA is stored in
specialized storage vesicles (219,220,221,222). Extracellular
stimuli then trigger the release of stored tPA to the cell surface.
The amount of tPA stored in storage vesicles in various cells, such
as endothelial, neuroendocrine, and adrenal chromaffin cells, is
large (220,222). In rats, the tissue stores of tPA were calculated
to be sufficient to maintain a steady-state plasma level of tPA for 2
days in the absence of protein synthesis (223). Compounds that
triggered release within a few minutes include bradykinin,
histamine, eledoisin, acetylcholine, -adrenergic agents,
platelet-activating factor, endothelin, calcium ionophore A-23187,
and acidosis (224,225). These compounds induce calcium influx
into the endothelial cell and activate G-proteincoupled receptors
(226). Some interventions that cause elevated tPA act through
decreased clearance, as discussed in the next section. Among
these are exercise and -adrenergic agents (227). In all situations
in which epinephrine levels are increased, such as stress, anxiety,
and exercise, tPA antigen levels increase (138,228).
DDAVP was widely used in the 1980s to study the release potential
for tPA in patients with idiopathic or recurrent thrombosis
(215,229,230,231). Earlier observations had suggested that DDAVP
acts through a release of a pituitary PAreleasing hormone (229).
Comparison of the effects of DDAVP and sodium nitroprusside
showed that nitroprusside produced an even greater increase of
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forearm blood flow but no increase of tPA, but that DDAVP indeed
stimulated tPA release from the vascular bed (232). The increase
of circulating tPA levels after intraarterial infusion of acetylcholine
and methacholine was shown to be mediated by muscarinic
receptors (232,233,234). Bradykinin and substance P both induced
tPA release after being infused into the forearm of volunteers
(235,236), and the releasable pool of tPA, rather than the
availability of PAI-1 to inhibit it, was the key factor (237).
Clearance
Free tPA and tPA in complex with inhibitors are rapidly removed
from the circulation and bound to receptors on endothelial cells
and hepatocytes. In normal individuals, the t 1/2 of tPA is
approximately 3 minutes (238,239). Studies on isolated rat
hepatocytes suggested that the complex was cleared more rapidly
than free tPA (240) but, in vivo in humans, clearance of tPA was
calculated to be faster in subjects with low PAI-1 (3.5 minutes)
versus high PAI-1 (5.3 minutes; P = 0.006), consistent with t 1/ 2 of
2.4 minutes for free tPA and t 1/ 2 of 5.0 minutes for tPA/PAI-1
complexes (P = 0.006) (241). Because of its clearance by the liver
and the dependence of clearance on liver blood flow
(242,243,244), the t 1/ 2 may be considerably prolonged in patients
with hepatic cirrhosis (239,245).
Early studies showed that the uptake of tPA by liver endothelial
and by Kupffer cells was inhibited by ovalbumin, leading to the
identification of the mannose receptor as a major tPA receptor
(246). Binding of ligands to the mannose receptor is pH sensitive
and dependent on Ca 2+ ions. Binding is inhibited by
mannose-albumin, mannan, D-mannose, and L-fucose, and
particularly by cluster mannosides of the composition M 6 L 5
(246,247,248). tPA has a high affinity (K d 1 to 4 nM) for the
mannose receptor compared to other glycoproteins, the K d for
most being 60 to 600 nM (248). In mice, the administration of
estradiol leads to increased expression of the mannose receptor
and increased clearance of tPA (249). Mutants of tPA have been
engineered for slower clearance by replacement of Asn117, which
is normally glycosylated, with Gln. Such mutants, including
TNKtPA, are cleared more slowly than wild-type tPA. It is not
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known whether the fucose residue at Thr61 in the EGF domain also
binds to the mannose receptor or to the previously described
fucose receptor (250).
The other major pathway for tPA clearance involves the
LRP/ 2 -macroglobulin receptor (251,252). The 600-kDa LRP, the
largest plasma membrane protein described, comprises 4,525
residues. It mediates the clearance of apolipoprotein Eenriched
chylomicron remnants, toxins, cytokines, complexes of
2 -macroglobulin with proteases from all subclasses, and free tPA
and tPA/PAI-1 and uPA/PAI-1 complexes. Complex formation of tPA
with PAI-1 increases the rate of clearance by LRP by at least one
order of magnitude compared with that of free tPA (253). The
binding sites for tPA/PAI-1 and for uPA/PAI-1 complexes are
situated in the second complement-type domain cluster of LRP
(254,255,256). The receptor-associated protein (RAP) inhibits
endocytosis of most ligands to LRP (251,254,257).
Other LRP-like multiligand receptors, such as glycoprotein 330 and
the 130-kDa very low density lipoprotein (VLDL) receptor, also can
mediate the clearance of free and PAI-1 complexed tPA
(258,259,260). For efficient uptake and clearance of several
ligands, LRP works in cooperative fashion with coreceptors, for
instance uPAR for the LRP-mediated degradation of uPA/PAI-1
complexes (261,262). Some monocytelike cell lines that expressed
LRP on the cell surface were unable to degrade tPA, leading to the
idea that a coreceptor was necessary for efficient degradation of
tPA by LRP (263).
Deficiency
Congenital deficiencies of tPA or uPA have not been described in
humans and were assumed to constitute a lethal condition, until
these genes were successfully deleted in mice (264).
Unexpectedly, mice with a disrupted tPA gene developed normally.
Microscopic examination revealed mild glomerulonephritis. In a
plasma clot lysis assay, fibrinolytic activity was lower than in
wild-type animals. After endotoxin-induced thrombogenic
stimulation, tPA -/ - mice had more extended thrombotic lesions than
tPA + /+ mice. The abnormalities found were rather mild, but mice
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with a double deficiency of tPA and uPA had severe spontaneous

thrombosis and exhibited impaired health, reduced survival, and
diminished fertility (264,265). It can be concluded that, in mice,
there is some redundancy of biological functions with respect to
PAs. On the other hand, it is obvious that a complete knockout of
the fibrinolytic activators leads to severe disease. It is not known
to what extent these results apply to human pathophysiology. The
double knockout has a similar phenotype to the PLG knockout,
consistent with tPA and uPA being the major mammalian activators
of PLG. These mice have had a profound impact on understanding
other functions of tPA, in areas as diverse as reproduction (266),
neuronal development (133,267) and learning (133,268).
P.346
URINARY-TYPE PLASMINOGEN ACTIVATOR

The urinary-type PA (EC 3.4.2.73; urokinase, uPA) is found in
urine at 40 to 80 g per L (269) and is synthesized by several cell
types, particularly cells with a fibroblastlike morphology, but also
by epithelial cells (270) and monocytes and macrophages
(271,272). Stimulation by endotoxin or tumor necrosis factor
causes cultured endothelial cells to produce uPA, and synthesis has
also been demonstrated by the human endothelium in vivo
(273,274). uPA activates PLG by the cleavage of Arg561Val562,
as does tPA, but importantly can do so in the absence of fibrin.
This characteristic has generated the view that tPA functions in
fibrinolysis in the vasculature, whereas uPA's primary role is in
processes such as degradation of extracellular matrix and cell
migration, with consequences for wound healing, inflammation,
embryogenesis, and invasion of tumor cells and metastasis
(275,276). These clear functions of the uPA system do not argue
against its importance in fibrin degradation, functions that have
emerged from both human and animal studies, as discussed in
subsequent text.
The complete primary amino acid sequence of uPA was determined
in 1982 (277). The cDNA and the genomic DNA of uPA have been
isolated and their nucleotide sequences established (278,279). The
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human uPA gene is 6.4 kb and on chromosome 10q24 (280). It

