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Ham-Wasserman Lecture

Role of the Plasminogen System in


Fibrin-Homeostasis and Tissue Remodeling
Désiré Collen, MD, PhD*

Plasminogen can be converted to plasmin either thrombolytic therapy of acute myocardial infarction
via the tissue-type plasminogen activator (t-PA) or and some other thromboembolic diseases. The
via the urokinase-type plasminogen activator (u- u-PA mediated pathway, in concert with the matrix
PA)/u-PA receptor (u-PAR) pathway. A dual role for metalloproteinase (MMP) system, plays a pleiotro-
these pathways is now well established: 1) t-PA is pic role in arterial neointima formation, atheroscle-
involved in fibrin homeostasis and 2) u-PA is rosis, angiogenesis, tumor growth metastasis, and
primarily involved in cell migration and tissue infarction. However, therapeutic interventions in the
remodeling. t-PA mediated activation is used for u-PA/MMP system remain to be further defined.

The plasminogen (fibrinolytic) system (Figure 1) com- the level of MMPs, by tissue inhibitors of MMPs
prises an inactive proenzyme, plasminogen, that can be (TIMPs). A dual role of the plasminogen system is pres-
converted to the active enzyme, plasmin, that degrades ently well established: 1) the t-PA mediated pathway is
fibrin and that activates matrix metalloproteinases primarily involved in fibrin homeostasis and 2) the u-PA
(MMPs), which in turn degrade extracellular matrix mediated pathway is primarily involved in phenomena
(ECM) (for references, cfr. 1, 2). Two physiological plas- such as cell migration and tissue remodeling. Conse-
minogen activators (PA) have been identified: tissue-type quently the terminology “fibrinolytic system” has be-
PA (t-PA) and urokinase-type PA (u-PA), which binds come inadequate and should be replaced by “plasmino-
to a cellular u-PA receptor (u-PAR). Inhibition of the gen system.” The elucidation of the biochemistry, the
plasminogen/MMP system occurs at the level of the PAs, (patho)physiology and the therapeutic applications of the
by specific plasminogen activator inhibitors (PAIs), at plasminogen system has been catalyzed by the emer-
the level of plasmin, primarily by α2-antiplasmin, or at gence of powerful molecular biological technologies,
including recombinant DNA techniques for the expres-
sion of heterologous proteins, and targeted gene manipu-
* Afdeling Moleculaire & Vasculaire Biologie, K.U. Leuven,
lation in vivo for the elucidation of the (patho)physio-
O & N Gasthuisberg, Herestr. 49, B-3000 Leuven, Belgium logical role of their translation products. This review
summarizes the main developments in the plasminogen
Acknowledgements: The experimental studies of the author field over the last two decades. Reference is primarily
referred to in this review could not have been performed made to review articles, in which more details and cita-
without essential contributions of many collaborators from tions to original work can be found.
several laboratories, which are gratefully acknowledged.

The author is a party to a royalty bearing licensing agreement Components of the Plasminogen System
on recombinant tissue-type plasminogen activator between the All enzymes of the plasminogen system are serine pro-
University of Leuven and Genentech Inc., South San Fransisco, teinases, i.e. their active site consists of a “catalytic triad”
CA and has an equity interest in ThromboGenics Ltd., a spin- composed of the amino acids serine, aspartic acid and
off company involved in the development of recombinant histidine. This active site is located in the COOH-termi-
staphylokinase for thrombolytic therapy. Neither of these
organizations nor the university have exerted any influence on
nal region of the molecules (serine proteinase part), while
the statements made in this review. the NH2-terminal regions contain one or more structural/

