1391
Tissue-Type Plasminogen Activator Mutants
Theoretical and Clinical Considerations
Nils U. Bang, MD
W'ITithin a short time after the first report on
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the successful cloning and expression of
TV
tissue-type plasminogen activator
(t-PA),l several laboratories began working on creating new t-PA derivatives through site-directed
mutagenesis.2-4 t-PA has unique properties that
make it an attractive choice for thrombolytic therapy. t-PA is "fibrin directed"; that is, it preferentially activates the fibrinolytic proenzyme plasminogen into plasmin on fibrin thrombi. In contrast,
streptokinase and urokinase, the older-used thrombolytic agents, indiscriminately activate thrombus
as well as plasma plasminogen.5 Nevertheless, work
on creating new t-PA mutants continued because
the following shortcomings of the native enzyme
were realized: 1) t-PA is not a very efficient plasminogen activator even on a fibrin thrombus; 2)
although t-PA was anticipated to cause minimal
bleeding complications, this prediction was not confirmed in major clinical trials in which t-PA was
given in the doses necessary for effective coronary
thrombolysis (100 mg per treatment); it proved as
likely to cause minor and major bleeding complications as streptokinase and in some patients caused
profound fibrinogen depletion and a serious coagulation defect6-8; 3) t-PA, even when given concomitantly with or followed by heparin, carried a high
risk of reocclusion (10-20%) once reperfusion has
been established; 4) the activity of infused t-PA is
attenuated because it is neutralized by a number of
plasma protease inhibitors, most notably plasminogen activator inhibitor 1 (PAI-1)9; and 5) t-PA
possesses a very short biologic half-life (<5 minutes) in humans and, therefore, must be administered in high doses as a constant intravenous infusion. The study by C.E.L. Lucore, S. Fujii and
See p 1204
B.E. Sobel10 in this issue of Circulation presents
pharmacodynamic data on t-PA and an interesting
t-PA mutant. The mutant has a sharply prolonged
tl/2 (30 minutes vs. 3 minutes for t-PA in the rabbit);
in other respects, it is comparable to t-PA in specific
From the Lilly Laboratories for Clinical Research, Eli Lilly
and Company; and Indiana University School of Medicine,
Departments of Medicine and Pathology, Wishard Memorial
Hospital, 1001 W. 10th Street, Indianapolis, IN 46202.
Received March 1, 1989; accepted March 1, 1989.
activity, fibrin affinity, and interaction with inhibitors, specifically PAI-1.
t-PA probably arose through exon shuffling, and
specific domains encoded by specific exons in t-PA
harbor specific and independent functions.2 The
major domains are called "finger" (F), epidermal
growth factor (EGF), kringle 1 (K1), kringle 2 (K2),
and serine protease (SP). The F and K2 domains
have been shown by several laboratories to be
involved in t-PA binding to fibrin.2 The EGF domain
may play a role in t-PA binding to cells. The KI
domain has not been assigned a function with
certainty except that it contains on residue Asn 117
a complex, high mannose branched carbohydrate
side chain that probably is responsible for the
effective binding, internalization, and catabolism of
t-PA by normal hepatocytes. The SP domain catalyzes the conversion of plasminogen into plasmin.3
The t-PA mutant examined by Lucore et al10 contains the F domain, a duplicated K2 domain, and
the SP domain, but the mutant lacks the EGF and
Kl domains. The lack of the EGF and Kl domains
explains why this and similar molecules reported
elsewhere possess prolonged biologic half-lives.3
Lucore et al10 elegantly show that the induction and
maintenance of a thrombolytic state, whether the
wild-type or mutant t-PA is used, depends entirely
on the concentration of free plasminogen activator
in the circulation. Thus, if the concentrations of
inhibitors (e.g., PAI-1) are elevated, the fibrinolytic
response to t-PA is "attenuated"; that is, active
t-PA will remain in the circulation for a shorter
time. On the other hand, if a t-PA mutant is introduced with substantially reduced clearance by hepatocytes and other cells during constant inhibitor
levels, the mutant will circulate at higher levels and
for longer time periods than the wild-type enzyme.
The interesting question of whether t-PA mutants
with prolonged t1,2 will result in therapeutic gains in
acute myocardial infarction cannot be answered
completely today. The mutant described by Lucore
et al'0 produced high enzyme levels for more prolonged time periods than did native t-PA. In theory,
if the patient is treated early enough, reperfusion
should be more rapid and additional salvage of
ischemic myocardium should occur. There is also a
distinct possibility that the incidence of reocclusion
could be reduced by this approach. Prolonged admin-
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Circulation Vol 79, No 6, June 1989
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istration of t-PA after successful reperfusion has
been reported to sharply lower the incidence of
reocclusion.11 However, the prolonged administration of t-PA also resulted in a substantial increase in
bleeding complications. Only prospective clinical
trials can resolve whether t-PA mutants of prolonged
t1/2 such as that described by Lucore et a110 significantly increase the benefit to risk ratio of thrombolytic therapy in acute myocardial infarction. A
separate issue is whether a plasminogen activator
with a prolonged t1/2 in some instances can be detrimental to the patient if and when bypass surgery is
contemplated in acute myocardial infarction.
Attempts to improve the functional properties of
t-PA through site-directed mutagenesis have been
less successful in other areas. Despite intensive
efforts,2-4 the creation of chimeric proteins with the
catalytic efficiency of urokinase and fibrin specificity of t-PA have not met with major success.
Although increased fibrin specificity has been shown
for certain mutants under in vitro conditions, the
gains in this area have not been impressive, and
whether such mutants will prove clinically superior
to t-PA is uncertain. Although certain mutants with
decreased affinity for the PAI-1 inhibitor can be
constructed, the manipulations involved also result
in loss in fibrin specificity and instability of the
protein.12'13 It is doubtful whether additional progress is going to be made with the current approach
of removing or substituting entire domains in the
t-PA protein. A complete understanding of the
three-dimensional structure of t-PA and its domains
appears to be the key to the construction of mutant
proteins in which discrete point mutations lead to
improved functional properties without serious
changes in the conformation of the entire molecule.
Ancillary therapy may also be of chief importance for improved results of enzymatic intervention in acute myocardial infarction. Many investigators suspect that the combinations recommended
today, for example, t-PA or streptokinase and heparin and/or aspirin, are suboptimal. Other candidate
drugs more efficacious than heparin or aspirin on
the arterial side of the circulation are activated
protein C14 and antiplatelet agents, for example,
thromboxane A2 receptor inhibitors in combination
with 5 -HT2 receptor antagonists,15 monoclonal
antibodies,16 or fibrinogenomimetic peptides17 capable of blocking the important step in platelet aggregation (the binding of fibrinogen to the GPIIb/IIJa
platelet receptor), and specific antithrombins such
as hirudin, and a variety of synthetic inhibitors.
Such approaches could improve our record in three
crucial areas of acute intervention in acute myocardial infarction: time to reperfusion, reduction of
reocclusion events, and reduction of bleeding complications.
(Circulation 1989;79:1391-1392)
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Tissue-type plasminogen activator mutants. Theoretical and clinical considerations.
N U Bang
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Circulation. 1989;79:1391-1392
doi: 10.1161/01.CIR.79.6.1391
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