contains 11 exons, and the intronexon organization of the uPA
gene closely resembles that of the tPA gene. uPA is produced as a
54-kDa, single-chain glycoprotein, 411 residues long and
containing three domains: an epidermal growth factor domain, a
kringle, and a protease domain (Fig. 18-1). The EGF domain
interacts with the uPAR found on many cells, as discussed in
subsequent text. The kringle domain has no affinity for fibrin, and
its function has been unclear; a recent report shows that it
stabilizes the interaction of uPA with uPAR (281). The protease
domain has the residues His204, Asp255, and Ser356 as the
catalytic triad, and sequence identity with tPA is approximately
40% (140). uPA is normally glycosylated at Asn302, whereas a
covalently attached single monosaccharide, fucose to Thr18 (282),
appears to affect the mitogenicity of uPA (283). There are two
phosphorylation sites on Ser138 and Ser303. The phosphorylated
form diminishes uPA's interaction with cells and PAI-1 (284).
Activation of single-chain urinary plasminogen activator (scuPA,
also called proUK) to the active enzyme, uPA (UK), is by cleavage
of Lys158Ile159 (285). Many enzymes can cleave this bond but
the most important to hemostasis are plasmin (286), factor XIIa,
and kallikrein (287). The activation of scuPA in PLG-deficient mice
was characterized as being due to kallikrein (288). Cleavage close
to this site results in inactive uPA. Thrombin cleaves
Arg156Phe157 in scuPA (287,289,290) and the inactive product
can be activated by release of the N-terminal dipeptide of its B
chain by cathepsin C or, albeit slowly, by plasmin (285,291).
Granulocyte elastase and cathepsin G cleave the Ile159Ile160
peptide bond; again, the resulting two-chain urinary-type
plasminogen activator (tcuPA) is inactive (292).
uPA was originally characterized as having two active forms:
HMW-UK and LMW-UK (293). These are, respectively, full-length
active uPA and a processed form, cleaved at Lys135Lys136 to
yield the amino-terminal fragment (ATF), which interacts with
uPAR, leaving 21 residues of the A chain and an intact B chain.
Cleavage within the uPAR binding region (e.g., of the Lys23Tyr24
bond) abolishes uPA receptor binding and therefore modulates cell
surface uPA activity (294,295).
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The conformation of uPA and of its domains has been studied by

NMR (296,297). Essentially, the domains are similar to those in
other serine proteases (Fig. 18-1) with few considerable
differences. The crystal structure of the catalytic domain of
recombinant, nonglycosylated human uPA has been solved at a
resolution of 2.5 (298). At six positions, insertions of extra
residues in loop regions create unique surface areas. The
interaction of Lys300 with Asp335 in the flexible loop region
297313 stabilizes the conformation of scuPA (299). The positively
charged residues 179184 in a surface loop interact with PAI-1
(297). A number of modified uPA molecules have been crystallized
and used as a basis for drug design (300,301).
Enzymatic Properties of Urinary-Type

Plasminogen Activator and Single-Chain
Urinary Plasminogen Activator
The question of whether scuPA is a true zymogen was hotly
debated for several years, a controversy that was fueled by the
problem of trace plasmin or uPA contamination, with reciprocal
activation of scuPA and PLG (302). The issue has been resolved by
studies on mutant forms of the proteins and by examining scuPA in
the absence of PLG. For instance, the Lys158Glu mutant of scuPA,
which cannot be cleaved by plasmin into uPA, still converted PLG
to plasmin (303). The emerging consensus is that scuPA has
approximately 0.4% of the activity of uPA (304,305,306,307).
Therefore, scuPA lies on the spectrum between PLG, a true
zymogen, and tPA, which is almost fully active in its single-chain
form. Lys300 and Asp355 are key structural elements of scuPA's
enzymatic activity. Lys300, situated in the flexible loop region
297313, forms a weak interaction with Asp355 adjacent to the
active site Ser356 and pulls Ser356 close to the position found in a
fully active protease. Site-directed mutagenesis of Lys300 to Ala
resulted in a 40-fold lower activity than in wild-type scuPA (308).
Likewise, a mutation of Asp355 to Asn resulted in a 270-fold
reduction of scuPA activity (299). When the flexibility of the
297313 loop was enhanced, intrinsic catalytic activity was
reduced. By contrast, when the loop was made less flexible,
intrinsic catalytic activity increased (309).
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Urinary-Type Plasminogen Activator

Receptor
A cellular receptor for uPA (uPAR; CD87) was first demonstrated on
freshly collected human monocytes (310) but is expressed on
several cell types. The gene for uPAR (gene code PLAUR) is on
chromosome 19 and consists of seven exons and six introns
extending over 23 kb (311,312). Constitutive expression of the
uPAR gene is dependent on the -141 to -61 region (313), and
expression can be increased by tumor promoters, growth factors,
cytokines, and hormones. Interestingly, it can be upregulated by
one of the plasmin reductases, phosphoglycerate kinase (75,314).
The 1.4-kb mRNA encodes a signal peptide of 22 and a protein of
313 residues, with a glycosylphosphatidyl inositol (GPI) anchor, by
which the receptor is attached to cell surface phospholipids (see
Fig. 18-6). The receptor consists of three homologous domains,
and high-affinity binding of uPA to the receptor is mediated by
region 1, but region 2 and 3 enhance this binding (315,316,317).
scuPA and uPA bind to uPAR receptor by the ATF (315). The
binding is of high affinity (K d 10 - 9 to 10 -11 depending on the cell
type) (318) and can localize and enhance uPAmediated PLG
activation on cell surfaces (71). The mechanism is a colocalization
of scuPA and PLG; uPAR does not play a catalytic role
P.347
(319). Binding can also elicit a number of intracellular signaling
responses, acting through the extracellular regulated kinase
(ERK)/mitogen activated protein kinase (MAPK) pathway (320).
Because uPAR is not a transmembrane protein, other components
must participate to transmit a signal to the intracellular
compartment. Several proteins are known to bind uPAR, including
vitronectin and integrins in complex with caveolin (321). Such
interactions regulate cell adhesion and are discussed further in the
context of PAI-1 in Chapter 19. On some cells, the complex
uPA/uPAR is associated with Endo 180, also known as
uPAR-associated protein (uPARAP) (322), and is involved in
collagen IV internalization (323). Receptor-bound uPA still is
susceptible to inactivation by PAI-1, but the inhibition is somewhat
less efficient than in free solution (319). The uPA/PAI-1
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enzyme/inhibitor complexes are internalized, and the uPAR is

recycled to the surface. This process requires the cooperation of
LRP (262,324).
FIGURE 18-6. A simplified diagram of the domain structure of

urinary-type plasminogen activator receptor (uPAR) and its
attachment to the cell-surface glycosylphosphatidylinositol
(GPI) membrane anchor. The N-terminal portion of the uPAR is
composed of three domains. Potential glycosylation sites are
indicated by . The C-terminal amino acids are depicted by
squares. The linkage between the protein and the glycolipid is
through a phosphoethanolamine that forms an amide bond with
the a-carboxyl group of the protein and a phosphodiester bond
with the glycan portion of the glycolipid. (PI-PLC,
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phosphatidylinositol-specific phospholipase C.) (From Ploug M,

Behrendt N, Lber D, et al. Protein structure and membrane
anchorage of the cellular receptor for urokinase-type
plasminogen activator. Semin Thromb Hemost
Urinary-Type Plasminogen Activator in the

