610 © 2003 Schattauer GmbH, Stuttgart
Review Article
Molecular and cellular modulation of fibrinolysis
Krasimir Kolev, Raymund Machovich
Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary
Summary
The structure of the fibrin network, the hemodynamic environ-
ment of the clot, the kinetic properties of the fibrinolytic
enzymes and the balance of their formation and inactivation
essentially determine the effectiveness of fibrinolysis in vivo.The
fibrin structure and the action of proteases, however depend
considerably on additional, apparently inert physiological and
pathological factors, which are restricted to more or less tran-
sient compartments in fluid-solid interface, such as thrombus
(fibrin with platelet membrane structures), endothelial cell sur-
face, the environment of polymorphonuclear cells (PMN). In
Keywords
Compartmental fibrinolysis, fibrin degradation, plasminogen
activation, tPA-cofactors, endothelial cells, polymorphonuclear
cells
Introduction
During fibrinolysis fibrin degradation products (FDP) are
formed, which are soluble in contrast to fibrin monomers that
polymerize to compose the skeleton of the clots. Fibrinolysis
generally means thrombolysis since in thrombosis fibrinolytic
therapy is applied. The term fibrinolysis, however, is a concrete
concept; it implies fibrin degradation catalyzed by proteases.
These enzymes are synthesized in a proenzyme (zymogen)
form. Thus, their activation is a prerequisite for their function.
Therefore, fibrinolysis is commonly interpreted as a combina-
tion of fibrin dissolution and zymogen activation. Although
there are several proteases with the capacity to degrade fibrin,
such as trypsin, elastase, cathepsin G, extracellular matrix me-
talloproteinases, in medicine plasmin is considered to be the
main fibrinolytic enzyme, which is formed from plasminogen in
the presence of plasminogen activators. In biochemical terms
Correspondence to:
Raymund Machovich
Department of Medical Biochemistry, Semmelweis University
Puskin u. 9., H-1088 Budapest, Hungary
Tel.: +36-1-2661030, Fax: +36-1-2667480
E-mail: mr@puskin.sote.hu
these compartments extreme changes in concentrations and
rate enhancements are observed. Components released by
endothelial cells, PMNs and platelets or molecules present in
circulating blood create a heterogeneous milieu that modulates
fibrinolysis.This review summarizes the effects, and where it is
possible, explains the mechanism of modulators of the fibri-
nolytic processes, such as cell membrane and cellular contents
of endothelium, PMN and platelets present in thrombi, the
action of normal and pathological blood plasma- and extracel-
lular matrix-components.
Thromb Haemost 2003; 89: 610 – 21
plasmin formation and fibrin solubilization are simple protease
activities; a single peptide bond in plasminogen is cleaved by
plasminogen activators, and the plasmin formed, thereafter
hydrolyses a few peptide bonds in fibrin, thereby it becomes
soluble (1). In vivo, however, the system is more complicated,
because these few reactions are controlled at multiple levels.
There are several feedback regulations for plasminogen activa-
tion, inhibitors act at two levels (plasmin formation and plasmin
action), and additionally cellular components modulate the plas-
min system (e.g. endothelial cells) or act independently in fibri-
nolysis (e.g. neutrophil leukocytes). The basic processes of
fibrinolysis are well described today (2), the role of its principle
components (plasminogen and its activators and inhibitors) is
supported by the phenotype of transgenic mice (3, 4), but the
influence of physiological and pathological molecular and cel-
lular components of the blood vessel is poorly understood and
frequently ignored by both researchers and clinicians.
Received October 30, 2002
Accepted after revision January 10, 2003

Fibrin degradation
Degradation of fibrin depends on the architecture of the fibrin
network and on the properties of the fibrinolytic enzymes, both
of which are modulated by several blood components.