Hematology 2001 1
Tissue-type plasminogen activator (t-
PA) consists of 530 amino acids, although
originally 527 were identified. It is com-
posed of several domains with homologies
to other proteins: a finger domain (residues
4-50), a growth factor domain (residues 50-
87), two kringles of about 80 residues, and
the protease domain (residues 276-527),
comprising the catalytic triad. Binding of t-
PA to fibrin is most likely mediated via the
finger and the second kringle domains.
Urokinase-type plasminogen activa-
tor (u-PA) is composed of an epidermal
growth factor domain, one kringle domain
and a protease domain containing the cata-
lytic triad. The epidermal growth factor
domain is responsible for the binding of u-
PA to its receptor, which is present on the
surface of a variety of cells. Single chain u-
PA is converted to two chain u-PA by cleav-
Figure 1. Schematic representation of the plasminogen (fibrinolytic) system.
age of the Lys158-Ile159 peptide bond.
The proenzyme, plasminogen, is converted to the active enzyme plasmin by
tissue-type plasminogen activator (t-PA) or by urokinase-type plasminogen
α 2-Antiplasmin (α 2-AP, α2-plasmin
activator (u-PA), which binds to a cellular u-PA receptor (u-PAR). Plasmin inhibitor) was originally isolated as a gly-
degrades fibrin and can convert latent matrix metalloproteinases (pro-MMPs) into coprotein containing 452 amino acids but it
active MMPs, which in turn degrade extracellular matrix (ECM). Pro-MMPs may
was later shown that native α2-AP contains
also be activated directly by u-PA, or by other MMP. t-PA mediated plasminogen
activation is primarily involved in fibrin homeostasis, while plasmin generation via 464 amino acids. It is unique among serpins
u-PA, complexed with u-PAR plays a role in tissue remodeling. Inhibition may by having a COOH-terminal extension of
occur at the level of the plasminogen activators by plasminogen activator inhibitors 51 amino acid residues, which contains a
(mainly PAI-1 and possibly PAI-2), at the level of plasmin by α2-antiplasmin and at
the level of the MMPs by tissue inhibitors of MMP’s (TIMPs). binding site that reacts with the lysine-bind-
ing sites of both plasminogen and plasmin.
The NH2-terminal Gln14 residue of α2-AP
2
functional domains (modules). The inhibitors of the plas- (Gln in the original numbering system) can cross-link
minogen system are members of the serpin (serine pro- to Aα-chains of fibrin, in a process which requires Ca2+
teinase inhibitor) superfamily. They have in their COOH- and is catalyzed by activated coagulation factor XIII.
terminal region a specific reactive site peptide bond (Arg- The two most important plasminogen activator in-
X or Lys-X), which is cleaved by their target enzyme, hibitors (PAIs) are PAI-1 and PAI-2. PAI-1 is stabilized
resulting in the formation of an inactive enzyme-inhibi- by a tight binding to the cell adhesion protein vitronectin.
tor complex. The physicochemical and genetic proper- PAI-2 exists in two different forms with comparable ki-
ties of the main components of the plasminogen system3 netic properties, a 47 kDa intracellular non-glycosylated
and of the MMP system, and their interactions2 are de- form with pI 5.0 and a 60 kDa secreted glycosylated
scribed in detail elsewhere. form.
Plasminogen consists of 791 amino acids as deter- The specific cell surface u-PA receptor (u-PAR) is
mined by cDNA sequencing, although originally 790 synthesized as a 313 amino acid polypeptide, which is
amino acids were identified by protein sequencing. It is posttranslationally processed at the COOH-terminus into
organized in seven structural domains, comprising a a protein of 283 amino acids anchored to the plasma
“preactivation peptide” (amino acid residues 1-77), 5 membrane by a glycosyl phosphatidylinositol (GPI)
sequential homologous kringle domains (disulfide moiety. It binds all forms of u-PA containing an intact
bonded triple loop structures of about 80 residues each), growth factor domain, with high affinity. It is composed
and the proteinase domain (residues 562-791). The of three distantly related homologous structural domains,
kringle domains contain lysine-binding sites that play a of which the NH2-terminal one binds u-PA.
crucial role in the specific binding to fibrin, cell sur-
faces and α2-antiplasmin. Plasminogen is converted to
plasmin by cleavage of a single Arg561-Val562 peptide
bond.

2 American Society of Hematology


Figure 2. Molecular interactions determining the fibrin-specificity of plasminogen activators.
Non-fibrin-specific plasminogen activators (streptokinase, two chain urokinase, anisoylated plasmino-
gen-streptokinase activator complex or anistreplase) activate both plasminogen in the fluid phase and
fibrin-associated plasminogen. Fibrin-specific plasminogen activators (t-PA, scu-PA and staphylokinase)
preferentially activate fibrin-associated plasminogen