Circulation
scuPA is found in plasma in concentrations of 2 to 4 ng per mL
(325,326), which seem to remain fairly stable. In contrast with tPA
and PAI-1, there is little circadian fluctuation in plasma uPA (327).
Venous occlusion did not increase uPA antigen once values were
corrected for the increase of hematocrit (136), but there are
reports of increases of uPA antigen after venous stasis (326),
DDAVP infusion (328), and strenuous physical exercise (329). The
activation of scuPA in plasma has not been studied in any detail,
and uPA activity is not normally detected in plasma, but both
leukocyte-associated and free scuPA are elevated in leukemia
(183) and other disorders, including liver disease (330). Plasma
uPA is cleared quickly, with a t 1/ 2 of approximately 7 minutes, in a
manner that depends on hepatic blood flow (331). The LRP system
binds and internalizes scuPA and uPA-PAI-1 complexes
(251,261,262). The asialoglycoprotein receptor, located exclusively
on parenchymal liver cells, also removes nonsialated uPA from the
circulation (331).
The activity of scuPA is increased by two orders of magnitude when
it is bound to uPAR on the surface of monocytes (332,333), which
is now understood to reflect colocalization with PLG rather than a
direct catalytic effect of uPAR (319). This local activity is relevant
to thrombus stability, into which monocytes migrate (334) and
express fibrinolytic activity (335). In freshly formed model
thrombi, there is considerable spontaneous generation of
fibrinolytic activity, most of which can be inhibited by antibodies to
uPA (336). Polymorphonuclear cells, primarily neutrophils,
generate this local uPA activity, apparently on uPAR (337); plasma
1 -antitrypsin is crucial in protecting the activity from neutrophil
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elastase (338). The integrin M 2 is important in the generation of

such local activity (339). When scuPA is present on uPAR, for
instance on monocytes, it can associate with PAI-1 and other
serpins, not in a classic covalent complex but in a reversible
manner (340). This has given rise to the ideas that receptor-bound
scuPA initiates proteolytic activity
P.348
and that conversion to uPA is primarily a way of achieving
inhibition by PAI-1 and other inhibitors and thereby regulating the
activity (341). Platelets also contain uPAR (342) and a further
70-kDa protein, distinct from uPAR bound uPA in resting platelets
with a K d of 43 nM (343). On other cells, too, cell-associated PLG
activation by uPA is not mediated exclusively by uPAR and other
receptors may come into play (344).
Although it does not bind to fibrin, scuPA has some fibrin
specificity in that it degrades fibrin with less systemic degradation
of fibrinogen and other plasma proteins than does uPA. It therefore
is a more potent thrombolytic agent than uPA (306,345). The
explanations for this include the different but complementary
mechanisms by which tPA and scuPA induce fibrinolysis (346). tPA
activates GluPLG, which is bound to an internal lysine, probably
A Lys157, which is exposed in nondegraded fibrin. As soon as
some fibrin is digested by plasmin, new C-terminal lysyl residues
are generated, increasing the binding of GluPLG (61,63) to fibrin.
scuPA binds with high affinity to PLG (K d approximately 65 nM) and
appears to activate selectively GluPLG, which is bound to
C-terminal lysines in partially degraded fibrin (347,348). The
generation of small amounts of fibrin-bound plasmin also converts
scuPA to uPA, explaining the typical lag phase observed for
fibrinolysis by scuPA. Efficient scuPA-mediated lysis occurs after
some fibrin has been degraded by tPA. Therefore, sequential
administration of tPA followed by scuPA resulted in increased
thrombolysis in a rabbit jugular vein thrombosis model; the
reverse order was less effective (349).
During coagulation, there is activation of the contact system
generating kallikrein also. Because kallikrein is an efficient
activator of scuPA (287,350), it is likely that scuPA is activated
wherever activation of the contact phase of coagulation has
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occurred. This process also takes place in the absence of PLG. In a

plasma milieu, clot lysis by scuPA is enhanced approximately
20-fold after dextran sulphate mediated activation of the contact
phase (350). It is not entirely clear if this in vitro generated
activity of kallikrein fully explains the contact system of PLG
activation (351). Part of the dextran sulfate mediated activation
may also be due to factor XII activity. Factor XIIa is a poor
activator of PLG but is present at high concentrations in plasma
(352).
Regulation of Expression
The expression of scuPA has been reviewed in detail (353). The
gene is regulated by several elements, with a typical TATA box 25
bp upstream of the transcription initiation site. A region of 500 bp
contains potential binding sites for the transcription factor Sp1.
Two putative AP-2 binding sites close to the transcription initiation
site are responsible for protein kinase A dependent induction of
uPA expression. Two NF-kB elements occur at -1,580 and -1,865
bp. The latter site acts as a repressor, as do two further negative
regulatory sites, one situated between -1,824 and -1,572 bp, the
other involving an enhancer-dependent cell-specific silencer region
between -660 and -536 bp. The promoter is strongly enhanced by
a region approximately 2,000 bp upstream of the transcription
initiation site. This enhancer consists of an upstream PEA/AP-1A
site and a downstream AP-1B site. All three sites are required for
induction of uPA gene transcription by a variety of extracellular
stimuli, such as phorbol ester, EGF, okadaic acid, and cytoskeleton
disruption (354). Synergism between PEA/AP-1 and AP-1B depends
on the integrity of an intervening cooperation mediator site that
contains several sequences for binding of urokinase enhancer
factors (355,356). In the 3 untranslated region UTR stretching
from bp 1,368 to 2,260 of the human uPA gene, fragments were
identified that exerted a positive or negative influence on chimeric
reporter genes. Fragments 1,999 to 2,190 behaved like
transcriptional enhancers, whereas fragments 1,532 to 1,723 had
an inhibitory effect on the expression of uPA (357). At least three
elements were found in the 5 UTR that confer instability to the
uPA mRNA (358).
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Urinary-Type Plasminogen
Activator/Urinary-Type Plasminogen
Activator Receptor Deficiency
There are no known deficiencies of uPA or uPAR in humans, but
mice with these disrupted genes reveal a number of interesting
insights. uPA -/ - mice, like tPA - / - mice, developed some fibrin
deposits but did not spontaneously clot; challenge with endotoxin
led to more venous thrombosis than in uPA + / + mice (264,265).
Lysis of plasma clots was normal (264), but monocyte-dependent
lysis was greatly decreased by the absence of uPA (359). This is
consistent with the earlier findings that fibrin resolution was
affected in mice deficient in uPA but not by deficiency in tPA or
uPAR (360). Much lower plasmin generation, and hence matrix
metalloproteinase activation, was observed in mice with combined
apoE and uPA deficiency, so that they were protected from the
atherosclerotic lesions that were induced in apoE - /- mice (361).
Wound healing in uPA - /- mice is greatly disturbed and, after
injury-induced depletion of vascular smooth muscle cells,
repopulation of the injured area with smooth muscle cells was slow
in uPA - / - mice (362). Perhaps surprisingly, the healing process
does not involve binding to uPAR (363), and there are several
other reports on uPAR-independent but uPA-dependent phenomena
(364) as well as some in which both uPA and uPAR deficiency
produce related effects (365). Some aspects of uPAR biology, such
as the delayed clearance of platelets in uPAR - /- mice (366),
macrophage infiltration into the vessel wall (367), and cancer cell
growth and invasion (368), are independent of uPA. Studies on
mice are helping to unravel which effects of uPA and uPAR operate
independently of each other and which effects require their
common functions.
OTHER PLASMINOGEN ACTIVATORS

Streptokinase
In 1933, it was shown that hemolytic streptococci produced an
extracellular protein that could induce lysis of human plasma clots
(369), and it has been a useful thrombolytic drug over several
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decades. Streptokinase and other bacterial activators have

presumably evolved for the purpose of invasion of the mammalian
host (370). SK is not an enzyme; rather, it acts by forming a 1:1
complex with PLG, which undergoes a change of conformation to
expose an active site in the altered PLG molecule (371). The
modified PLG in the equimolar complex (SK-PLG') autocatalytically
converts to plasmin, and the activated complex is a potent PA
(372). It retains the fibrin binding of the PLG moiety with
consequent relative protection from inhibition by 2 -antiplasmin
(373). The C-terminus of SK regulates the PLG binding and
activation, and mutational studies have revealed the essential
features (374).
SK is a 47-kDa protein, containing no cysteines. NMR and
crystallographic studies revealed a three-domain structure
(375,376). The binding site for a kringle in PLG is located at the
tip of a fully exposed hairpin loop in the -domain of SK
(377,378). The physicochemical properties of SK and the methods
for its assay are reviewed (379); a new reference method that
allows direct comparison with other PA has been developed (380).
P.349
SK is highly antigenic. Most apparently healthy persons have
anti-SK antibodies in their blood, owing presumably to previous
infections with -hemolytic streptococci, and anti-SK titers may
rise several hundredfold after administration to humans (381,382).
After intravenous injection of SK into humans, dogs, or mice, SK is
cleared from the circulation with a t 1/2 of 15 to 30 minutes (383).
Clearance mostly takes place in the liver, primarily by the liver
2 -macroglobulin receptor (383), now known to be identical to LRP
(251).
Anisoylated PlasminogenStreptokinase
Activator Complex
Plasmin generated in whole blood or plasma is immediately
inactivated, but plasmin bound via its high-affinity LBS to fibrin is
inactivated at a much lower rate by 2 -antiplasmin. In the search
for more efficient thrombolytic agents, researchers acylated the
active site center of plasmin and of the SKPlg' activator complex,
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in which a conformational change also exposes the active