Fibrin architecture
When fibrinogen (a soluble dimer of three polypeptide chains,
Aα, Bβ and γ) is converted to fibrin, thrombin cleaves the
N-terminal 16 aminoacid-residue peptide of the Aα-chain of
fibrinogen exposing the glycyl-prolyl-arginyl (GPR) “knob” for
interaction with the acceptor pocket in the end γ-domain of a
second fibrin monomer. Thus the two central GPR “knobs” of
the first fibrin monomer stabilize the end-to-end interaction of
the second monomer with a third one. The result is formation of
long double-stranded protofibrils composed of monomers in
half-staggered alignment, as evidenced recently by crystallo-
graphic analysis (5, 6). Following this initial step of fibrin poly-
merization, thrombin cleaves the N-terminal 14 aminoacid-
residue peptide of the Bβ-chains allowing for lateral assembly
of the protofibrils in bundles and because of the flexibility of the
monomers also for branching of the fibers resulting in a spatial
fibrin network. The final structure of the polymerized fibrin
(fiber diameter, distance between the branching points and pore
size) depends on relatively small number of factors, when fibrin
formation is examined in ideal systems (using diluted solutions
of fibrinogen and thrombin). These are actually the factors that
determine the rates of the sequential steps described above
(cleavage of the Aα and Bβ peptides, polymerization interac-
tions): concentration of fibrinogen and thrombin. The largest in
vivo variations (probably orders of magnitude) are expected for
the thrombin concentration because of the complex physiologi-
cal mechanisms that control its activity. When increasing con-
centrations of thrombin are applied for the conversion of pure
fibrinogen to fibrin, following initial increase (up to 0.05 U/mL
thrombin) the average fiber diameter decreases from 198 nm (at
0.05 U/mL thrombin) to 99 nm (at 2 U/mL thrombin) (7). There
is direct evidence that modulation of the thrombin activity
results in similar changes in clots prepared from blood plasma,
too (8, 9). The concentration of fibrinogen in blood plasma fluc-
tuates within a narrow range (5–20 µmol/L), but even these
variations modify the fibrin structure (7). The situation in vivo,
however, is much more complex. In environment overcrowded
with macromolecules such as blood plasma the physicochemi-
cal behavior of fibrinogen can not be simply described by
parameters gained in vitro for equilibria and kinetics in dilute
ideal solutions (10). Thus, in plasma environment fibrinogen
participates in reactions (e.g. hydrolysis by thrombin) or bind-
ing interactions (e.g. to platelets) as in a 10-fold more concen-
trated ideal solution (11). Consequently the morphology of the
fibrin network in the presence of “inert” macromolecules is
expected to differ from the structure formed under the same
Fibrinolytic modulators 611
conditions, but lacking the molecular “crowd”. Direct evidence
supports this prediction for the proteins present at high concen-
trations in blood plasma: albumin (12, 13), immunoglobulins
(14-18). An additional physiological consequence of the molec-
ular crowding in blood is the self-association of fibrinogen in
plasma. A recent sedimentation equilibrium study (11) evi-
dences that the dominant form of fibrinogen at its typical plas-
ma concentration is a dimer in the presence of 40 g/L albumin.
This observation can also contribute to our understanding of the
variations in fibrin structure in the presence of inert molecules.
The different sensitivity of the various fibrin structures to fibri-
nolytic enzymes is discussed later in this review and it provides
the link between the alterations in the blood level of these
“inert” modifiers and the hyper- or hypofibrinolysis in certain
disease states.
A previous review (19) summarizes the earlier data on the
self-association of fibrinogen and also on its interactions with
other plasma proteins (plasminogen, factor XIII, apolipopro-
tein(a), α2-macroglobulin, β-thromboglobulin, transferrin,
fibronectin, thrombospondin, hyaluronate). Among these the
first two factors play well-defined roles in fibrinolysis.
Following activation by thrombin factor XIIIa (FXIIIa) cat-
alyzes the formation of γ-glutamyl-ε-amino-lysine covalent
bonds between the γ-chains (and a small fraction of the α-
chains) of adjacent fibrin monomers, which modifies the con-
formation of the plasmin-susceptible peptide bonds and
decreases the rate of their hydrolysis (20-22). At a slower rate
FXIIIa crosslinks α2-plasmin inihibitor to fibrin (23). Thus, the
maturation (the post-polymerization modifications) of the fibrin
clot renders the structure resistant to proteolysis with plasmin.
The complexity of the fibrin network formation and matura-
tion is even greater in whole blood: the blood cells (ery-
throcytes, polymorphonuclear and mononuclear leukocytes,
platelets) are occluded in the clot and exert opposite effects on
its structure. The fibrin network formed at red blood cell count
corresponding to the normal hematocrit values has twice larger
pore size than the fibrin formed under the same conditions, but
lacking erythrocytes (24). Platelets bind to fibrin (25, 26) and
their contractile system begins to pull on the fibers. The platelet
content of 10 mL whole blood is compacted in 400 µL of arte-
rial thrombi, whereas the fibrin content of the same thrombi cor-
responds to the fibrinogen concentration in blood plasma (27).
After activation the platelets release their cytosolic content and
lose the majority of their phospholipid (28). Simple calculations
based on the average amount of lipid, protein and platelet count
(29, 30) indicate that 300 mg/L phospholipid circulates in the
blood within platelets. When the platelets are trapped in the
ageing thrombi, the phospholipid content of arterial thrombi
(7.5 g/L) is even higher than their fibrin content (1.7 g/L).
Fibrin(ogen), however, binds to phospholipids and this bound
form displays different susceptibility to thrombin and plasmin
(31-36). Similar calculations can be done for the intracellular
612 Kolev, et al.
proteins, e.g. within platelet-rich thrombi the concentrations
of actin and myosin are in the micromolar range (approximate-
ly 20 µM and 0.5 µM, respectively). Intracellular proteins are
also reported to modify the fibrin structure, e.g. in the presence
of actin thinner fibrin fibers are formed (37).
Despite the numerous facts summarized above a unifying
theory accounting for the modulating effects of variable blood
components on the fibrin structure is still missing.
Fibrinolytic enzymes
There are several proteases with the capacity to degrade fibrin
in vitro. Among them, the most efficient is plasmin, followed by
mini-plasmin (des-kringle1-4-plasmin), trypsin, microplasmin
(plasmin lacking all kringle domains) and PMN-elastase (38).