Phenotype of Mice Deficient in Plasminogen presence of fibrin (KM between 0.15 and 1.5 µM). This
System Components increased affinity appears to be the result of a “surface
Targeting of genes via homologous recombination in assembly” of plasminogen activator and plasminogen on
embryonic stem cells has allowed the generation of de- the fibrin surface. In this reaction t-PA binds via the
ficiencies of specific gene products in transgenic mice. finger domain and kringle 2 to fibrin, and plasminogen
Mice with single deficiency of t-PA, u-PA, PAI-1, u-PAR, binds primarily via the “lysine-binding site” in kringle
plasminogen or α2-AP survived embryonic development 1. Thus one way of regulating fibrinolysis is at the level
and were apparently normal at birth, while no effects on of plasminogen activation localized at the fibrin surface.
health and survival were observed in most gene-inacti- Plasmin is very rapidly inactivated by α2-AP (k1~ 107 M-
1
vated animals. However, plasminogen deficient and com- sec-1); the initial half-life of free plasmin in the blood is
bined t-PA:u-PA deficient mice developed chronic ul- therefore estimated to be approximately 0.1 sec. Plas-
cerations and rectal prolapse and suffered a progressive min with an occupied lysine-binding site is however in-
wasting syndrome and a significantly shortened life span activated 50 times more slowly by α2-AP. Reversible
due to generalized thrombosis and organ failure (for ref- blocking of the active site of plasmin with substrate also
erences cfr. 4). Contrary to patients with low or absent markedly reduces the rate of inactivation by α2-AP.
plasma PAI-1 or α2-AP levels, PAI-1 or α2-AP deficient From these findings one can extrapolate that plas-
mice did not reveal spontaneous or delayed rebleeding, min molecules generated on the fibrin surface, which
even after trauma. Thus the plasminogen system is im- are bound to fibrin through their lysine-binding sites and
portant in maintaining vascular patency, and t-PA and u- involved in fibrin degradation, are protected from rapid
PA are the only physiologically significant plasminogen inactivation by α2-AP. Plasmin released from the fibrin
activators in vivo which appear to cooperate in fibrin surface would, however, be rapidly inactivated.
surveillance in the mouse. Control of plasminogen activation: Rapid removal
of t-PA from the blood occurs by clearance in the liver
The Plasminogen System and Fibrin Homeostasis via two different recognition systems.6 Hepatocytes bind
t-PA via the low density lipoprotein receptor-related pro-
Physiological fibrinolysis tein (LRP) and endothelial cells via a mannose-depen-
Molecular mechanism: Physiological fibrinolysis dent receptor. PAI-1 reacts with t-PA with a second or-
appears to be regulated by specific molecular interac- der rate constant of about 107 M-1s-1. PAI activity is very
tions between components of the plasminogen system.5 rapidly cleared from the circulation via the liver.7
t-PA has a weak affinity for plasminogen in the absence The synthesis and secretion of t-PA and PAI-1 by
of fibrin (KM = 65 µM) but a much higher affinity in the endothelial cells is highly regulated. Histamine and

