HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Distinct dose-dependent effects of plasmin and TPA on coagulation
and hemorrhage
Daphne Stewart, Mansze Kong, Valery Novokhatny, Gary Jesmok, and Victor J. Marder
All thrombolytic agents in current clinical
usage are plasminogen activators. Although effective, plasminogen activators
uniformly increase the risk of bleeding
complications, especially intracranial
hemorrhage, and no laboratory test is
applicable to avoid such bleeding. We
report results of a randomized, blinded,
dose-ranging comparison of tissue-type
plasminogen activator (TPA) with a directacting thrombolytic agent, plasmin, in an
animal model of fibrinolytic hemorrhage.
This study focuses on the role of plasma
coagulation factors in hemostatic competence. Plasmin at 4-fold, 6-fold, and 8-fold
the thrombolytic dose (1 mg/kg) induced
a dose-dependent effect on coagulation,
depleting antiplasmin activity completely,
then degrading fibrinogen and factor VIII.
However, even with complete consumption of antiplasmin and decreases in fibrinogen and factor VIII to 20% of initial
activity, excessive bleeding did not occur.
Bleeding occurred only at 8-fold the
thrombolytic dose, on complete depletion of fibrinogen and factor VIII, manifest
as prolonged primary bleeding, but with
minimal effect on stable hemostatic sites.
Although TPA had minimal effect on coagulation, hemostasis was disrupted in a
dose-dependent manner, even at 25% of
the thrombolytic dose (1 mg/kg), manifest
as rebleeding from hemostatically stable
ear puncture sites. Plasmin degrades
plasma fibrinogen and factor VIII in a
dose-dependent manner, but it does not
disrupt hemostasis until clotting factors
are completely depleted, at an 8-fold
higher dose than is needed for thrombolysis. Plasmin has a 6-fold margin of safety,
in contrast with TPA, which causes hemorrhage at thrombolytic dosages. (Blood.
2003;101:3002-3007)
© 2003 by The American Society of Hematology
Introduction
Thrombolytic therapy with plasminogen activators is a highly
effective modality for achieving both vascular reperfusion and
clinical benefit in patients with arterial occlusions, for example,
acute myocardial infarction, peripheral arterial occlusion, and
ischemic stroke.1 However, such therapy is unfortunately accompanied by a significant risk of intracranial hemorrhage (ICH), which
occurs in 1% to 2% of patients who receive continuous treatment
for 2 to 24 hours.2,3 ICH is especially prevalent in patients who are
older and smaller, those who are hypertensive or have a history of
transient ischemic attack or stroke, and those treated with tissue
plasminogen activator (TPA).4,5 Patients treated for ischemic stroke
are especially prone to symptomatic or lethal ICH, with an
occurrence of 10% to 16% after receiving TPA or streptokinase
(SK), about 10-fold greater than in patients who receive only
anticoagulation.6 To date, the use of every effective thrombolytic
agent causes this adverse outcome, and even newly approved TPA
mutants7,8 or reduced-dosage regimens of TPA9 share this risk
for ICH.