Under normal or pathological conditions, primarily plasmin (2),
and partly PMN-elastase (39, 40) are described as fibrinolytic
proteases, and plasmin is used as a therapeutic agent in throm-
bolysis. It is applied via activation of plasminogen with plas-
minogen activators [urokinase type plasminogen activator
(uPA), tissue type plasminogen activator (tPA), streptokinase]
and only recently its direct administration is put forward in
practice (41). Because of its lower catalytic efficiency (38, 42)
PMN-elastase seems to play a role only under special conditions
such as inflammation, extracellular matrix degradation, tumor
metastasis formation, and very probably it is not involved in
physiological fibrinolysis.
Plasmin cleaves selected peptide bonds in the Aα-, Bβ- and
γ-chains of fibrinogen yielding two major final degradation
products: fragment E (~50 kDa, the central domain of the
molecule) and fragment D (~100 kDa, the terminal domains)
(43). The same bonds are hydrolyzed in fibrin, but because of
the isopeptide bridges between adjacent monomers, D-dimers
are released. Complete solubilization of fibrin requires the pro-
teolytic cleavage of only 25% of the total fragment E-fragment
D connections, the rest of the soluble products being larger
protofibril fragments (44). Not only the type of the final soluble
products varies with the degree of covalent crosslinking, but
also the rate of cleavage of the susceptible bonds (22). The
structural basis of the plasmin-resistance in crosslinked fibrin
has not been elucidated yet, but kinetic analysis shows that the
affinity of plasmin (and other fibrinolytic proteases) is signifi-
cantly lower for the covalently modified substrate (38). Recent
morphological observations have shown that plasmin catalyzed
solubilization proceeds by transverse cutting across the
protofibril bundles rather than progressive shell-wise digestion
from the outside to the inner layers of the fibers (45). Thus, it is
not surprising that scanning electron microscopy (46) and con-
focal microscopy (45) show that the disappearance of thinner
fibers in the course of fibrinolysis precedes that of thicker fibers
as predicted by earlier kinetic analysis (38). If plasma clots are
prepared in the presence of activated protein C (a negative
regulator of thrombin generation), the average diameter of the
fibrin fibers is significantly decreased (9), which could con-
tribute to the profibrinolytic effects of protein C. Loose fibrin
gels (with low density of branches) are dissolved faster than
tight gels (45). Because the factors that influence the architec-
ture of the fibrin network usually couple the thinner fibers with
tighter porosity and thicker fibers with looser structure, the
overall impact on the course of fibrinolysis is difficult to predict.
Correct understanding of the interrelations between fibrin archi-
tecture and lysis rate will help the interpretation of certain
pathological states. The fibrin gel formed in the normal blood
plasma milieu is a balanced substrate of the lytic system.
Hypofibrinolysis can develop with thinner fibers, if the decrease
in the gel pore size dominates (nephrotic hypoalbuminemia
(47), monoclonal gammopathies (15, 18), Dusart syndrome
(48)), with thicker fibers (relatives of patients with premature
coronary heart disease) (49) or with slightly thicker fibers stabi-
lized by additional non-covalent interactions (antiphospholipid
syndrome) (50). Hyperfibrinolysis can be observed in gels with
thinner fibers and relatively normal pores (as seen in plasma
clots formed in the presence of activated protein C) (9). A uni-
fying quantitative theory that accounts for all steps from fibrino-
gen cleavage to fibrin digestion is still missing.
The necessity for such a theory is well exemplified by the
data on the impact of immunoglobulins on fibrinolysis. The
immunoglobulin G (IgG) modifies the fibrin structure, at 40 g/L
concentration it decreases the mass-length ratio of fibrin fibers
by a factor of 7 (15). Monoclonal immunoglobulins exert even
stronger effects in the same direction; they inhibit fibrin
monomer polymerization (14, 17) and the result is even thinner
fibers (18). On the basis of fiber size only one may predict
increase in the fibrinolytic rate in the presence of IgG, but just
the opposite is seen in tPA-induced clot lysis (18, 50); probably
because of the lower gel porosity, which develops in parallel
with the thinner fibers or the weaker cofactor properties of the
thinner fibers in plasminogen activation (45, 51). IgG isolated
from normal human blood plasma prolongs the time of fibrin
solubilization by plasmin, too, under both static and flow con-
ditions (50). It means that in vivo the fibrinolytic events are
slower than expected from in vitro measurements in purified
systems. IgG from patients suffering in antiphospholipid
syndrome with thrombotic complications exert even stronger
antifibrinolytic effect, for which the Fab portion of the IgG is
responsible. The priority of specific antibody-antigen inter-
action over mass action is illustrated by a case of hyperglobu-
linemia, in which the IgG does not exert its normal fibrin-stabi-
lizing effect and the patient presents with bleeding tendency
(50). A coherent understanding of these effects with respect to
the involved antibodies is still awaited.