plasmin(ogen) site. Coupling of p-anisoyl to the active site serine
of the PLG portion of SKPlg' led to the development of the
anisoylated PLGSK activator complex (APSAC, Eminase). Its
catalytic site is blocked, but its affinity to fibrin via the LBS is
preserved. APSAC deacylates with a t 1/ 2 of approximately 40
minutes at 37C (384). As it binds to fibrin, it is feasible to
administer this drug in the form of a bolus injection. Evaluation in
experimental animal models showed that APSAC had better clot
specificity than SK, in that it caused less fibrinogenolysis and
consumption of PLG and 2 -antiplasmin and had a lesser
hypotensive effect in vivo (385). Clinical studies showed that
APSAC is an effective thrombolytic agent, but the systemic effects
are comparable with SK.
Staphylokinase
Staphylokinase (SAK) a protein with an M r of 15,000, produced by
Staphylococcus aureus, has been known for more than 40 years to
have thrombolytic properties (386,387). As recombinant
techniques made production possible in larger quantities, there
was renewed interest in its thrombolytic efficacy (388). Like SK,
SAK is not an enzyme but forms a stable stoichiometric complex
with PLG (372). In the absence of fibrin, it does not activate PLG.
Trace amounts of plasmin, such as may be found on the surface of
a thrombus, convert the SAKPLG complex to SAKplasmin
complex that is a highly fibrin-specific PA. The complex binds to
fibrin via the LBS of PLG, and the bound activator complex is
somewhat protected from inactivation by 2 -antiplasmin. The
molecule is quite antigenic, and extensive mutagenesis has been
undertaken to decrease this property (389). Coupling of SAK
mutants to polyethylene glycol of M r 20,000 was shown to increase
the t 1/ 2 of the complex severalfold, which would permit single
bolus thrombolytic therapy in humans (390). Several clinical
studies with SAK have been reported. In peripheral arterial
occlusions and in patients with myocardial infarction, SAK
compared favorably with tPA (391,392).
Vampire Bat Plasminogen Activator
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Many hematophagous animals, such as leeches, hookworms,

mosquitoes, and the vampire bat, produce substances that
interfere with the clotting or fibrinolysis mechanisms. In the
1960s, a potent PA was found in the saliva of the common vampire
Desmodus rotundus (393). The bat lives exclusively on blood and
uses the enzyme in its saliva to keep blood in a fluid state. The D.
rotundus salivary plasminogen activator (DSPA) was cloned
independently by two research groups and shows more than 70%
identity with human tPA (394,395). Three major forms were
identified: DSPA-, which contains a finger, EGF, kringle, and
protease domain; DSPA-, which lacks the finger domain; and
DSPA- which has just a kringle and a protease domain.
DSPA- comprises 441 residues and, like SAK, has higher fibrin
specificity than tPA (396). In the absence of fibrin, hardly any PLG
activation occurs (397,398). PLG activation by DSPA is barely
stimulated by D-dimer or the DDE complex, whereas these fibrin
fragments strongly enhance tPA activity (399). The crystal
structure of the catalytic domain of DSPA-
has been solved at
2.9 resolution (400) and shows strong similarity to tPA, which is

also active in the single-chain form. Experiments in animals
revealed that the thrombolytic potential of DSPA was at least equal
to, and the clearance rate approximately 10 times slower than,
that of tPA (401,402,403). The hope that high fibrin specificity
would result in lesser bleeding at comparable thrombolytic
potential was not fully realized (401), but this agent has several
promising features, long clearance time, and fibrin specificity,
which permits bolus administration and may be useful in stroke as
well as acute myocardial infarction (404).
INHIBITORS OF THE FIBRINOLYTIC

SYSTEM
Several inhibitors control and modulate the expression of
fibrinolytic activity. These act at the PLG activation step or on
plasmin (see Fig. 18-7), and they prevent excessive systemic
fibrinolytic activity. The principal inhibitor of tPA and uPA is PAI-1
(see Chapter 19), whereas 2 -antiplasmin inhibits plasmin. Both
are members of the serpin family, which also includes antithrombin
and heparin cofactor (see Chapter 13). The serpins all function in
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the same way: They present to the target protease a reactive

center loop, the P 1 -P 1 of which mimics a substrate. A stable
inactive 1:1 complex is formed between protease and serpin,
lowering the effective protease concentration. Dissociation of the
covalent complex, which occurs at different rates for different
proteaseserpin pairs, results in a cleaved form of the serpin,
which lacks the short peptide from P 1 to the original C- terminus.
The serpins have marked specificity for particular proteases, but
the specificity is not absolute, so that other inhibitors present at
high concentration provide a back-up inhibitory system.
2 -Antiplasmin
2 -Antiplasmin, also called plasmin inhibitor, is the primary
fast-acting inhibitor of plasmin (405). It is a single-chain molecule
of M r of 70,000, comprising 452 residues, the additional mass
accounted for by approximately 13% carbohydrates on four
potential asparagine glycosylation sites. The plasma concentration
of 2 -antiplasmin is 70 mg per L (i.e., 1 M), approximately half
the concentration of PLG on a molar basis. It is synthesized in the
liver and consequently decreased in patients with advanced
impairment of hepatic function. The t 1/2 of the native inhibitor is
approximately 3 days, whereas the plasmin/ 2 -antiplasmin complex
is cleared with a t 1/ 2 of approximately 0.5 days (406). The cDNA
has been cloned (407), the organization of the gene determined
(408), and the amino acid sequence derived. The gene for
2 -antiplasmin (gene symbol PLI) has been localized to
chromosome 17p13 (409). The inhibitory activity of 2 -antiplasmin
resides in its reactive center loop, in which the P 1 -P 1 is ArgMet
(see Fig. 18-8); as with
P.350
other serpins, mutation of Arg to Ala destroys inhibitory function
(410). The rate of interaction of wild-type 2 -antiplasmin with
plasmin is very fast, with a rate constant of 4 10 7 per M per
second (411), comparable with the interaction of tPA and PAI-1.
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FIGURE 18-7. Activators and inhibitors of the blood

fibrinolytic system. scuPA, single-chain urinary plasminogen
activator; sctPA, single-chain tissue plasminogen activator;
HMWK, high-molecular-weight kininogen; z -AP
2 -antiplasmin; 2 M 2 -macroglobulin; C1-INH, complement C1
esterase inhibitor; HRG, histidine-rich glycoprotein; PAI-1,
plasminogen activator inhibitor 1.
Plasma 2 -antiplasmin can occur in four different forms, depending

on limited proteolysis at N- and C-termini. This processing appears
not to affect the inhibitory capacity of 2 -antiplasmin which resides
in the core of the protein. The N-terminus of 2 -antiplasmin was
originally identified as Asn (407), but it was later recognized that
there is also a proform with an additional 12 residues at the
N-terminus (412). Plasma 2 -antiplasmin was shown to have about
equal amounts of the two forms (413), whereas HepG2 cells
produced the Met-form, which, after addition to plasma, was
cleaved to the Asn form (414). An enzyme that removes the 12
residue N-terminal fragment has been purified and characterized
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(415). The importance of the N-terminal processing lies in the