We recently reported on an alternative to plasminogen activatortype thrombolytic agents, namely, plasmin, a direct fibrinolytic
enzyme that does not require plasminogen to achieve thrombolysis.10 First tested for efficacy more than 40 years ago, plasmin was
ineffective when administered by the intravenous route11 because it
was neutralized by plasma antiplasmin.12 However, our regional
administration of plasmin by catheter directly to the thrombus
avoids antiplasmin neutralization, and rabbit arterial and venous
thrombolysis models clearly document thrombolytic activity of
plasmin.10,13 In addition, our studies show a significant advantage
of plasmin over TPA in its safety from fibrinolytic hemorrhage.10
Thrombolytic doses of plasmin (2 or 4 mg/kg) did not cause
bleeding, whereas thrombolytic doses of TPA (1 or 2 mg/kg)
induced fibrinolytic hemorrhage.10 Our explanation for safety using
plasmin is that antiplasmin neutralizes plasmin that appears in the
circulation, whereas TPA circulates, binds to hemostatic plugs at
sites of vascular injury, degrades fibrin, and induces fresh
hemorrhage.10
Although plasmin clearly shows safety from bleeding with
therapeutic (thrombolytic) dosages, important questions still remain regarding its pharmacophysiology. For example, does a
higher dosage of plasmin consume antiplasmin, thereby allowing
free plasmin to circulate, degrade clotting factors, and induce
hemorrhage? If so, our hypothesis regarding the mode of action of
plasmin would be strengthened. In addition, such results would
indicate that laboratory monitoring of antiplasmin could provide a
useful predictive marker for bleeding. Because there is no currently
applicable laboratory assay available for predicting hemorrhage in
a patient receiving a plasminogen activator, such a monitoring tool
for thrombolytic therapy with plasmin would represent a unique
From the Vascular Medicine Program, Los Angeles Orthopaedic Hospital, The
David Geffen School of Medicine at UCLA, University of California Los Angeles,
CA; and Department of Pharmacology, Bayer Corporation, Research Triangle
Park, NC.
V.N. and G.J. are employed by the company whose thrombolytic agent
(plasmin) was studied in the present work.
Submitted August 20, 2002; accepted November 11, 2002. Prepublished online as
Blood First Edition Paper, November 21, 2002; DOI 10.1182/blood-2002-08-2546.
Supported by research funding provided by the Los Angeles Orthopaedic
Foundation and by a research grant from Bayer Corporation to Los Angeles
Orthopaedic Hospital.
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Reprints: Victor J. Marder, Los Angeles Orthopaedic Hospital/UCLA, 2400
South Flower St, Los Angeles, CA 90007; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2003 by The American Society of Hematology
BLOOD, 15 APRIL 2003 䡠 VOLUME 101, NUMBER 8
BLOOD, 15 APRIL 2003 䡠 VOLUME 101, NUMBER 8
advantage for its use. Further, does bleeding follow antiplasmin
consumption or does bleeding occur only after further consumption
of clotting factors such as fibrinogen and factor VIII? Observations
regarding these laboratory end points could provide insights for the
pathophysiology of plasmin-induced bleeding and for its prevention.
Accordingly, this study uses an animal model of fibrinolytic
hemorrhage14 to evaluate escalating dosages of plasmin, above the
2-mg/kg dose that dissolves experimental thrombi,13 to determine
if a threshold dose for bleeding exists and if such bleeding can be
predicted or at least explained by changes in blood coagulation or
fibrinolytic parameters. As control, and to test what dose of TPA
can be administered without inducing bleeding, TPA at progressively lower doses, below that which is optimal for thrombolysis (1
mg/kg),13 were compared for bleeding manifestations and for blood
coagulation parameters.
Materials and methods
Human plasminogen was purified from plasma by affinity chromatography
on lysine–Sepharose 4B (Amersham Pharmacia, Uppsala, Sweden),15,16
eluted with 0.1 M glycine and 0.03 M lysine at pH 3.0, and stabilized by
addition of ⑀-aminocaproic acid (EACA; 0.02 M final concentration). After
adjustment to pH 7.5, the plasminogen was activated to plasmin with a
100:1 molar ratio of plasminogen to streptokinase (American Diagnostica,
Greenwich, CT) in 0.05 M glycine, 0.015 M L-lysine, 0.01 M EACA, and
10% glycerol. After 16 hours at 4°C, activation was stopped with sodium
chloride and EACA, final concentrations 0.5 M and 0.25 M, respectively.