Hematology 2001 3
thrombin bind to specific receptors and activate phospho- rupture of an atheromatous plaque in the wall of a coro-
lipase C which acts on phosphatidyl-inositol bisphos- nary artery. Occlusive thrombosis results in loss of blood
phate to produce diacylglycerol, which activates mem- flow to vital organs producing local oxygen deprivation,
brane-bound protein kinase C, that regulates t-PA syn- cell necrosis and loss of organ function. The hypothesis
thesis. Synthesis and secretion of PAI-1 can be modu- underlying thrombolytic therapy of thromboembolic dis-
lated by various agonists.7,8 ease is that early and sustained recanalization prevents
Most cells bind plasminogen via its lysine binding cell death, reduces infarct size, preserves organ func-
sites with a high capacity (> 107 sites per cell) but a rela- tion, and reduces early and late mortality. One approach
tively low affinity (Kd of 1 µM). Gangliosides as well as to treatment consists of the pharmacological dissolution
membrane proteins with COOH-terminal lysine residues of the blood clot by intravenous infusion of plasmino-
such as α-enolase, also bind plasminogen (for references, gen activators that activate the plasminogen system. Pri-
cfr. 9). Endothelial cells bind t-PA and plasminogen via mary angioplasty, when applied under optimal circum-
annexin II and thereby may play a role in maintaining stances, may provide a similar or better alternative, al-
blood fluidity. Lp(a) competes with plasminogen for though the relative clinical benefits of the pharmaco-
binding and may play a role in the regulation of fibrin- logical and interventional approaches remain debated.
olysis at the endothelial cell surface. Thrombin The modern era of thrombolytic therapy started
activatable fibrinolysis inhibitor (TAFI) may have an around 1980 with the demonstration by De Wood et al
antifibrinolytic effect by removing COOH-terminal that myocardial infarction in its early stage was invari-
lysine residues from the fibrin surface. ably associated with thrombotic coronary artery occlu-
sion and the demonstration by Rentrop et al that infu-
Pathophysiology of Fibrinolysis sion of streptokinase within the infarct-related coronary
artery early after symptom onset induced rapid recanali-
Impaired fibrinolysis and thrombosis: A deficient fibrin- zation. Randomized clinical trials with short term intra-
olytic response may be caused by impaired release of t- venous streptokinase demonstrated moderate but signifi-
PA from the vessel wall or by an increased rate of neu- cant potency for coronary artery recanalization and cul-
tralization (for references cfr. 10). A causal relationship minated in 1986 in the GISSI trial, which demonstrated
between deficient synthesis and/or release of t-PA and a significant overall reduction in mortality with intrave-
thrombosis has however not been conclusively estab- nous streptokinase (for references cfr. 11).
lished in man. Transgenic mice which totally lack func- In a parallel development in the early 1980s, eluci-
tional t-PA lyse experimental pulmonary emboli at a dation of biochemical mechanisms that regulate physi-
markedly reduced rate, but are healthy under basal con- ological fibrinolysis provided the conceptual framework
ditions. for fibrin-selective thrombolysis with t-PA, which fu-
The PAI-1 concentration in plasma is increased in eled the hope that more specific and efficacious throm-
several diseases including venous thromboembolism, bolytic agents could be developed.5 With the develop-
obesity, sepsis and coronary artery disease. There is a ment of recombinant DNA technology, recombinant
clear correlation between the circadian variation in the human t-PA could concurrently be produced in suffi-
time of onset of myocardial infarction, with the highest cient amounts to test its clinical efficacy against the non-
incidence at about 8 a.m., and the circadian rhythm of fibrin-selective streptokinase.
plasma PAI-1 activity, which is also highest early in the Initially coronary patency studies (TIMI-1 and
morning. ECSG-1) supported the higher efficacy of fibrin-selec-
Enhanced fibrinolysis and bleeding: Increased lev- tive recombinant t-PA (rt-PA) over non fibrin-selective
els of t-PA or deficiency of α2-AP or PAI-1 may cause a streptokinase, and several mechanistic trials of the TIMI
bleeding tendency. Homozygous α2-AP deficiency may organization led by E. Braunwald, the ECSG organiza-
be associated with a severe and heterozygosity with no tion chaired by M. Verstraete and the TAMI group of E.
or only a mild hemorrhagic diathesis (for references cfr. Topol and R. Califf validated and extended the hypoth-
10). Excessive fibrinolysis due to decreased PAI-1 lev- esis of fibrin-selective thrombolytic therapy for acute
els has been reported in a few cases and was apparently myocardial infarction (for references cfr. 12). However,
associated with bleeding complications. two subsequent megatrials (GISSI-2 and ISIS-3) could
not confirm that this translated into a mortality benefit.
Thrombolytic Therapy Finally, the GUSTO trial 13 and its angiographic
substudy14 conclusively demonstrated that brisk full per-
Major developments since 1980: Acute myocardial inf- fusion of the infarct vessel (TIMI grade 3 flow) with
arction is the first cause of death and disability in West- early and persistent coronary artery recanalization are
ern societies. It is caused by thrombosis, triggered by the primary determinants of clinical benefit.15 The ben-