The cellular components of thrombi introduce further com-
plexity in the fibrinolytic process. When platelets are activated,
they also release plasminogen activator inhibitor-1 (PAI-1) and
thrombospondin, and present phospholipid surface for blood

coagulation factors in a thrombus. They are inhibitory on fibri-
nolysis. PAI-1 inhibits plasminogen activation (52). Phospho-
lipids hinder plasmin activity on fibrin substrate and inhibit the
diffusion of plasmin into the fibrin network (Kolev et al., unpub-
lished). Thrombospondin, a 450 kDa protein, is released from
the α-granule of platelets and despite its low level in plasma
(~250 pM) it increases extremely in a thrombus. Thrombospon-
din not only interacts with fibrinogen and plasminogen, it also
inhibits plasminogen activation and plasmin activity. It forms
an equimolar complex with plasmin with a rate constant of
k” ≈ 6 × 103 M–1s–1) (53), a rate similar to that of a reaction with
established physiological relevance (the thrombin-antithrombin
reaction without heparin). Myosin [Kolev et al., in press (53a)]
and actin (54) inhibit fibrin degradation by plasmin. The role
of intracellular proteins as modulators of fibrinolysis can be
reliably considered only on the basis of quantitative evaluation
of their effects.
The role of other cellular constituents of the thrombi (the
polymorphonuclear leukocytes, PMN) is underscored by the
phenotype of transgenic animals with deficient components of
the plasmin-dependent fibrinolysis. Knock-out mice deficient in
plasminogen survive (55, 56), but following induced venous or
arterial thrombosis more PMNs infiltrate the clot in plasmino-
gen deficient mice than in normal control animals and the iso-
lated PMNs from the knock-out mice exert higher fibrinolytic
activity than the control (57). These data imply that PMNs may
compensate, at least partly, impairment in the classic fibrino-
lysis and may represent an auxiliary system for clot dissolution
under physiological conditions. It is well-known that PMNs
infiltrate thrombi and take part both in their initiation (express-
ing tissue-factor) (58) and removal via phagocytotic activity
(59) or extracellular proteolysis based on released elastase and
cathepsin G (58, 60). These ideas are implanted in the concepts
of non-plasmin fibrinolysis (60, 61).
Termination of fibrinolysis
The main inhibitor of plasmin is α2-plasmin inhibitor, synthe-
sized by the liver and secreted into the blood circulation, where
its concentration is approx. 1 µM (the concentration of plas-
minogen in blood is approx. 2 µM). Plasmin forms an equi-
molar complex with α2-plasmin inhibitor and in this structure
the enzyme is inactivated (62). The rate of inactivation is high;
k” ≈ 4.3 × 106 M-1s-1. It is remarkable that when plasmin is
bound to fibrin, the rate of inactivation by α2-plasmin inhibitor
slows down (k” ≈ 1 × 104 M–1s–1) and is completely protected
against α2-macroglobulin and α1-protease inhibitor (63). In the
case of another fibrinolytic enzyme action, the effect of fibrin
is more impressive, the rate of PMN elastase inactivation by
α1-protease inhibitor decreases by more than three orders of
magnitude in the presence of fibrin (63). The conclusion from
these experimental results is that enzymes bound to their sub-
strate, the fibrin, are quasi “protected” against their inhibitors.
Fibrinolytic modulators 613
Consequently, fibrinolytic agents designed for clinical applica-
tion should preferably generate plasmin on fibrin surface. The
soundness of this concept is supported by the direct application
of plasmin in local thrombolysis, when plasmin, escaping from
fibrin into the circulation, is rapidly inactivated by α2-plasmin
inhibitor, and thus bleeding complications are less frequent than
with tPA (41).
Besides α2-plasmin inhibitor, plasmin activity is inhibited
by α2-macroglobulin, α1-protease inhibitor and antithrombin as
well, whereas PMN elastase by α1-protease inhibitor and α2-
macroglobulin. The relative efficiency of the plasma protease-
inhibitors in the inhibition of plasmin and elastase is calculated
from the second-order rate constant of enzyme inactivation
determined in vitro (63) and the physiological concentration
of an inhibitor. The most efficient inhibitor for plasmin is α2-
plasmin inhibitor (4 × 106 M–1s–1), followed by α2-macroglobu-
lin, α1-protease inhibitor and antithrombin (all approximately
105 M-1s-1), the latter reaction, however, is accelerated by
heparin by an order of magnitude (64). PMN-elastase is most
efficiently inactivated by α1-protease inhibitor (4 × 109 M–1s–1),
followed by α2-macroglobulin. The in vivo significance of these
reactions is not known yet, only the functional capacity of the
enzymes can be estimated. For example, the life span of plasmin
on the surface of the fibrin compartment is expected to be 420 s
in blood plasma environment (63), but it can be prolonged in a
real thrombus infiltrated with PMNs, because antithrombin and
α2-plasmin inhibitor are degraded by PMN-elastase.
Sources of fibrinolytic enzymes
In vivo primarily plasmin and partly leukocyte elastase may
play a role in fibrinolysis, therefore in the next section the func-
tion of the plasmin and the non-plasmin systems, their mutual
interactions and the effects of other interfering components are
discussed.
Plasminogen activation
Although plasminogen activation by both endogenous and
exogenous activators is an apparently simple process (a single
peptide bond in the zymogen is hydrolyzed by proteases), in
vivo multiple factors should combine to yield efficient plasmin
generation.