The pH was adjusted to 8.5 and SK-activated plasminogen was applied to a
benzamidine–Sepharose 6B (Amersham Pharmacia Biotech, Piscataway,
NJ) column equilibrated with 0.05 M Tris (tris(hydroxymethyl)aminomethane), 0.5 M sodium chloride, 0.25 M EACA buffer at pH 8.5. Plasmin was
eluted with 1.0 M EACA, pH 7.5, and SK was removed by octyl–Sepharose
4 FF (Amersham Pharmacia) chromatography at pH 3.4 after equilibration
with 0.1 M glycine, 0.03 M lysine. Polyacrylamide gel electrophoretic
analysis with sodium dodecyl sulfate (SDS-PAGE) showed a single
nonreduced band and bands corresponding to heavy and light chains under
reduced conditions. Final preparations showed N-terminal lysine, contained
18 to 23 caseinolytic U/mg17 and were not contaminated by SK, as
measured by enzyme-linked immunosorbent assay (ELISA).
The experimental system for assessing fibrinolytic hemorrhage used the
rabbit ear puncture model based on the “rebleed phenomenon.”14,18 The
experimental protocol was approved by the Animal Care and Use Committee at Los Angeles Orthopaedic Hospital. Thirty New Zealand white rabbits
(Hazelton Research Products, Denver, PA) weighing 3.5 to 4.5 kg were
assigned to 6 treatment groups in a randomized, prospective manner.
Following these observations, baseline measurements were obtained in an
additional group of 5 rabbits, which received saline only, in an open,
unblinded manner.
Animals were anesthetized with 40 mg/kg ketamine HCl (Phoenix
Pharmaceuticals, St Joseph, MO) and 5 mg/kg xylazine (Phoenix Pharmaceuticals; 2.7-3.25 mL total volume) by intramuscular injection. After
anesthesia was achieved, the skin of both ears and the neck was shaved and
a depilatory (Nair; Sally Hansen Division, Del Laboratory, Farmingdale,
NY) was applied to the ears. The left external jugular vein was exposed
surgically, catheterized with a 30-cm 7F 3-lumen catheter (Braun, Bethlehem, PA), and attached to syringes containing saline, anesthesia mixture,
and experimental infusion. Anesthesia was maintained by intermittent
injections of the ketamine HCI/xylazine mixture every 3 to 10 minutes as
required. Primary bleeding times were determined after full-thickness
punctures (3.5 mm width) of the ear with a number 11 surgical blade
(Feather Safety Razor, Medical Division, Osaka, Japan), at 30 and 10
minutes and immediately prior to initiation of TPA or plasmin infusion. The
duration of bleeding was measured by absorption of blood onto 11-cm
circular filter paper discs (Whatman International, Maidstone, United
Kingdom) at 30-second intervals. Thrombolytic was administered by
COAGULATION AFTER PLASMIN ADMINSTRATION
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continuous pump infusion (Sage Instruments, Orion Research, Boston,
MA) of a 10-mL sample over 50 to 55 minutes. Each rabbit received 1 of 30
coded samples, prospectively randomized and blinded to the observer,
containing TPA (Genentech, South San Francisco, CA; 1, 2, or 4 mg) or
human plasmin (16, 24, or 32 mg). Plasmin used in these experiments had a
pH of 3.7 prior to infusion. All infusates were prepared at Bayer
Corporation, stored at ⫺80°C, then thawed at 32°C just before infusion.
After initiation of the infusion, ear bleeding times were performed at 10, 30,
60, 70, 120, and 180 minutes. During the 3-hour observation interval after
initiation of infusion, all lesions were monitored for renewed bleeding. No
infused animal died prior to being killed at 180 minutes with intravenous
sodium pentobarbital (48 mg/2 mL, Kentosol injection; Med-Pharmex,
Pomona, CA). Figure 1 illustrates the experimental plan, showing the times
for ear puncture bleeding times (arrow) and blood samples (*) relative to
agent or saline infusion.