accessibility of Gln (at position 2 in the processed form); the
proform terminus blocks access to the cross-linking site (412).
Gln2 in 2 -antiplasmin is cross-linked by factor XIIIa to the A
chain of fibrinogen or fibrin, at Lys303 (416,417). Fibrin with
bound 2 -antiplasmin is markedly more resistant to lysis than
fibrin alone, and this observation was the basis of discovering the
first deficiency of 2 -antiplasmin (416). The cross-linking site can
be saturated, as elegantly shown by studies with wild-type
2 -antiplasmin and an active site Arg to Ala mutant (418).
Antibodies specific for cross-linked 2 AP greatly stimulated lysis
of fibrin (419).
Other studies focused on the C-terminus of 2 -antiplasmin which is
extended relative to other serpins by approximately 50 residues
(407). Two forms are detectable in normal human plasma (420).
The native, full-length form binds PLG, whereas a processed form
retains inhibitory capacity but cannot bind PLG (421). The two
forms were distinguished by two- dimensional
immunoelectrophoresis, with PLG incorporated in the gel in the
first dimension (422). The ratio of the PLG
P.351
binding to nonbinding form remains relatively constant in plasma
samples, at approximately 65:35, even in patients with advanced
liver cirrhosis (330).
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FIGURE 18-8. Interaction of 2 -antiplasmin with plasmin and

with the chain of fibrinogen. For Lysplasmin, only the
high-affinity lysine binding site (LBS) 1 is indicated. Hdenotes the N-termini and -OH the C-termini of the three
proteins depicted. Lys residues in the C-terminus of
2 -antiplasmin interacts with the LBS 1 of plasmin, and the
N-terminal Gln2 is covalently cross-linked to Lys303 in the
chain of fibrin by factor XIIIa. tPA, tissue-type plasminogen
activator; UK, urokinase. (From Bachmann F. Fibrinolysis. In:
Verstraete M Vermylen J, Lijnen HR, et al., eds. Thrombosis
and haemostasis. Leuven: Leuven University Press, 1987:227,
with permission.)
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The ability of 2 -antiplasmin to bind PLG interferes with the

fibrin-PLG interaction, because both involve LBS-1, as illustrated in
Figure 18-8. It is also crucial to the fate of plasmin formed on the
fibrin surface, to which both tPA and PLG bind by virtue of their
lysine-binding sites. Plasmin formed on fibrin is therefore relatively
protected from the action of 2 -antiplasmin (423). This concept
arose from experiments using lysine analogues, where 2 AP was
shown to be approximately 100 times less effective toward plasmin
in the presence of lysine analogues (424). The exact Lys residues
responsible for binding the C-terminal region of 2 -antiplasmin to
LBS-1 in PLG are not yet defined. Binding of recombinant
C-terminal peptide (55 residues) showed that binding to isolated
kringles was most affected by mutation of Lys452 but that other
internal Lys residues tether the kringles (425). A different study,
in which each Lys from position 429 to the C-terminal Lys452 was
separately mutated, indicated that Lys436 had the greatest effect;
their modeling suggested that Lys452 is too close to Phe448 for it
to be able to interact with the LBS (426).
The importance of 2 AP in stabilizing fibrin has been revealed in
several in vitro systems, including clot lysis (427,428). A mutant
of tPA that is relatively resistant to 2 AP was very efficient in
lysing both clots and model thrombi (429). Deficiency of 2 AP
results in delayed bleeding, but initial hemostasis is normal, and
this is characteristic of fibrinolytic defects (217). Inheritance was
autosomal recessive in a well-characterized Japanese family
(430,431). An interesting defect in 2 AP Enschede was explained
by the abnormal protein behaving as a substrate for plasmin rather
than as an inhibitor (432), as a result of the extended (by one Ala
residue) reactive center loop (433). Mice deficient in 2 AP were
essentially normal in many respects, but they did show enhanced
endogenous fibrinolytic activity without overt bleeding (434). In
experimental models, the lack of 2 AP had effects on liver
regeneration (435), platelet aggregation (436), and pulmonary
heart failure (437).
2 -Macroglobulin
2 -Macroglobulin is a major inhibitor of wide specificity and is the
backup inhibitor of several proteases, including plasmin (438). It
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comes into play when the fibrinolytic system is fully activated and
when the capacity of 2 -antiplasmin is exhausted; it also forms
complexes with tPA and uPA (183). 2 -Macroglobulin is a
glycoprotein of M r 725,000 that consists of four identical chains of
M r 160,000 and contains approximately 8% carbohydrate (Table
18-1). Its plasma concentration is 2.5 g per L (3 M). The tetramer
is arranged as a pair of dimers, each consisting of two monomers
linked together via a disulfide bond. The complete sequence of this
1,451-residue molecule has been established and the gene locus
(gene symbol A2M) attributed to chromosome 12p13.3-p12.3
(439,440). The inhibitor contains two reactive sites. The sequence
Arg-Val-Gly-Phe-Tyr-Glu (681,682,683,684,685,686) acts as a bait
region that contains sites for proteases of different specificities
(441). Cleavage results in a conformational change and activates
the thioester site at positions 949952. Electron microscopic
studies showed that the catalytic domain of the rod-shaped
plasmin molecule is entrapped inside the 2 -macroglobulin cavity,
whereas its N-terminal kringle domains protrude outside one end
between the two armlike features of the transformed
2 -macroglobulin structure (442). It is noteworthy that proteases
inhibited by 2 -macroglobulin can still act on small peptide
substrates, but their position in the inhibitor cavity makes larger
targets nonaccessible.
PLASMINOGEN ACTIVATOR INHIBITOR 1

This is the principal inhibitor of both tPA and uPA and is discussed
in detail in Chapter 19; the emphasis in this chapter is therefore to
place it in the context of regulation of the PLG/plasmin system in
the vasculature. In addition to its inhibition of tPA and uPA, PAI-1
has a role in regulating cell adhesion processes relevant to tissue
remodeling, mediated by its binding to the adhesive glycoprotein
vitronectin, rather than by proteinase inhibition (443).
The 52-kDa glycoprotein consists of 379 residues. It is unusual
among the serpins in its tendency to lose activity through
spontaneous insertion of the reactive center loop into the body of
the molecule, forming an extra strand of -sheet A. This form was
termed latent because, after denaturation and refolding, activity
was regained (444); there are no physiologic conditions under
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which this can occur. The noncovalent binding of vitronectin

stabilizes the active form of PAI-1 (445), which is present in
plasma at low concentrations, approximately 20 ng per mL
(446,447,448). Because it has a t 1/ 2 of less than 10 minutes, its
rate of synthesis must be high. The variation between normal
individuals is rather high, ranging from 1 to 40 ng per mL,
corresponding to 1 to 40 U per mL, the unit being the activity that
neutralizes one unit of tPA in 10 minutes.
PAI-1 is produced by most cultured cells, and early publications
identified it as a platelet protein (449), a product of
megakaryocytes (450), endothelial cells (451), or hepatocytes
(452); more recently, the production by adipocytes has been
stressed (453,454,455). The source of resting plasma PAI-1 is not
certain. Studies showing that normal endothelium or hepatocytes
do not express PAI-1 (456) point to adipocytes as the normal
source, whereas elevations in the acute-phase are likely to result
from hepatocytes (457). Platelets constitute the major pool of
circulating PAI-1. Although it is less active than plasma PAI-1,
platelet PAI-1 accounts for about half the circulating PAI-1 activity,
by virtue of the abundance of the protein in platelets (447). Large
amounts of platelet PAI-1 accumulate in thrombi (458,459), and
this directly correlates with their lysability (459).
PAI-1 inhibits tPA (tc and sc) and uPA (460). It does not inhibit
scuPA, which is largely inactive, but it does associate with scuPA
noncovalently, as discussed in the section on uPA (340). PAI-1 in
plasma is in excess over tPA; therefore, most of the tPA is present
in a complex with PAI-1. The second-order rate constants for the
interaction with tPA and uPA are greater than 10 7 M -1 S - 1 , a little
lower for sctPA (see Table 18-5).
Hereditary deficiency of PAI-1 in humans is quite rare but leads to
a lifelong bleeding disorder, presumably caused by premature lysis
of hemostatic plugs at sites of vascular trauma, and consistent
with this, bleeding occurs after a delay (461,462,463,464). The
oral administration of fibrinolytic inhibitors such as AMCA has been
effective in normalizing hemostatic function (463,464).
Mice with a disrupted PAI-1 gene demonstrate normal
development, fertility, and survival. The lack of inhibitor activity
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induces a mild hyperfibrinolytic state and confers a greater