Native plasminogen can exist in at least three conformations
(65): a closed one, which is less sensitive substrate for plas-
minogen activators, and two more extended forms, which are
more susceptible to activation. The closed conformation is
maintained by interaction between kringle5 and Lys residues
in the N-terminal portion of the zymogen and is stabilized by
chloride ions in blood plasma (66-68). For efficient activation
these interactions should be disrupted either by proteolytic
removal of the N-terminal portion or competitive displacement
of its lysine residues from their binding site (69, 70). Thus, com-
614 Kolev, et al.
ponents with the capacity to influence native plasminogen struc-
ture are modulators of fibrinolysis via plasminogen activation.
Such a proteolytic modulator can be the plasmin itself (if pres-
ent in trace amounts), which removes the N-terminal 77-amino
acids, or other proteases, e.g. elastase, which hydrolyzes sever-
al bonds and thereby removes several kringle structures from
the N-terminal of the proenzyme. Conformational changes in
plasminogen are caused by lysine residues in fibrin, 6-amino-
hexanoate (ε-aminocaproic acid) or lysine-containing synthetic
peptide substrates for plasmin (71, 72).
Although all plasminogen activators attack the same peptide
bond in plasminogen, their activation mechanisms are somehow
different; uPA is more effective than tPA, the efficiency of the
latter, however, is elevated by at least an order of magnitude in
the presence of cofactors, the role of which is discussed later in
this review. Knock-out mice deficient in these endogenous acti-
vators survive (73) and this observation supports the existence
of auxiliary pathways compensating their action (a tentative
possibility is through the action of activated factor XII) (74).
Streptokinase, an exogenous plasminogen activator synthesized
by hemolytic streptococcus, is also used in medicine (75).
Streptokinase is a protein (≈45 kDa) without any known
enzyme activity, however it forms a complex with human plas-
minogen causing a conformational change in the zymogen, and
thereby its hidden active center appears and thus the complex
acts as a plasminogen activator. Recently, a fibrin-dependent
streptokinase has been described; removing an N-terminal por-
tion of streptokinase induces a change in the specificity of the
modified streptokinase/plasminogen complex (76). The clinical
significance of this fibrin-dependent enzyme has not been deter-
mined yet.
There are several feedback controls in plasminogen activa-
tion. Plasmin converts native (Glu-) plasminogen to des1–77-
plasminogen (Lys-), which is easily activated. The proenzyme
of uPA (single-chain uPA) is activated by plasmin (besides
kallikrein) and the single-chain tPA is also converted by plasmin
to the more active, two-chain tPA. Fibrin, the main substrate of
plasmin, binds both plasminogen and tPA (77) and accelerates
plasminogen activation (78, 79). Its cofactor properties, how-
ever, are even stronger following initial partial degradation by
plasmin (based on the exposure of new carboxyterminal
lysines). The efficiency of fibrin as a cofactor of plasminogen
activation depends on the fiber architecture, too; thicker fibers
are a better cofactor (51). The cofactor function of fibrin, how-
ever, is further modulated. Carboxypeptidase B (also known as
thrombin activatable fibrinolysis inhibitor, TAFIa), formed from
its precursor by thrombin/thrombomodulin complex, eliminates
the cofactor effect of fibrin (80) and directly moderates the
plasmin activity (81). Moreover the plasminogen activators are
well controlled by the plasminogen activator inhibitor system,
which remains out of the scope of this survey, but has been
extensively and recently reviewed (1, 52).
Many factors can modulate plasminogen activation, espe-
cially by tPA, via conformational modification of the zymogen
and/or of the enzyme. In some cases it is clear, that the effector
is a cofactor for tPA like fibrin. Several others also facilitate
plasminogen activation, but the formation of ternary complex of
zymogen, enzyme and cofactor is not proved. Besides fibrin
(82), cofactor or cofactor-like functions are described for some
extracellular matrix components (83), myosin (84), actin (85),
polylysine (86), a part of prothrombinase complex (87) and
plasmin-digested coagulation factor X (88), certain denatured
proteins (89-91), prostromelysin-1 (92) and endothelial cell
membrane components (93-95). Their in vivo function is not
well understood yet, perhaps their primary biological role is the
initiation of fibrinolysis and/or induction of extravascular plas-
min formation. It is remarkable that neither of these molecules
influences plasminogen activation by uPA or streptokinase, and
that the denatured proteins adopt cofactor function only after
partial digestion with plasmin. The native albumin, antithrom-
bin, prothrombin, α2-macroglobulin, apoferritin, immunoglobu-
lin G are not sensitive to plasmin-degradation and do not display
cofactor function in contrast to their denatured form (90). At
present it is an open question why (in terms of both structure
and biological function) after denaturation certain proteins
become a tPA cofactor in plasminogen activation. Perhaps the
function of the fibrinolytic enzymes is not only fibrin solubiliza-
tion, but also activation of matrix metalloproteinases, digestion
of extracellular tissue and thus a more general role can be attrib-
uted to them in the removal of “old”, “damaged”, “unnecessary”
proteins. The actions of these modulators are rather complex
and difficult to be explained. For example, the overall effect of
myosin being a substrate for plasmin, a cofactor for tPA (84)
and an inhibitor of fibrin degradation [Kolev et al., in press
(53a)] seems to be contradictory. Considering that in an arterial
thrombus, the cofactor nature of myosin is negligible because of
the presence of fibrin (which is a more efficient cofactor than
myosin), myosin released by platelets, infarcted smooth muscle
cells or infiltrating mononuclear cells is inhibitory on the over-
all course of fibrinolysis. On the other hand, at sites, where the
primary function of plasmin is not fibrin solubilization (e.g. in a
compartment without fibrin), myosin facilitates plasmin forma-
tion and the degradation of certain proteins (e.g. extracellular
matrix components) and this may play a role in tumor invasion,
metastasis or inflammation.