Blood samples were obtained at 30 minutes and immediately prior to
the start of infusion, and at 10, 60, 70, 90, 120, and 180 minutes after the
start. Blood (4.5 mL) was collected on citrate anticoagulant (0.5 mL, 0.25
M sodium citrate) through a dedicated lumen of the external jugular vein
catheter and kept at 4°C until the end of each experiment. After centrifugation at 3000g at 4°C for 30 minutes, plasma was tested immediately for
factor VIII activity, fibrinogen concentration, and functional antiplasmin
activity according to established procedures.19-21
Data were grouped according to coded samples and analyzed without
knowledge of the specific agent infused by ␹2 and by t test or repeated
measure analysis of variance (ANOVA) comparison of means.
Results
Figure 2 schematically illustrates the ear puncture primary bleeding
times (PBTs) and rebleeding episodes for animals that received
TPA (0.25, 0.5, or 1.0 mg/kg) or plasmin (4, 6, or 8 mg/kg).
Five animals that were infused with saline control showed no
change in PBT, mean of 50 tests 2.25 ⫾ 0.4 minutes (not shown).
TPA at 1 mg/kg showed a nonsignificant prolongation of the PBT
during infusion (8.2 ⫾ 20.0 versus 2.3 ⫾ 0.9; P ⫽ .26), with return
to pretreatment values after infusion (Table 1). Plasmin at 4 mg/kg
had no effect on PBT; at 6 mg/kg, the posttreatment PBT was slightly
prolonged. At 8 mg/kg, plasmin strikingly prolonged the PBT, but
only after the infusion was completed (25.5 ⫾ 41.0 minutes).
Ear puncture sites were continuously scrutinized for renewed
bleeding for a maximum of 180 minutes. All 10 rabbits infused
with TPA at 1 or 0.5 mg/kg and 4 of the 5 rabbits receiving only
0.25 mg/kg showed renewed bleeding from previously stable ear
puncture sites at 36%, 30% and 12%, respectively, of 50 sites.
None of the animals exposed to plasmin at 4 mg/kg or 6 mg/kg
manifested rebleeding. Rebleeding from stable hemostatic sites
occurred in 3 of 5 rabbits that received plasmin at 8 mg/kg, at 16%
of the ear puncture sites.
Figure 3 shows the absolute total duration of bleeding for each
rabbit. Bleeding for more than 50 minutes occurred more often at
Figure 1. Schematic representation of the experimental design. Diagram shows
the bleeding times (arrows) and blood samples (*) relative to the 50- to 60-minute
infusion of plasmin or TPA. Infusions into rabbits (4 kg body weight) were performed
in a randomized, blinded manner, using coded vials containing plasmin (16, 24, or 32
mg) or TPA (1, 2, or 4 mg) in a total volume of 10 mL. Data were pooled by group for
statistical analysis, without knowledge of treatment. Control infusions of 10 mL saline
to 5 animals were performed in an open-label manner at the end of the study.
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STEWART et al
Figure 2. Schematic representation of bleeding. Bleeding is
diagramed before, during, and after infusion of plasmin (left) or TPA
(right) to groups of 5 rabbits. PBTs were performed on cohorts of 5
animals at ⫺30 minutes, ⫺10 minutes, and 0 minutes before infusion
and at 10, 30, 60, 70, 90, 120, and 180 minutes after the start of
infusion. The vertical shaded rectangle represents the infusion period
from 0 to 55 to 60 minutes. The heavy lines indicate when bleeding
occurred, either the primary bleeding times shown at the extreme left
margin of each line, or renewed bleeding at these same sites over the
180-minute observation period. Intervals of stable hemostasis are
indicated by the light horizontal lines.
higher doses of TPA, in 1 of 5, 3 of 5, and 4 of 5 rabbits receiving
TPA at 0.25, 0.5, and 1 mg/kg, respectively, suggesting a dosedependent pattern of bleeding. Plasmin caused excessive bleeding
only at the highest dose (3 of 5 animals), suggesting a distinctive
threshold, rather than a dose-dependent, pattern of bleeding.