4 American Society of Hematology


eficial effect of fibrin-selectivity with respect to bleed- celerated” alteplase with intravenous heparin produced
ing is less convincing, but in aggregate, fibrin-selectiv- somewhat over 50% complete recanalization (TIMI
ity is a desirable property of thrombolytic agents, as dis- grade 3 flow) at 90 minutes compared to around 30%
cussed in more detail elsewhere.11 with streptokinase and aspirin. The 30 day mortality in
Currently used thrombolytic agents: Thrombolytic over 40,000 patients was 6.3% for rt-PA and 7.3% for
agents that are generally approved for use in patients streptokinase (p = 0.001). A combined end point of death
with acute myocardial infarction include streptokinase, or disabling stroke was also significantly lower in the
recombinant tissue-type plasminogen activator (rt-PA or accelerated alteplase group than in the streptokinase-only
alteplase), and the rt-PA derivatives reteplase and groups (6.9% versus 7.8%, p = 0.006). This difference
tenecteplase (TNK-rtPA). The beneficial effects of
thrombolytic therapy in acute myocardial infarction have
been well established in placebo controlled clinical tri-
als (for references cfr. 16) and it has become routine Table 1. Current indications/contraindications and currently
used regimens for thrombolytic therapy in acute myocardial
treatment. It is given to more than 750,000 patients per infarction.
year worldwide, while at least three times that number
could potentially benefit from this treatment. The cur- A. Indications and contraindications
rent indications and contraindications to thrombolytic Indications
therapy in patients with acute myocardial infarction and Patients with chest pain consistent with the diagnosis of acute
the currently used regimens for coronary thrombolysis myocardial infarction and at least 0.1 mm of ST-segment
elevation in at least two contiguous ECG leads in whom
are also briefly summarized in Table 1. treatment can be initiated within 12 hours of pain onset,
Streptokinase is a bacterial protein that, when added provided there are no contraindications to thrombolytic
to human plasma, forms a complex with plasminogen; therapy.
this complex activates other plasminogen molecules to Contraindications
plasmin. The streptokinase-plasmin(ogen) complex is in- History of a serious bleeding tendency.
sensitive to circulating proteinase inhibitors and activates Recent acute internal hemorrhages.
circulating and fibrin-bound plasminogen relatively in- Major surgery, trauma, or delivery within 10 days.
discriminately, producing the so-called “systemic lytic Traumatic cardiopulmonary resuscitation.
state,” characterized by fibrinogen degradation and α2- Vascular puncture in a noncompressible site.
AP depletion in circulating blood. The standard dose in Uncontrolled hypertension.
patients with acute myocardial infarction is 1.5 million U Previous use of streptokinase is a contraindication for its
intravenously infused over 60 minutes. Streptokinase repeated administration.
causes transient hypotension in many patients and signifi-
cant allergic reactions in a small percentage of patients. Its B. Currently used regimens
administration causes a rapid rise in anti-streptokinase an- Streptokinase and aspirin
tibody titer after about 4 to 7 days, which is sufficient to Streptokinase 1.5 million U IV over 30 to 60 minutes,
neutralize (in vitro) a standard dose of streptokinase and combined with acetylsalicylic acid (ASA) 160 to 325 mg daily
to make repeated treatment of uncertain efficacy. started as soon as possible and continued indefinitely.
t-PA is a human protein produced by recombinant Alteplase and intravenous heparin*
DNA technology (recombinant t-PA, rt-PA, alteplase). t- Alteplase (recombinant tissue-type plasminogen activator; rt-
PA is a poor enzyme in the absence of fibrin, which en- PA) 100 mg IV over 90 minutes (15 mg bolus, 0.75 mg/kg not
exceeding 50 mg over 30 minutes, and 0.5 mg/kg not
hances the activation rate of plasminogen by at least 100 exceeding 35 mg over the next hour) combined with 160 to
times. Activation of the fibrinolytic system thus seems to 325 mg ASA and immediate intravenous heparin (5000 U
be triggered by and largely confined to fibrin. The pre- bolus and 1000 U per hour, preferably monitored with
activated partial thromboplastin time).
ferred dosage regimen of fibrin-selective alteplase at
Selection of regimen
present consists of a weight-adjusted, accelerated (“front-
In GUSTO, the accelerated alteplase regimen was associated
loaded”) regimen over 90 minutes (15 mg bolus, 0.75 mg/ with a statistically significant lower mortality than streptoki-
kg over 30 minutes [not to exceed 50 mg], and 0.5 mg/kg nase (6.3% vs 7.3%, p= .001) but with a slightly higher
over 60 minutes [not to exceed 35 mg]), because of the incidence (0.1%) of survival with disabling stroke.
survival benefit of this accelerated regimen over streptoki- The t-PA congeners (reteplase and tenecteplase) have been
nase demonstrated in the GUSTO trial.13,17 shown in comparative megatrials to be equivalent to alteplase
but they can be administered as a double or single bolus,
Comparative trials of streptokinase and alteplase respectively.
have shown significant differences in efficacy for early
coronary artery recanalization (for references cfr. 11). * Recent evidence (ASSENT III) suggests that low molecular
In the angiographic substudy of the GUSTO trial,14 “ac- weight heparin may be preferable over unfractionated heparin.