resistance to the prothrombotic effect of endotoxin injected into
the footpad (465). After arterial injury, wound healing in PAI - / mice was accelerated with rapid migration of smooth muscle cells
from the uninjured tissue (466). In an arterial thrombosis model,
time to development of an occlusive thrombus was approximately
twice as long in
-/-
than in
-/-
mice (467). In another model of
carotid artery injury, platelet-rich thrombi were generated and

residual thrombi evaluated 24 hours later. Partially or completely
occlusive thrombi were found in 34% of
mice (468).
-/-
mice and 65% of
- /-
TABLE 18-5 SECOND-ORDER RATE CONSTANTS FOR THE

INHIBITION OF TISSUE-TYPE PLASMINOGEN ACTIVATOR
AND URINARY-TYPE PLASMINOGEN ACTIVATOR BY
SERPINS
Second-order rate constant of

inhibition/M/s
sctPA
tctPA
uPA
PAI-1
10 7
3 10 7
5 10 7
PAI-2
10 3
2 10 5
10 6
Protease nexin
1.510 3
3 10 4
1.5 10 6
Protein C
inhibitor
10 3
10 3
C1-inhibitor
1.3
2 -Antiplasmin
13
[approximate,
[approximate,
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equals]10 2
equals]10 2
sctPA, single-chain tissue-type plasminogen activator;

tctPA, two-chain tissue-type plasminogen activator; uPA,
urinary-type plasminogen activator; PAI-1, plasminogen
activator inhibitor-1; PAI-2, plasminogen activator
inhibitor-2; C1-inhibitor, complement 1 esterase inhibitor.
From Kruithof EKO. Inhibitors of plasminogen activators. In:
Kluft C, ed. Tissue-type plasminogen activator (tPA).
Physiological and clinical aspects, Vol I. Boca Raton, FL:
CRC Press, 1988:189210; Lobov S, Wilczynska M,
Bergstrom F, et al. Structural bases of the redox-dependent
conformational switch in the serpin PAI-2. J Mol Biol
2004;344:13591368; Huisman LGM, Van Griensven JMT,
Kluft C. On the role of C1-inhibitor as inhibitor of
tissue-type plasminogen activator in human plasma. Thromb
Haemost 1995;73:466471, with permission.
P.352
The opposite phenomenon, high plasma PAI-1, has been the
subject of many studies that examined potential links with
thrombosis. Mice overexpressing human PAI-1 in endothelium are
subject to age-dependent development of arterial thrombosis
(469). In humans, high circulating PAI-1 is clearly associated with
a variety of pathologies, including cardiovascular disease
(470,471,472) and cancer (473). Many studies have been
conducted to investigate causal significance, and it appears that
high PAI-1 does not independently predict disease, once other
factors like obesity, diabetes, and elevated triglycerides are taken
into account (474). A guanine insertion/deletion polymorphism at
position 675 in the PAI-1 promoter (475) is associated with
differences in circulating PAI-l (476). Despite promising early
findings (477), the current view is that the predictive power of
studying this polymorphism is low (474,478). In contrast to these
studies on subtle variation in plasma PAI-1, Gram-negative
septicemic patients have plasma PAI-1 concentraions that are
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sometimes raised as much as 50-fold over normal, and this is

associated with high mortality (479).
Chemical Inhibitors of Plasminogen

Activator Inhibitor 1
Compounds that inhibit the synthesis or action of PAI-1 have been
sought because of the relation between elevated PAI-1 and
disease. They might be useful for enhancement of constitutive
fibrinolytic activity or as adjuvant drugs in the thrombolytic
treatment of thrombotic disorders. Gemfibrozil and clofibric acid
act at the level of PAI-1 synthesis, decreasing it by approximately
40% (480). Also acting at the level of PAI-1 synthesis, a butadiene
derivative T-686, administered to mice for 8 days before an
injection of lipopolysaccharide, protected mice from its lethal
effect in a dose-dependent fashion (481). In a rat thrombosis
model, it limited thrombus growth (482) and also attenuated
development of atherosclerotic lesions in rabbits subjected to a
high-cholesterol diet (483).
A 14amino acid peptide, corresponding to the PAI-1 reactive
center loop (residues 333 to 346), rapidly inhibited PAI-1 function
and prevented the formation of a complex between tPA and PAI-1.
It also enhanced considerably in vitro lysis of platelet-rich clots
(484). Sideroxylonal C, a phloroglucinol dimer isolated from the
flowers of Eucalyptus albens, inhibited PAI-1 in an in vitro assay
without having a considerable effect on tPA (485). AR-H029953XX
is a derivative of flufenamic acid, known to inhibit the C1-inhibitor.
It prevents complex formation of tPA with PAI-1 through binding to
Arg76 or Arg118, or both (486). A series of diketopiperazine-based
inhibitors of PAI-1, XR334, XR1853, and XR5082, inhibited the
action of PAI-1 on tPA and tcuPA (IC 50 : 5 to 80 M) and slowed the
time to blood vessel occlusion in a rat carotid artery injury model
(487). XR5118 binds to a region in PAI-1 (residues 110 to 145)
known to interact with tPA, and it stimulated endogenous lysis of
rabbit jugular vein thrombi and diminished thrombus growth (488).
XR5118 binds to PAI-1 at -sheet A, which causes an inactivating
conformational change (489).
Plasminogen Activator Inhibitor 2
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The gene for PAI-2 (gene symbol PLANH2) spans 16.5 kb and is
located on chromosome 18q22.1 (490). The cDNA of 1.9 kb
encodes a protein of 415 residues. A segment of more than 5 kb
has been sequenced in the 5-flanking region and contains many
regulatory elements important for constitutive and for stimulated
gene expression (491,492).
PAI-2 was initially identified as a urokinase inhibitor in human
placenta (493); it later was purified from human placenta and from
the monocytoid cell line U-937 (494,495). PAI-2 is the
predominant PA inhibitor of squamous epithelia in epidermis,
esophagus, cornea, oral mucosa, tongue, and vagina (496). It is
incorporated into the cornified envelope during terminal
differentiation of the keratinocyte via transglutaminase-catalyzed
crosslinks (497). Monocytes are an important source of PAI-2
(498) and may increase fibrin stability on migration into thrombi,
especially because the PAI-2 is cross-linked to fibrin (499).
PAI-2 belongs to a subgroup of serpins called the
ovalbumin-related serpin family (ov-serpins). Members of this
subgroup lack a cleavable N-terminal signal sequence (490), and
accordingly much of the protein is intracellular. In addition to this
nonglycosylated form of apparent mass 47 kDa, some secreted,
glycosylated 60-kDa PAI-2 is found in cultured U937 cells (500).
All of the potential glycosylation sites on Asn75, Asn115, and
Asn339 appear to be used. Peripheral blood monocytes also secrete
some nonglycosylated PAI-2 by a mechanism distinct from the
secretion system for interleukin 1 and other nonclassically
secreted proteins (499,501). PAI-2 exhibits an unusually long
sequence between helices C and D, 33 residues that probably
protrude from the molecule. The crystal structure for PAI-2 is of a
form from which this loop was deleted (502). The loop contains
three glutamines in positions 83, 84, and 86 that are essential for
transglutaminase-mediated cross-linking of PAI-2 to trophoblast
membranes, the cornified envelope of epidermis (503), and fibrin
(499,504). Residues between 66 and 98 act as a protein-binding
domain, which binds annexins I, II, IV, and V, among others (505).
Purified PAI-2 polymerizes spontaneously at room temperature by
inserting the reactive center loop into the A-sheet of another
molecule, a loop-sheet polymerization that has some similarity to
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that which occurs in the Z-mutant of 1 -antitrypsin (506). This