The mean duration of bleeding at each ear puncture site,
induced before, during, or after infusion of TPA or plasmin, is
shown in Figure 4. TPA disrupted hemostasis at sites induced prior
to or during infusion, with no effect at sites induced after the
infusion (60 minutes and thereafter). By contrast, plasmin had no
effect on preinfusion ear puncture sites or on those induced early
(10 minutes) into the infusion. Rather, plasmin caused bleeding
from puncture sites induced toward the end (30 minutes) and after
the infusion.
To assess whether blood coagulation contributed to these
patterns of bleeding, antiplasmin, factor VIII, and fibrinogen were
monitored over the course of each experiment (Figure 5). TPA
caused minimal changes in antiplasmin activity or fibrinogen and
only a mild decrease in factor VIII to 60% of initial concentration.
There was no difference in coagulation factor concentration at TPA
doses between 0.25 mg/kg and 1 mg/kg. Results with plasmin were
different, showing a dose-dependent decrease in all 3 laboratory
parameters, reaching nadir levels at the end of infusion (60
minutes). Antiplasmin activity produced apparent negative nadir
values, probably reflecting the presence of catalytically active
plasmin entrapped in ␣2-macroglobulin.22
The disparate dose-dependent effects of TPA and plasmin on
coagulation and bleeding are shown in Figure 6. Progressively
higher doses of TPA showed no dose-dependent effect on fibrinogen, but did show a stepwise increase in bleeding (P ⫽ .051 and
P ⫽ .055, respectively, for 1 mg/kg and 0.5 mg/kg versus 0.25
mg/kg). On the contrary, plasmin caused a dose-dependent decrease in fibrinogen concentration, but a threshold effect on
hemostasis, excessive bleeding occurring only at the highest dose.
Excessive bleeding with TPA occurred despite maintenance of
fibrinogen concentrations above 150 mg/dL, whereas plasmin at 4
or 6 mg/kg decreased the fibrinogen concentration to 100 mg/dL
without causing bleeding. Plasmin caused bleeding only when the
fibrinogen concentration was less than the lower limit of quantification (25 mg/dL). The timing of the nadir fibrinogen values caused
by plasmin (8 mg/kg; Figure 5) coincided with the time that ear
punctures showed prolonged bleeding, near to or after the end of
infusion (Figure 2).
Discussion
This study clearly distinguishes between TPA and plasmin relative
to the relationship of plasma coagulation factors with bleeding.
TPA caused bleeding regardless of the plasma concentration of
antiplasmin, fibrinogen, or factor VIII (Figure 6), consistent with
prior observations that show minimal effect of TPA on coagulation23 and no predictive value of laboratory coagulation assays for a
bleeding event caused by TPA.2,24-26 By contrast, plasmin caused
no excessive bleeding when fibrinogen and factor VIII were
Table 1. Mean ⴞ SD of PBT relative to start of plasmin or TPA infusion
Plasmin
TPA
4 mg/kg
6 mg/kg
8 mg/kg
0.25 mg/kg
0.5 mg/kg
1 mg/kg
Before treatment
2.1 ⫾ 0.6
2.2 ⫾ 0.4
2.6 ⫾ 1.4
1.8 ⫾ 0.7
2.4 ⫾ 0.9
2.3 ⫾ 0.9
Treatment
2.3 ⫾ 1.9
3.0 ⫾ 1.0
5.6 ⫾ 8.0
2.1 ⫾ 1.3
3.2 ⫾ 1.2
8.2 ⫾ 20.0
After treatment
2.6 ⫾ 1.4
3.6 ⫾ 2.8*
25.5 ⫾ 41.0†
1.9 ⫾ 1.1
3.0 ⫾ 1.2
2.6 ⫾ 1.2
Pretreatment times were ⫺30, ⫺10, and 0 minutes; treatment times, 10 and 30 minutes; and posttreatment times, 60, 70, 90, 120, and 180 minutes.