Hematology 2001 5
was maintained after one year. The slightly but signifi- bodies against the platelet GPIIb/IIIa receptor and small
cantly (p = 0.03) increased risk of intracranial hemor- synthetic arginine-glycine-aspartic acid (RGD)-contain-
rhage with rt-PA remains, however, a concern. ing peptides, are presently being explored (for references
Toward improved thrombolytic therapy: Throm- cfr. 18). The concomitant administration of potent platelet
bolytic therapy could be improved by: 1) earlier and ac- GPIIb/IIIa antagonists with aspirin, heparin and reduced
celerated treatment to reduce the duration of ischemia; dose reteplase was found to be comparable but not bet-
2) the use of plasminogen activators with increased ter than full dose reteplase in the recent GUSTO V trial.
thrombolytic potency to enhance coronary thromboly- Another approach consists of the use of selective throm-
sis, which can be administered by bolus injection; and bin inhibitors, including hirudin and its derivatives, or
3) the use of more specific and potent anticoagulant and synthetic thrombin inhibitors. An ongoing megatrial,
antiplatelet agents to accelerate recanalization and pre- HERO 2, comparing hirulog with heparin, both in com-
vent reocclusion (for references cfr. 16). bination with thrombolytic therapy has demonstrated a
There is compelling evidence that patients should marginal benefit of the former.
receive thrombolytic therapy as soon after the onset of Staphylokinase, a potential highly fibrin-selective
symptoms as possible. Continued and intensified edu- thrombolytic agent: Staphylokinase is a single polypep-
cation of the public, paramedical personnel and physi- tide chain of 136 amino acids without disulfide bridges
cians, together with the development of rapid and effi- secreted by certain strains of Staphylococcus aureus.
cient triage systems and out of hospital treatment are Like streptokinase, staphylokinase is not an enzyme but
essential to achieve these goals. it forms a 1:1 stoichiometric complex with plasmin(ogen)
Variants of rt-PA with reduced clearance, altered that activates other plasminogen molecules. Staphylo-
binding to fibrin, and resistance to plasma protease in- kinase added to human plasma containing a fibrin clot
hibitors have been constructed. Two variants, reteplase will react poorly with plasminogen in plasma, but will
and tenecteplase have been approved for clinical use. react with high affinity with traces of plasmin at the clot
Reteplase is a deletion mutant, consisting of the kringle surface, where the plasmin.staphylokinase complex ef-
2 and protease domains of rt-PA, given as a double bo- ficiently activates plasminogen to plasmin. Both plas-
lus in patients with acute myocardial infarction, which min-staphylokinase and uncomplexed plasmin bound to
is probably equipotent to alteplase as demonstrated in fibrin are protected from rapid inhibition by α2-AP,
the GUSTO III trial. Tenecteplase, an rt-PA mutant in whereas their unbound counterparts, liberated from the
which Thr103 is substituted by Asn, Asn117 by Gln, and clot or generated in plasma, are rapidly inhibited by α2-
the sequence Lys296-His-Arg-Arg by Ala-Ala-Ala-Ala antiplasmin. The process of plasminogen activation is
has an 8-fold slower clearance and a 200-fold enhanced thereby confined to the thrombus, preventing excessive
resistance to PAI-1 as compared to alteplase. Given as a plasmin generation, α2-AP depletion and fibrinogen deg-
single bolus it is equivalent to alteplase, as demonstrated radation in plasma. The biochemical pathways govern-
in the ASSENT II trial. These trials indicate that rt-PA ing these fibrin-selective interactions are summarized
mutants can be produced with significantly reduced elsewhere (for references cfr. 20).
plasma clearance, but with similar thrombolytic potency In an effort to reduce the antigenicity of
as rt-PA.17 Because these variants have the same cata- staphylokinase, over 350 plasmids encoding mutants of
lytic machinery as rt-PA, they probably should not have SakSTAR (product of the gene cloned from the genomic
been expected to outperform rt-PA. In order to obtain DNA of a lysopenic S. aureus strain) were constructed
thrombolytic agents with an increased thrombolytic po- and expressed in E. coli, and the expression products
tency for coronary recanalization, it will probably be were purified and characterized.21 A comprehensive
necessary to turn to other plasminogen activators with analysis of combination variants led to the identifica-
higher specific activity and different mechanisms of fi- tion of SakSTAR (K35A,E65Q,K74Q, D82A,S84A,
brin-selectivity. Staphylokinase could constitute such an T90A,E99D,T101S,E108A,K109A,K130T,K135R) with
alternative, as further discussed below. a maintained fibrinolytic potency and fibrin selectivity
Aspirin and heparin have a limited impact on the in a human plasma milieu, and a markedly reduced re-
speed of coronary thrombolysis and on the resistance to activity with anti-SakSTAR antibodies in pooled immu-
lysis, and do not consistently prevent reocclusion. This nized patient plasma.
could have been anticipated on the basis of the un- Staphylokinase disappears from plasma in a biphasic
selective inhibition by aspirin of the synthesis of both mode with a t1/2α of 6.3 min and plasma clearance of
proaggregating and antiaggregating prostaglandins, and 270 ml/min. The clearance can, however, be reduced 5-
of the relative inefficacy of heparin to inhibit clot-asso- to 30-fold by selective chemical substitution with single
ciated thrombin. Several more specific approaches to polyethylene glycol molecules with Mr 5,000 to 20,000.
reduce platelet aggregation including monoclonal anti- A recombinant staphylokinase variant, substituted with