Modulators of plasminogen activation are clearly associated
with certain pathological states. High level of lipoprotein(a) is
known as a risk factor for coronary and cerebrovascular diseases
(96, 97). Lipoprotein(a) shows homology with plasminogen,
primarily because of the multiple copies of plasminogen
kringle4 structures. In blood plasma, plasminogen interacts
weekly with fibrinogen, but when fibrinogen is converted to
fibrin, the affinity of plasminogen increases (98, 99). Lipopro-
tein(a) through its similar nature competes with plasminogen

for binding with fibrin and cell membrane structures (100-102).
It is interesting and should be considered in pathological condi-
tions that the affinity of lipoprotein(a) to fibrin is higher by 1 to
2 orders of magnitude in the presence of homocysteine (100).
This effect may contribute to the thrombophilia developed in
hyperhomocysteinaemia.
Another example for pathological modification of fibrinolysis
is the metabolic transformation of acetone to methylglyoxal. An
isoenzyme of cytochrome P450, which catalyzes methylglyoxal
formation, is induced in diabetes mellitus. Although the high
level of glucose in untreated diabetes mellitus does not interfere
essentially with the fibrinolytic system, the elevated level of
methylglyoxal (an efficient modifier of lysine, arginine and cys-
teine residues in plasminogen) inhibits fibrinolysis. The rate of
plasminogen activation by tPA, uPA or streptokinase decreases
and the activity of the generated plasmin is impaired (103).
In addition to the role of endothelium in blood coagulation
(104), endothelial cells are the source for tPA, uPA, PAI-1 and
their surface assembles fibrinolytic components, so that plas-
minogen activation can proceed efficiently (105). Plasminogen
binds to endothelial cells. Although its affinity (Kd ≈ 10–7 M)
(93) is not as high as that of plasmin (Kd ≈ 10–9 M and 2 × 10-8 M)
(106), at the blood plasma concentration of plasminogen
(approx 2 × 10–6 M) there is always zymogen present on the
endothelial surface, where its activation by tPA is accelerated,
because endothelial cell surface works as a cofactor for tPA
(93–95) via a tPA receptor (107). Plasmin binds to endothelial
cells with high affinity and prevents fibrin accumulation,
removes peptide1-77 from bound native plasminogen, and
although it does not cause intracellular Ca2+ increase in contrast
to other proteases such as thrombin or elastase, plasmin does
induce contraction of brain capillary endothelial cells (108, 109)
and degrades protease-activated receptors (110, 111). In addi-
tion, thrombomodulin on the endothelial surface facilitates
procarboxypeptidase B (TAFI) activation by thrombin, and the
enzyme formed removes lysine residues from fibrin (which are
responsible for the cofactor function of fibrin in plasminogen
activation by tPA) (80).
Besides the previously mentioned role of the PMNs in the
removal of fibrin, the leukocyte elastase affects blood coagula-
tion and plasminogen activation as well. Elastase digests many
blood coagulation factors (e.g. Factor V, -VII, -VIII, -IX, -XII,
-XIII); as a result thrombin formation is impaired (39). Elastase
hydrolyzes peptide bonds between kringle structures in plas-
minogen, thus des-kringle1-4-plasminogen (miniplasminogen,
plasminogen with kringle5 only) and separated kringles may be
formed (112). The isolated kringle structures (known as angio-
statin) play a role in angiogenesis (113), which is out of the
scope of this survey. Miniplasminogen is easily converted to
miniplasmin by plasminogen activators, and does not need a
cofactor for its activation (114). Miniplasmin formed is an effi-
cient fibrinolytic enzyme, especially in the degradation of
Fibrinolytic modulators 615
crosslinked fibrin (38, 42). Thus, in the presence of elastase the
balance in the blood coagulation-fibrinolytic system is shifted
extremely into the direction of fibrinolysis (additional details
are summarized in an earlier review, Ref. 39).
Since elastase has an extremely efficient inhibitor, the α1-
protease inhibitor (α1-antitrypsin), which has a high concen-
tration in blood plasma (≥40 µM), elastase function in the
circulating blood is limited. However, when PMN is activated,
not only elastase and cathepsin G, but also myeloperoxidase is
released, and oxidizes the reactive site Met358 of the α1-protease
inhibitor, thus the myeloperoxidase system inactivates the elas-
tase inhibitor efficiently (115).
