Portada Simposios - Supplements - Haematologica

XLII Reunión Nacional de la AEHH y XVI Congreso de la SETH. Simposios
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acute myocardial infarction include streptokinase, recombinant
tissue-type plasminogen activator (rt-PA or
alteplase), rt-PA derivatives such as reteplase and
TNK-rtPA, anisoylated plasminogen-streptokinase activator
complex (APSAC or anistreplase), two-chain urokinase-type
plasminogen activator (tcu-PA or urokinase),
recombinant single-chain u-PA (scu-PA, pro-u-PA
or prourokinase), and recombinant staphylokinase
and derivatives. Fibrin-selective agents (rt-PA and derivatives,
staphylokinase and derivatives and to a lesser
extent scu-PA) which digest the clot in the absence of
systemic plasminogen activation are distinguished
from non fibrin-selective agents (streptokinase, tcu-PA
and APSAC) which activate systemic and fibrin-bound
plasminogen relatively indiscriminately (for references,
cf. 1,2). In this contribution, we will review the development
of novel fibrin-specific thrombolytic agents.
Molecular interactions regulating
fibrin-specific fibrinolysis
Tissue-type plasminogen activator
Human t-PA is a single-chain serine proteinase (M r
70 kDa), consisting of 530 amino acids 3 . The t-PA
molecule contains four domains: 1) an NH 2 -terminal
region of 47-residues (residues 4 to 50) which is
homologous with the finger domains mediating the
fibrin affinity of fibronectin; 2) residues 50 to
87 which are homologous with epidermal growth
factor; 3) two regions comprising residues 87 to
176 and 176 to 262 which share a high degree of
homology with the five kringles of plasminogen and
4) a serine proteinase domain (residues 276 to 527)
with the active site residues His 322 , Asp 371 and Ser 478 .
The t-PA molecule comprises three potential N-glycosylation
sites, at Asn 117 , Asn 184 and Asn 448 (3).
t-PA is a poor enzyme in the absence of fibrin, but
the presence of fibrin strikingly enhances the activation
rate of plasminogen. Kinetic data support a mechanism
in which fibrin provides a surface to which
t-PA and plasminogen adsorb in a sequential and ordered
way yielding a cyclic ternary complex. Formation
of this complex results in an enhanced affinity
of t-PA for plasminogen, yielding up to three orders
of magnitude higher efficiencies for plasminogen activation
4 . In agreement with this mechanism, the increase
in fibrin stimulation which occurs after formation
of fibrin X-polymers, is associated with an
enhanced binding of t-PA and plasminogen. This increased
and altered binding of both enzyme and
substrate to fibrin is mediated in part by COOH-terminal
lysine residues generated by plasmin cleavage.
Interaction of these COOH-terminal lysines with lysine
binding sites on t-PA and plasminogen, may
allow an improved alignment as well as allosteric
changes of the t-PA and plasminogen moieties, thus
enhancing the rate of plasminogen activation 5 .
Plasmin formed at the fibrin surface has both its
lysine binding sites and active site occupied and is
thus only slowly inactivated by 2 -antiplasmin
(half-life of about 10-100 s); in contrast, free plasmin,
when formed, is rapidly inhibited by 2 -antiplasmin
(half-life of about 0.1 s) 6 .
Staphylokinase
Staphylokinase is a single polypeptide chain of
136 amino acids without disulfide bridges secreted
by certain strains of Staphylococcus aureus. Like streptokinase,
staphylokinase is not an enzyme but it forms
a 1:1 stoichiometric complex with plasmin(ogen)
that activates other plasminogen molecules.
When staphylokinase is added to human plasma
containing a fibrin clot, it will react poorly with plasminogen
in plasma, but it will react with high affinity
with traces of plasmin at the clot surface. At the clot
surface, the plasmin.staphylokinase complex efficiently
activates plasminogen to plasmin. Both plasmin.staphylokinase
and uncomplexed plasmin
bound to fibrin are protected from rapid inhibition
by 2 -antiplasmin, whereas their unbound counterparts,
liberated from the clot or generated in plasma,
are rapidly inhibited by 2 -antiplasmin. Thereby
the process of plasminogen activation is confined
to the thrombus, preventing excessive plasmin generation,
2 -antiplasmin depletion and fibrinogen degradation
in plasma. The biochemical pathways governing
these fibrin-selective interactions are summarized
elsewhere 7,8 .
Fibrin-specific agents
Currently available thrombolytic agents have several
limitations. At best, TIMI 3 flow within 90 minutes
is obtained in somewhat over 50 % of patients,
acute coronary reocclusion occurs in about 10 % of
patients, coronary recanalization requires on average
45 minutes or more, intracerebral bleeding occurs
in 0.3 % to 0.7 %, and the residual mortality is at
least 50 % of that without thrombolytic treatment.
Furthermore, the most commonly used agents streptokinase
and alteplase are routinely administered by
intravenous infusion over 60 to 90 minutes, which in
emergency conditions is less convenient than bolus
injection. Thrombolytic therapy could be improved
by earlier and accelerated treatment to reduce the
duration of ischemia, by the use of plasminogen activators
with increased thrombolytic potency to enhance
coronary thrombolysis, which can be administered
by bolus injection and by the use of more
specific and potent anticoagulant and antiplatelet
agents to accelerate recanalization and prevent reocclusion
(for references cfr. 1).
Mutants and variants of rt-PA
By deletion or substitution of functional domains,
by site-specific point mutations and/or by altering
the carbohydrate composition, mutants of rt-PA
have been produced with higher fibrin-specificity,
more zymogenicity, slower clearance from the circu-