6 American Society of Hematology


a single polyethylene glycol molecule with Mr 5,000, predilection site of plaques have been observed between
has been investigated in a pilot study in patients with mice with a single deficiency of alipoprotein E (apoE)
acute myocardial infarction22 and a formal phase II trial or with a combined deficiency of apoE and t-PA, or of
in 500 patients has recently been concluded. In essence, apoE and u-PA, suggesting that plasmin is not essential
bolus doses of 0.0185 to 0.0375 mg/kg were found to be for subendothelial infiltration by macrophages. However,
comparable to front-loaded alteplase in terms of coro- destruction of the media with resultant aneurysmal dila-
nary artery recanalization at 60 minutes. tation and rupture of the vessel wall were more frequent
and severe in mice lacking apoE or apoE and t-PA than
The Plasminogen System and Tissue Remodeling in mice lacking apoE and u-PA.24 Macrophages were
Proteinases play an essential role in cell migration and absent in the media of uninvolved arteries and were only
tissue remodeling, occurring in many biological pro- able to infiltrate into and destroy the media of athero-
cesses. They degrade extracellular matrix components, sclerotic arteries after they degraded the elastin fibers.
a prerequisite for endothelial, smooth muscle, inflam- A dramatic increase of free u-PA activity (which is mini-
matory, or cancerous cells to migrate to distant sites, and mal in quiescent arteries) was generated by the infiltrat-
activate cytokines, or liberate sequestered growth fac- ing plaque macrophages, which abundantly expressed
tors. Recent gene targeting and gene transfer studies in MMP-3, MMP-9, MMP-12 and MMP-13, colocalizing
the mouse have revealed a pleiotropic role of the plas- with u-PA in plaque macrophages and suggesting that
minogen and the matrix metalloproteinase (MMP) sys- plasmin is a likely activator of proMMPs in vivo.24
tems in arterial neointima formation, in atherosclerosis,
aneurysm formation and myocardial ischemia, in angio- Myocardial ischemia
genesis, tumor growth and metastasis and in infection. Recently, a mouse model of chronic myocardial infarc-
These studies will be briefly reviewed here but have been tion has been used to evaluate the role of the plasmino-
discussed in more detail elsewhere.23 gen system in cardiac healing.25 Following ligation of
the left anterior descending coronary artery, wild type
Neointima formation or t-PA deficient mice heal their ischemic myocardium
u-PA, t-PA and, to a lesser degree, PAI-1 activity in the within two weeks via scar formation, i.e. the ischemic
vessel wall are significantly increased after injury, coin- myocardium becomes infiltrated by leukocytes, endot-
cident with the time of smooth muscle cell proliferation helial cells and fibroblasts with resultant deposition of
and migration; expression of MMP-3, MMP-7, MMP-9, collagen. In a fraction of these mice, rupture of the is-
MMP-12 and MMP-13 is induced in injured, transplanted chemic myocardium occurs shortly after infarction due
or atherosclerotic arteries (for references cfr. 23). to excessive u-PA-generated plasmin proteolysis by in-
Neointima formation and neointimal cell accumulation filtrating wound cells. In sharp contrast, mice lacking u-
after injury were significantly reduced in mice deficient PA or plasminogen are protected against ventricular wall
in u-PA, plasminogen or combined t-PA:u-PA due to rupture, but fail to heal the ischemic myocardium, which
impaired migration but not proliferation of medial and remains largely devoid of infiltrating leukocytes, endot-
neointimal smooth muscle cells (for references cfr. 23). helial cells and fibroblasts. Thus u-PA-generated plas-
u-PAR deficient arteries developed a similar degree of min proteolysis is required for healing, but needs to be
neointima formation as wild type arteries, suggesting that carefully balanced to avoid tissue destruction and ven-
sufficient pericellular plasmin proteolysis is present in tricular wall rupture (for references cfr. 23).
the absence of binding of u-PA to its cellular receptor.
Similar levels of proMMP-2 and active MMP-2 but sig- Angiogenesis
nificantly lower levels of active MMP-9 were present in Migration of endothelial cells involves proteolysis of the
extracts of plasminogen deficient arteries than of wild extracellular matrix. When endothelial cells migrate, they
type arteries. Since MMP-9 is primarily expressed by significantly upregulate u-PA, u-PAR, and, to a lesser
leukocytes, which are involved in the healing of the in- extent, t-PA at the leading edge of migration (for refer-
jured arteries, the lower active MMP-9 levels may con- ences cfr. 26). Although PAI-1 is also increased, its ex-
tribute to the impaired medial and adventitial remodel- pression at different locations and times allows a net in-
ing and to the reduced neointima formation. crease in fibrinolytic activity. Surprisingly however, mice
deficient in u-PA and/or t-PA, PAI-1, u-PAR, plasmino-
Atherosclerosis gen or α2-AP develop normally without overt vascular
Expression of t-PA, u-PA and several MMPs in plaques anomalies.
is increased, but a causative role of the plasminogen (Plg) Migration of endothelial cells alongside a denuded
and/or MMP system in atherosclerosis has not been con- vessel does not require u-PA-generated plasmin, whereas
clusively demonstrated. No differences in the size or the invasion of endothelial cells through an anatomic bar-