Hemodynamic and vascular
compartmentation in fibrinolysis
The anatomic localization of thrombi creates variable hemo-
dynamic environment for fibrinolysis. An occlusive thrombus
localized in a smaller artery branch near the bifurcation point is
exposed to a relatively higher pressure than a thrombus lo-
calized downstream in the same branch or in a vein. In the
latter cases zones of stagnation can also be formed upstream
the thrombus. Thus, in different loci circulating plasma-born
molecules can penetrate into the clots via two mechanisms:
perfusion, if high pressure drop exists on the surface of clot, or
diffusion, if no hydrostatic pressure acts on the surface.
Although the pore size of the fibrin gel (from 0.1 µm for tight
gels up to 5 µm in loose gels) (116) can not hinder the diffusion
of molecules with the size of the fibrinolytic enzymes (around
5 nm) (117), the diffusion rate of such molecules is rather low
(1 cm diffusion is estimated to take place in 10 days) (118).
Pressure-driven permeation (in the range typical for the arterial
circulation) accelerates the tPA-induced, the uPA-induced and
the plasmin-catalyzed clot lysis by a factor of 50, 25 and 10,
respectively (119, 120). Consequently, even at identical plasma
concentrations of fibrinolytic enzymes and zymogens, the time-
course of clot dissolution will vary greatly in the separate
vascular compartments depending on the flow conditions. The
perfusion into the clots depends not only on the pressure values,
but on the gel architecture, too: the faster permeation into coarse
fibrin results in fast fibrinolysis, whereas the slow permeation
into fine fibrin is associated with slow fibrinolysis (119). The
blood current over the fibrin surface maintains a continuous
supply of plasminogen and activators, which bind to fibrin and
form a sharp active zone (approximately 50 µm deep), where
their concentration increases multiple times and plasmin is
generated (121). Under these conditions the clot dissolution
proceeds through the sequential solubilization of the outer
shells of fibrin and inward movement of the reactive lytic zone.
Recently introduced models of the transport and catalytic events
in this boundary layer of a fluid and a solid phase provide tools
for the quantitative characterization of the exogenously initiated
clot lysis (38, 119, 122). If however endogenous plasmin (uni-
616 Kolev, et al.

formly dispersed within the clot) digests fibrin and shear forces
act on the surface, a different pattern of dissolution is observed:
at a certain stage of fibrin degradation a particulate disassembly
of the clot occurs (123). This pattern of dissolution may cause
thromboembolic complications in vivo and it is remarkable that
some fibrinolytic proteases (e.g. leukocyte elastase) do not
cause such disaggregation, apparently because different peptide
bonds are cleaved in fibrin (123). The latter pattern is a poten-
tial advantage, if a therapeutic agent can reproduce it.
A compartmental approach
to fibrinolysis
Similarly to blood coagulation, reactions of fibrinolysis occur
on the interface of fluid-phase and solid-phase structures,
generally in transiently formed compartments, where the
molecular concentrations and reaction rates are changed
extremely.
Enzymology of proteases in a water phase is extensively
worked out and widely used for several decades. The fibrinoly-
tic proteases, however, act on an interface between fluid and
solid phase. The enzymology of this system is poorly under-
stood because of its very complex character. The fluid phase
protease (plasmin, elastase) binds to a nonspecific site of fibrin
surface, thereafter the enzyme approaches the susceptible site
through lateral movement along the fiber, and/or can move into
the fiber.
When blood coagulation is initiated almost all reactions take
place in compartments formed transiently by proenzymes,
enzymes, cofactor proteins, phospholipid surfaces of platelets,
in the presence of Ca2+. As a result, the ultimate product, the
thrombin converts fibrinogen to fibrin. During maturation the
fibrin network is further modified by covalent crosslinks, by
entrapped blood molecular components and by infiltrating cel-
lular elements. The platelets in the thrombi not only contribute
to the acceleration of blood coagulation, but also prevent the

Figure 1: Endothelial cell surface as a compartment for plasminogen activation and plasmin inactivation. Endothelial cell (EC) surface
is an assembly site for several factors of the blood coagulation-fibrinolytic system, from which only few are shown in this scheme:
plasminogen (Pg), tissue plasminogen activator (tPA), plasmin (P), α2-plasmin inhibitor (α2PI). Their affinities, the Kd values for tPA and
endothelial tPA-receptor (tPA-R), Pg and EC, P and EC are from Refs. 124, 107 and 106, respectively.The catalytic efficiency kc (kcat/
KM, × 103 µM-1s-1) of the activation reaction is a measured value for the fluid phase (82), whereas it is estimated for the solid support
(cell or fibrin surface) (95). For the efficient activation of both free (Pgf) and bound plasminogen (Pgb) the conversion of native
plasminogen to des1-77- plasminogen is a prerequisite (69, 70, 95), but not shown in this figure. Plasmin (Pf) generation in fluid phase is
slow compared to plasmin (Pb) formation on solid phase.When plasmin is formed in water-phase, it is immediately inactivated by α2PI
with a second order rate constant (k”, ×104 M-1s-1) (63), while the cell-surface (125) or fibrin-bound enzyme (63) is protected against
inactivation. Bound plasmin is proteolytically active in fibrin degradation and conversion of Glu- to Lys-plasminogen. For clarity many
reactions (even relevant to fibrinolysis) are not shown.The indicated rate enhancements are approximative, because measurements
depend on the type of endothelial cells (arterial, vein, capillary) and even for the best-studied plasminogen activation by tPA almost
three-order of magnitude variations can be found in kinetic parameters published for various, more or less physiologically relevant
conditions (126).