polymerization depends on disulfide bond formation between Cys
residues 37 (in the C-D loop) and 161, which can occur as a result
of the mobility of the C-D loop (507). Alternatively,
P.353
monomeric PAI-2, with a reduced Cys37, can form disulfide bonds
with other molecules such as vitronectin (508).
PAI-2 is an efficient inhibitor of tcuPA, with a second-order rate
constant of 10 6 M - 1 S -1 ; it reacts approximately five times more
slowly with tctPA and quite poorly with sctPA (Table 18-5)
(460,495). In normal plasma, there are no measurable levels of
PAI-2, but high levels are found in pregnancy, with a steady rise
until approximately 33 weeks, to reach approximately 250 ng per
mL (509). Lesser increases during pregnancy were observed during
intrauterine growth retardation (510,511,512). PAI-2 might
therefore be a marker for decreased placental function (511,512).
Different forms of PAI-2 occur in plasma in pregnancy (513), and it
is not clear whether these represent PAI-2 disulfide-bonded or
cross-linked to other proteins.
PAI-2 is also found in the plasma of patients with acute
myeloblastic leukemia of the M 4 and the M 5 type (514), and in
patients with sepsis, in whom PAI-1 is also increased (515).
Speculation remains on the real function of PAI-2. Its intracellular
location suggests roles other than inhibition of PA, and it was
found to protect cells from the rapid cytopathic effects of
alphavirus infection (516). It also acts intracellularly as a
retinoblastoma protein (Rb)-binding protein (517). Its local activity
appears to be relevant to a number of cancers (518,519). Mice
with a disrupted PAI-2 gene did not present any phenotypic
abnormalities (520); it will be interesting to see if appropriate
inflammatory challenges reveal a phenotype.
C1-Inhibitor
C1-inhibitor is a highly glycosylated 105-kDa serpin, consisting of
478 residues (Tables 18-1 and 18-2). The 16 kb gene is located on
chromosome 11q12-q13.1 (521,522,523). It inhibits the activated
subcomponents, C1r and C1s, of complement C1, and factors XIIa
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and XIa, plasma kallikrein, and plasmin. Its high plasma

concentration (1.7 M) gives it the capacity to inhibit tPA when it
is present in excess over PAI-1 (138,330,524). By virtue of its
inhibitory effect on components of the contact system, it probably
plays a role in controlling contact phasedependent fibrinolysis and
the conversion of scuPA to uPA.
Histidine-Rich Glycoprotein
Histidine-rich glycoprotein (HRG) is named for the region between
residues 330 and 389, which are similar to the histidine-rich
regions in human and bovine kininogen (525). The 75-kDa protein
has 507 residues and approximately 14% of carbohydrate. HRG
competitively inhibits PLG associations by binding LBS-1 of PLG (K d
1 M) It is suggested, on the basis of their relative concentrations,
that PLG (2 M) and HRG (1.5 M) would circulate predominantly
in a 1:1 reversible complex (526), which would decrease the PLG
available for binding to fibrin. However, 2 -antiplasmin had a
greater effect than HRG on binding of PLG to fibrin (527). It has
recently been proposed that HRG tethers PLG to the cell surface,
which would enhance the cells' migratory potential (528). Only one
family with congenital heterozygous HRG deficiency has been
described. The proband, but none of the other five family members
with low HRG levels, had thrombotic disease (529).
Thrombin-Activatable Fibrinolysis
Inhibitor
The generation of C-terminal lysyl residues is an important
feedback mechanism for the binding of PLG to fibrin and the
enhancement of fibrinolysis. These residues can be removed by
TAFIa (EC 3.4.17.20; synonyms: carboxypeptidase B, U, or R),
further regulating fibrinolysis (530), as discussed fully in Chapter
20. The zymogen TAFI is activated by thrombin/ thrombomodulin
and functionally connects the fibrinolytic and coagulant pathways
(531).
TAFI is primarily produced by the liver and circulates at
approximately 75 nM, but the normal variation is wide (530,532).
The more important variable is the circulating active enzyme,
which has a very short t 1/ 2 and only a small proportion has to be
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activated for full function (533). The function of TAFIa was shown
originally in clot lysis assays; potato tuber carboxypeptidase
inhibitor relieves the inhibition (531,534). The effect of TAFIa has
also been shown in model thrombi made from whole blood (535)
and in animal studies, where TAFIa activity increased the time
taken to restore blood flow in a canine coronary artery thrombosis
model (536). Inhibitors of TAFIa enhanced endogenous fibrinolysis
and also thrombolysis by tPA (537,538). The homozygous-deficient
mouse shows enhanced plasma clot lysis but is comparable to
wild-type mouse in many thrombus models, mostly those in which
time taken to occlusion rather than thrombus resolution is
assessed (539).
There are some reports of elevated TAFI as a mild risk factor for
thrombosis, and it is elevated in inflammation, correlating with
other acute-phase markers (540). Several polymorphisms in the
TAFI gene have been reported. These seem to explain the wide
normal range of concentrations but do not correlate strongly with
disease (541). Some methods show bias toward particular
polymorphic variants of TAFI (542,543); associations with disease
therefore need to be interpreted with caution.
TAFIa activity appears to be the reason for increased fibrinolysis in
hemophilia, where thrombin generation may be sufficient for fibrin
formation but suboptimal for TAFI activation. Most thrombin is
formed after clot formation, mainly via the intrinsic pathway by
activation of factor XI by thrombin. This leads to a positive
feedback mechanism and optimal activation of pro-TAFI (544).
Factor IXdeficient plasma clots lysed prematurely, but the defect
could be reversed by the addition of factor IX or of
thrombomodulin to the plasma (545). Similarly, addition of TAFI,
thrombomodulin, or factor VIII to hemophilia A plasma restored
normal fibrinolysis (546). Consistent with this, incorporation of
antifactor XI antibodies or inhibition of TAFI in a rabbit model
resulted in an almost twofold increase of endogenous thrombolytic
activity (547).
Lipoprotein (a)
Apolipoprotein (a), a component of lipoprotein (a) [Lp(a)], is
linked by a disulfide bond to apolipoprotein B-100. Apo(a) has a
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wide size distribution, which depends on the number of kringle

repeats; these are similar to kringle 4 of PLG (548). The total mass
of Lp(a) therefore varies between 800 and 1,300 kDa. There is also
a wide range of plasma Lp(a) concentrations, from 10 to 1,000 mg
per L, and a skewed distribution, median value 50 mg per L.
Besides apo(a), a gene cluster on chromosome 6q26-27 also
contains PLG and two apo(a)-related genes (11,549,550).
Lp(a) binds to fibrin, extracellular matrix, platelets, and cells by
its kringles; predictably, binding is inhibited by lysine and ACA.
Lp(a) therefore competes with PLG and tPA for binding to fibrin
and may thus exert an antifibrinolytic effect (551,552,553). PLG
binding and activation on platelets (554) as well as on fibrin
(555,556,557,558) is attenuated by Lp(a). Transgenic mice
expressing the human apo(a) gene resolved experimental thrombi
poorly when treated with tPA (559). These observations are
consistent with the association of high Lp(a) with coronary artery
disease (560,561,562). It is now clear that Lp(a) size is a key
factor; only the smaller forms bind with high
P.354
affinity to fibrin (563,564,565). Individuals with the combination of
high Lp(a) levels and predominance of the small isoform showed
the lowest fibrinolytic activity (565).
BALANCE OF THE SYSTEM