Hematology 2001 7
rier of extracellular matrix may (ischemic myocardium, coccus neoformans pathogen, which disseminated
polyoma tumor model) or may not (cornea, skin heal- widely and ultimately infected the brain, leading to death.
ing) require plasmin proteolysis. Whether these differ- This pattern of wide dissemination and death with strain
ences relate to the composition and/or thickness of the 52D has only been seen in profoundly immuno-
extracellular matrix, or to the expression pattern of pro- incompentent mice (for references cfr. 23).
teinases by endothelial cells during these different con- A number of invasive bacteria can interact with the
ditions, remains to be determined. host plasminogen system by expressing endogenous plas-
minogen activators and by binding plasminogen directly
Tumor growth and dissemination. through bacterial cell-surface receptors, thereby allow-
Pericellular plasmin proteolysis has been proposed to ing them to utilize the plasminogen activators of the host
play a role in tumor invasion and metastasis by facilitat- for activation. Studies in plasminogen-deficient mice
ing the migration of malignant cells through anatomic indicate that plasminogen is required for efficient dis-
barriers via degradation of extracellular matrix constitu- semination of the spirochete Borrelia burgdorferi within
ents. u-PA generally exerts a positive effect on tumori- the tick and for enhancement of spirochetemia in mice.29
genesis, although its mode of action (e.g. on the growth, A similar requirement of host-derived plasminogen by
dissemination or progression of the tumors) may differ Yersinia pestis for its dissemination was recently re-
according to the models studied. ported. Bacterial strains expressing a plasminogen acti-
Based on its ability to block u-PA proteolysis, PAI- vator (pla+) escaped elimination by the host immune
1 would be anticipated to impair tumorigenesis. The role system and were almost a million-fold more pathogenic
of PAI-1 in tumor growth and metastasis remains, how- than pla- strains (not expressing such plasminogen acti-
ever, controversial as epidemiologic studies indicate that vator) in wild type but not in plasminogen deficient hosts.
PAI-1 is a marker of poor prognosis for survival of pa-
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Hematology 2001 9