initiation of fibrinolysis through the release of PAI-1 and phos-
pholipids, which impair the function of the plasmin system. If
however the blood coagulation and platelet activation are termi-
nated, the thrombus compartment is the ideal stage for action of
the fibrinolytic system. There plasminogen activation by tPA is
facilitated by fibrin and the fibrin-bound plasmin is protected
against the endogenous plasmin inhibitors.
On the endothelial cell surface the reactions of blood
coagulation and fibrinolysis are well balanced under normal
condition. Endothelium is an assembly site for fibrinolytic com-
ponents, and many reactions are accelerated on this solid phase
support (Fig. 1). That is, endothelium becomes a compartment
where tPA and uPA are synthesized, released and controlled by
PAI-1. Both the released tPA and the blood-born plasminogen
bind to the endothelial surface, which affects plasminogen acti-
vation as a cofactor for tPA. The generated plasmin binds with
high affinity to endothelial cell and converts Glu-plasminogen
to Lys-plasminogen and the uPA-zymogen to active enzyme,
thereby further plasmin generation is accelerated and plasmin
prevents fibrin accumulation on endothelium. Similar compart-
mentation of plasminogen activation on cellular surfaces has
been reported for other cell types (127) and the enzymological
aspects of this process have been recently reviewed (128).
When PMNs are activated, the fibrinolytic serine proteases
(elastase, cathepsin G) can escape the very efficient inhibitor,
the α1-protease inhibitor, only in a compartment, where myelo-
peroxidase, the inactivator of the inhibitor, is also present or the
Fibrinolytic modulators 617
limitations of diffusion ensure the predominance of protease
over inhibitor in the zone around activated PMNs (Fig. 2). A
compartment formation has been described for fibrinogen
bound to activated PMN, where proteases are inaccessible to
plasma protease inhibitors with molecular mass higher than
40 kDa (131). Additionally a non-oxidative mechanism for
quantum proteolysis has been developed theoretically and sup-
ported experimentally (132, 133). If a single granule of PMN
is extruded, the concentration of PMN-elastase at the point
of release will be 5.33 mM. Its diffusion in environment with
physiological concentrations of inhibitors will result in a sphe-
rical zone of proteolysis with a 1.33 µm radius for a period of
12.4 ms (this is a proteolytic quantum restricted in time and
space). Thus, the sequential release of granules determines the
size and duration of proteolysis around activated PMNs.
A potential link between the endothelium- and the PMN-
related compartments is the fibrin(ogen) itself. The adhesion
receptors, integrins on the surface of the leukocytes recognize
and bind fibrinogen (through its D-domain) (134), which on its
turn can form bridges to the endothelial cells through integrin
(135, 136), non-integrin (137, 138) binding sites or even trans-
glutaminase-mediated covalent bonds (139). This fibrin(ogen)-
dependent leukocyte-endothelium compartmentation in vivo is a
potential target of the plasmin and PMN-elastase action espe-
cially in inflammation, but its exact biological significance
remains to be elucidated.

Figure 2: Pericellular fibrinolytic compartment around activated polymorphonuclear cells. Activated polymorphonuclear cell (PMN)
may release elastase into the blood (Elf), where its inactivation by α1-protease inhibitor (α1PI) is extremely rapid.The fibrin (F)-bound
elastase (Elb) is inactivated by α1PI at a several order of magnitude lower rate than free elastase. PMN may release myeloperoxidase
(MPO), too, which inactivates α1PI efficiently. In addition, on the surface of this cell, plasminogen (Pg) activation by tPA is facilitated
(129, 130) and the formed plasmin (P) degrades fibrin. Moreover PMNs are able to digest and take up fibrin, and the fibrin degradation
products (FDP) appear in the cell (57). Numerical values for elastase inactivation by α1-protease inhibitor (k”, ×104 M-1s-1) are from
Ref. 63.
618 Kolev, et al.

The abundant data summarized above justify the concept of Acknowledgements

compartmental fibrinolysis. A unifying theory that can quantita- Grants: The pertinent research of the authors is supported by grants from the
tively treat the discrete steps in each compartment at all stages Hungarian Scientific Research Fund (T031891), the Hungarian Ministry of
Health (ETT 287 & 288/2000), the Hungarian Ministry of Education (FKFP
of fibrin dissolution is anticipated. The recent advances in 0013/99) and the Wellcome Trust (069520/Z/02/Z).
heterogeneous phase enzymology and the newly introduced
mathematical models support the hope that in the near future a
tool for characterization of fibrinolysis in complex environment
will be available.

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