The fibrinolytic system is normally kept quiescent as a result of
two factors: the relative abundance of inhibitors over proteases
and the requirement for fibrin or cell surfaces for initiation of
activity. In normal individuals, steady-state levels of PLG in
plasma are rather stable, whereas both the PA and their inhibitors
are subject to change, suggesting that regulation of fibrinolytic
activity occurs mainly via up- and downregulation of the
expression of the PA and their inhibitors. This general statement
relates both to the relative concentrations of PA, many orders of
magnitude lower than the PLG concentration, and to the very short
half-lives of PA compared to PLG, minutes rather than days (Table
18-2).
The fibrinolytic system is geared to remove fibrin from the
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circulation in a controlled way, and therefore to prevent excessive

fibrin accumulation. Circulating PLG is not normally converted to
plasmin, which would cause generalized proteolysis and
degradation of clotting factors, particularly factor V, factor VIII,
VWF, and platelet glycoproteins. Instead, fibrin acts as a focus for
the generation of activity, which is relatively protected from
inhibitors. Although fibrin deposition plays an important role in
hemostasis, wound healing, and inflammatory processes, fibrin
formation must always be limited in time, and fibrin should be
removed once it has fulfilled its role. The primary role of the
PLGplasmin system is not to lyse large formed blood clots, but to
prevent excessive build-up of fibrin formation. That said, the
system has been harnessed usefully for therapy, mainly by
administering activators. Recently there has been a resurgence of
interest in therapeutic use of plasmin (566).
Fibrin acts to assemble the fibrinolytic components, which is
central to the control of the system. At the earliest stages of fibrin
formation, tPA and PLG bind to the forming fibrin strands. Once
small amounts of tPA and PLG are bound to fibrin in the form of a
ternary complex, the catalytic efficiency of tPA for PLG is several
hundred times higher than it is in the absence of fibrin. Plasmin
generation causes proteolytic cleavage of fibrin that starts at the
C-terminal portion of the chain of fibrin and produces new
C-terminal lysyl residues. Partially digested fibrin binds up to 10
times more GluPLG than undegraded fibrin. scuPA binds to and
appears to activate selectively GluPLG bound to C-terminal
lysines in the partially degraded fibrin. Trace amounts of plasmin
also activate scuPA to uPA, and there is reciprocal activation of
PLG to plasmin.
In healthy individuals, there is a molar excess of active PAI-1 in
plasma over tPA (Table 18-2). Consequently, most of the tPA in
plasma is in the form of an inactive complex with PAI-1 (241).
Small amounts of free tPA occur in the circulation as a result of the
high synthetic rate of both proteins and the finite time it takes for
complex formation to occur. Even though PAI-1 in plasma is kept
in its active conformation by its interaction with vitronectin, its
tendency to lose activity does increase the possibility of plasma
tPA expressing some of its activity. Normally, tPA activity is
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detectable at approximately 0.1 to 0.5 U per mL of plasma, even

when blood is collected in an acidified anticoagulant to prevent
complexing of any free tPA by PAI-1 (567). The maximum activity
normally seen is equivalent to 1 g per L; tPA has a specific
activity of 500,000 to 700,000 U per mg (568). This low tPA
activity is generally close to the borderline for detection, which is
why procedures like euglobulin preparation, which removes most
plasma inhibitors, but notably only about half the PAI-1 (447), are
used. Even after the levels of free tPA increase severalfold, after
strenuous exercise, venous occlusion, or intravenous injection of
DDAVP to healthy volunteers, no circulating plasmin forms because
PLG activation does not occur at these still physiologic tPA
concentrations. This does not apply during the treatment of acute
myocardial infarction with recombinant tPA, during which
circulating plasma concentrations of tPA are achieved that are up
to 1,000-fold higher than those observed in normal plasma.
Changes in the balance of tPA and PAI-1 are a clear example of
how fibrinolytic activity can be initiated. Indeed, these components
of the system fluctuate remarkably during a 24-hour period (569).
tPA activity is lowest at night and in the early morning and then
increases up to threefold during the day. PAI-1 antigen and
activity are highest in the early morning hours and then gradually
decline during the day, reaching their lowest level in the early
afternoon. Consequently, if a drug (or placebo) is given at 8 AM,
the activity of the fibrinolytic system invariably is higher a few
hours later, and only trials in which a comparison to placebo is
made are valid. Various factors may interfere with the normal
circadian rhythm (327).
There is a large literature on the predictive value of tPA antigen
and PAI-1 as risk factors for acute myocardial infarction, stroke,
and peripheral vascular disease (472,477,478,570). High tPA
antigen is found to be associated with disease in a number of
studies, and this probably reflects the tPA/PAI-1 complex
(241,571).
It is generally assumed that PLG, present in plasma at
approximately 2 M, is not rate-limiting for fibrinolysis. Even
though the concentration is so much higher than the activators
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(Table 18-2), it is indeed possible for it to become a limiting

factor. The need to consider local PLG concentration, especially
when the activators are used therapeutically, has been clearly
shown (572). The effect on variation in available PLG is highlighted
in studies on TAFIa (573). The effect of TAFIa on tPA-initiated lysis
is usually stressed, in line with tPA's known fibrin dependence, but
TAFIa also has strong effects on uPA and scuPA-induced lysis,
pointing to its primary effect being on PLG binding (535).
Global circulating (a rare observation, unless thrombolytic agents
are administered to patients at high doses) and local fibrinolytic
activity are modulated by serpins. tPA and uPA are efficiently
inhibited by PAI-1. More than 90% of blood PAI-1 is located in the
platelets and promptly released on platelet activation. Although
most of the platelet PAI-1 is inactive, there is still enough active
PAI-1 in platelets to stabilize platelet-rich thrombi, as occur in the
arterial circulation in which the blood pressure is high and
premature lysis of a hemostatic plug is undesirable. Plasmin bound
to fibrin is partly protected from inactivation by 2 -antiplasmin
This assures that fibrinolysis proceeds on the surface of a clot.
However, free plasmin that spills over into the general circulation
is rapidly inactivated by 2 -antiplasmin After massive activation of
the fibrinolytic system has taken place, such as in thrombolytic
therapy, the amount of 2 -antiplasmin does not suffice to inhibit
all circulating plasmin, and 2 -macroglobulin acts as a scavenger
protease inhibitor.
The fibrinolytic system is a highly modulated enzyme system, and
nature has taken many steps to limit its action in time and space
but has also provided the necessary feedback loops that enhance
the fibrinolytic system on the local level.
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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

two major PAs that occur in the circulating blood, tissue-type

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

a C-terminal B chain, which is the protease domain. The kringle

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

extravascular space of most tissues, and some, such as

FIGURE 18-1. Structural elements of some serine proteases.

TABLE 18-1 CHROMOSOME LOCATION AND GENE ORGANIZATIO

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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

mRNA, messenger ribonucleic acid; tPA, tissue-type plasminogen activ

Heavy chain of tetramer.

Variable, depending on the number of kringle 4 inserts.

TABLE 18-2 PROPERTIES OF PROTEIN CONSTITUENTS OF TH

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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

z, protease zymogen; tPA, tissue-type plasminogen activator; p, prote

Estimated value, calculated from difference of molecular weight deter

amino acids derived from complementary DNA.

M r of nonglycosylated form as listed in Swiss protein base ExPASy.

Heavy chain of homotrimer.

Many isoforms due to 13 up to 37 type 4 kringle motifs.

Heavy chain of heterotetramer (formed by two heavy and two 10-kDa

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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

kringle 5 (34,35). Kringle 2 displays only a weak interaction with

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

FIGURE 18-2. Sequence of the human plasminogen molecule.

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

Activation of Plasminogen to Plasmin

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

FIGURE 18-3. Model of the interaction site of plasminogen

Several -aminocarboxylic acids, such as 6-aminohexanoic acid

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

constant K M for activation with tPA or uPA, by approximately one

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

FIGURE 18-4. Activation of native Gluplasminogen. In the

FIGURE 18-5. A sketch of the conformational change in

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

plasminogen induced on occupation of a weak lysine binding

Many modulators increase or decrease the rate of PLG activation;

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Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice