Portada Simposios - Supplements - Haematologica

108 Haematologica (ed. esp.), volumen 85, supl. 2, octubre 2000
lation and resistance to plasma proteinase inhibitors
(for references, cf. 9). During thrombolytic therapy
there is a vast excess of t-PA over PAI-1 in the circulation,
but critical lysis occurs at the surface of an arterial
thrombus where the local PAI-1 concentration
can be very high. Therefore, mutants with resistance
to PAI-1 may be useful to reduce reocclusion. In addition,
mutants with prolonged half-life may allow
efficient thrombolysis by bolus administration at a
reduced dose. Several mutants and variants of t-PA
are presently evaluated at the preclinical level in animal
models of venous and arterial thrombosis and
in pilot studies, mainly in patients with acute myocardial
infarction. These agents include reteplase
(Rapilysin ® or Ecokinase ® ), TNK-rt-PA, the vampire
bat (Desmodus rotundus) salivary plasminogen activator,
and lanoteplase.
Reteplase is a single-chain non-glycosylated deletion
variant consisting only of the kringle 2 and the
proteinase domain of human t-PA; it contains amino
acids 1-3 and 176-527 (deletion of Val 4 -Glu 175 );
the Arg 275 -Ile 276 plasmin cleave site is maintained.
The plasminogenolytic activity of reteplase and of
rt-PA in the absence of a stimulator does not differ,
but the activity of reteplase in the presence of CNBr
fragments of fibrinogen as a stimulator was 4-fold
lower as compared to rt-PA, and its binding to fibrin
was 5-fold lower. Reteplase and rt-PA are inhibited
by PAI-1 to a similar degree. In patients, an initial
half-life of 14-18 min was observed for reteplase, as
compared to about 3-4 min for wild-type rt-PA. In
the GUSTO-III trial, no clinical benefit of reteplase
over alteplase could be demonstrated, leading to the
conclusion that both agents are equivalent 10 .
In TNK-rt-PA, replacement of Asn 117 with Gln
(N117Q) deletes the glycosylation site in kringle
1 whereas substitution of Thr 103 by Asn(T103N) reintroduces
a glycosylation site in kringle 1, but at a different
site; these modifications substantially decrease
the plasma clearance rate. In addition, the amino
acids Lys 296 -His 297 -Arg 298 -Arg 299 were each replaced
with Ala, which confers resistance to inhibition by
PAI-1. TNK-rt-PA has a similar ability as wild-type
rt-PA to bind to fibrin, and lyses fibrin clots in a plasma
milieu with enhanced fibrin-specificity. In patients
with acute myocardial infarction, TNK-rt-PA has a
half-life of 17 ± 7 min, as compared to 3.5 ± 1.4 min
for wild-type rt-PA 11 . In the TIMI-10B trial, a phase
2 efficacy trial, a single bolus of 40 mg TNK-t-PA yielded
similar TIMI-3 flow rates at 90 minutes as accelerated
rt-PA, with faster and more complete reperfusion
12 .
Different molecular forms of the Desmodus salivary
plasminogen activator (DSPA) have been purified,
characterized, cloned and expressed. Two high
molecular weight forms, DSPA1 (43 kDa) and DS-
PA2 (39 kDa) exhibit about 85 % homology to human
t-PA, but contain neither a kringle 2 domain
nor a plasmin-sensitive cleavage site. DSPA lacks
the finger-domain and DSPA lacks the finger and
epidermal growth factor domains. DSPA1 and DS-
PA2 exhibit a specific activity in vitro that is equal
to or higher than that of rt-PA, a relative PAI-1 resistance
and a greatly enhanced fibrin specificity with a
strict requirement for polymeric fibrin as a cofactor.
In several animal models of thrombolysis, DSPA1
has a 2.5 times higher potency and four-to eight-fold
slower clearance than rt-PA (for references, cf. 2,9).
Lanoteplase is a deletion mutant of rt-PA (without
the finger and growth factor domains) in which glycosylation
at Asn 117 is lacking. Given as a single bolus
of 120 U/kg in the “Intravenous n-PA for Treating
Infarcting Myocardium Early (InTIME-1)” trial, higher
infarct-related vessel patency rates were obtained
than with alteplase 2,9 .
Staphylokinase and variants
Recombinant staphylokinase was compared to accelerated
weight-adjusted alteplase in two open randomized
studies, each in 100 patients with acute
myocardial infarction (for references cfr. 7,8). In
both studies recombinant staphylokinase was found
to be at least equipotent to alteplase in terms of
complete arterial recanalization within 90 minutes.
Staphylokinase was highly fibrin-selective, as revealed
by virtually unaltered levels of plasma fibrinogen,
plasminogen and 2 -antiplasmin. No strokes, allergic
reactions or other side effects were recorded.
Thus intravenous staphylokinase, combined with heparin
and aspirin, is a potent, rapidly acting and
highly fibrin-selective thrombolytic agent in patients
with acute myocardial infarction.
However, most patients develop high titers of neutralizing
specific IgG after infusion of staphylokinase,
which would predict therapeutic refractoriness upon
repeated administration. Efforts have been undertaken
to reduce the immunogenicity of staphylokinase
by site-directed mutagenesis. Wild type staphylokinase
(SakSTAR variant), was found to contain
three non-overlapping immunodominant epitopes,
at least two of which could be eliminated, albeit
with partial inactivation of the molecule, by site directed
substitution of clusters of two or three charged
amino acids with alanine.
In an effort to optimize the activity/antigenicity ratio,
a comprehensive site-directed mutagenesis study
was carried out, yielding SakSTAR (K35A, E65Q,
K74Q, D82A, S84A, T90A, E99D, T101S, E108A,
K109A, K130T, K135R, K136A, 137) with a maintained
fibrinolytic potency and fibrin-selectivity in a
human plasma milieu, and a markedly reduced reactivity
with anti-SakSTAR antibodies in pooled immunized
patient plasma. Intra-arterial administration in
patients with peripheral arterial occlusion induced
SakSTAR-neutralizing activity exceeding 5 g/ml
plasma in only 2 of 7 patients. Thus staphylokinase
variants with markedly reduced antibody induction
and intact thrombolytic potency can be generated 13 .