*P ⫽ .02 versus before treatment.
†P ⫽ .027 and .04 versus before treatment and after treatment.
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COAGULATION AFTER PLASMIN ADMINSTRATION
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Figure 3. Total bleeding induced by TPA or plasmin. Results shown for 5 animals
that received 0.25, 0.5, or 1 mg/kg TPA (left panel) or 4, 6, or 8 mg/kg of plasmin (right
panel). Total bleeding is calculated for individual animals as the sum of bleeding
detected from all 10 puncture sites (Figure 2).
present, but induced excessive bleeding when the factors were
absent (below the limit of quantification). Thus, for the first time, a
clear profile of plasma coagulation assays is linked to protection
from bleeding on administration of a thrombolytic agent, albeit
only with plasmin.
In addition to these different effects on coagulation, plasmin and
TPA exhibited markedly different patterns of bleeding. First, TPA
caused bleeding at therapeutic dosage13 and even at 25% of this
amount; plasmin caused no bleeding except at supratherapeutic
(8-fold) dosages. Second, TPA caused bleeding in a dosedependent manner, increasing progressively with incremental doses
between 0.25 mg/kg and 1 mg/kg, whereas plasmin showed normal
hemostasis at doses of 2 mg/kg,10 4 mg/kg, or 6 mg/kg and
excessive bleeding only at the threshold dose of 8 mg/kg (Figure 6).
Third, TPA caused bleeding only during administration, whereas
plasmin (at 8 mg/kg only) caused bleeding toward the end and after
termination of infusion (Figure 4). Fourth, TPA caused rebleeding,
that is, renewed oozing from previously stable ear puncture sites,
whereas plasmin mostly affected primary hemostasis (Figure 2).
The distinct effects of plasmin and TPA on coagulation and
bleeding can be explained by their biochemical properties. TPA
avidly binds to fibrin on thrombi and at vascular injury sites, where
its enzymatic activity is physiologically focused, but causes only
limited degradation of plasma coagulation factors.27-29 The dose-
Figure 4. Total bleeding induced by TPA or plasmin. Total bleeding induced by
TPA (1 mg/kg) or plasmin (8 mg/kg) relative to the time during which agent was
infused. Results are expressed as mean of 5 animals for either treatment group. The
preinfusion ear puncture bleeding times were performed at ⫺30, ⫺10, and 0 minutes
before starting TPA or plasmin. During the 55- to 60-minute infusions, ear punctures
were induced at 10 and 30 minutes; postinfusion bleeding times were performed at
60, 70, 90, and 120 minutes. TPA (u) caused increased bleeding from the sites
induced before or during infusion, most evident at the 10-minute site, whereas
plasmin (f) caused bleeding from midpoint of infusion and at all subsequent ear
puncture sites. Results for bleeding at 180 minutes are not shown because
experiments were terminated just after these observations.
Figure 5. Concentrations of plasma antiplasmin, factor VIII, and fibrinogen.
Plasma antiplasmin (top), factor VIII (middle), and fibrinogen concentrations (bottom)
in animals exposed to TPA (open symbols) or plasmin (solid symbols) over the course
of the 180-minute observation. Mean results for each cohort of 5 animals expressed
as percent of pretreatment value at ⫺30 minutes, normalized to 100%. Data are
corrected for results obtained with saline alone, which was associated with a 5%
decrease in all results during the 60-minute infusion and with a 10% decrease in
values of samples obtained from 60 to 180 minutes. Results for TPA were not different
at dosages of 0.25 mg/kg (E), 0.5 mg/kg (䡺), or 1.0 mg/kg (䉫). Plasmin showed a
dose-dependent decrease in all 3 assays on exposure to increasing dosages of 4
mg/kg (䉬), 6 mg/kg (f), and 8 mg/kg (F). Antiplasmin activity was fully depleted with
plasmin at 6 mg/kg, a dose that decreased fibrinogen and factor VIII to 20% to 30% of
initial activity. Only at a dose of 8 mg/kg did plasmin cause depletion of fibrinogen and
factor VIII to levels at or below the lower limit of quantification.
dependent nature of TPA bleeding reflects progressively higher
local accumulations on hemostatic plugs, thus inducing bleeding
from stable ear puncture sites, even from those incurred before the
TPA infusion was started. In patients, this effect is indirectly
reflected by the correlation of blood TPA concentration with
bleeding risk.30 Once the TPA infusion is terminated, the agent is
rapidly cleared31 and at these dosages, newly induced, postinfusion
trauma sites do not bleed excessively (Figure 4).
The effects of plasmin can now be explained as follows.
Plasmin is rapidly neutralized by antiplasmin32 and is thus prevented from circulating and binding to fibrin of hemostatic plugs.
Thus, stable ear puncture sites are immune from disruption by
infused plasmin. However, we show that at a high enough dose of
plasmin (8 mg/kg), antiplasmin activity was depleted and plasminsensitive substrates in blood such as fibrinogen and factor VIII
were degraded in a dose-dependent manner (Figure 6). When
fibrinogen and factor VIII were completely depleted, a threshold of
hemostatic protection was breached, allowing bleeding to occur
(Figure 6). Such bleeding was observed only toward the end and
after plasmin infusion (Figure 2). So long as fibrinogen and factor
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STEWART et al
Figure 6. Fibrinogen concentrations compared with total bleeding. Nadir
fibrinogen concentrations (top) are compared with total bleeding (bottom) in animals
receiving TPA or plasmin. Control values for nadir fibrinogen concentrations, at 0
mg/kg of plasmin or TPA, were determined in plasma samples obtained before
infusion (at ⫺30 and 0 minutes). Control values for total bleeding were determined in
the 5 rabbits that received saline (mean of 50 determinations, 2.25 ⫾ 0.4 minutes).
Decrease in plasma fibrinogen concentration with plasmin was dose-dependent and
more profound than with TPA, which did not alter the fibrinogen concentration
significantly from the pretreatment level. Nevertheless, TPA induced bleeding in a
stepwise manner at increasing dosages, unrelated to the plasma fibrinogen concentration. Plasmin did not induce excessive bleeding at 4 or 6 mg/kg. At a plasmin dose
of 8 mg/kg, the nadir fibrinogen concentration dropped below the lower limit of
quantification, in association with the appearance of excessive bleeding.
VIII concentrations remained below the lower limit of quantification (Figure 5), bleeding continued for the ensuing 120-minute
observation period (Figure 2).
There are clinical implications for our observations regarding
the safety, efficacy, and monitoring of thrombolytic therapy using
plasmin. As to efficacy, plasmin would not be applicable for
systemic administration, such as in patients with acute myocardial
infarction, because safe dosages of the agent would be neutralized
by antiplasmin. However, regional administration of plasmin such
as in patients with thrombosed arteriovenous shunts, arterial graft
occlusions, deep vein thrombosis, or coronary artery thrombosis
after failed percutaneous intervention, should be achievable without the risk of ICH that accompanies regional use of TPA,
urokinase, or staphylokinase.33-35 Because the dose of plasmin that
causes excessive bleeding was more than 6-fold greater (Figure 6)
than the effective thrombolytic dose,13 additional dosages of
plasmin could be infused locally to maximize thrombolytic efficacy. This potential of safe administration of supratherapeutic
amounts of plasmin contrasts with the situation using plasminogen
activators, where even reduced dosages are associated with bleeding36 and higher doses of TPA-type agents proportionately increase
bleeding risk.2
Our concept of plasmin use for thrombolysis, namely, regional
infusion for efficacy and dose selection that preserves coagulation
for safety, contrasts with the approach used by Nagai et al37 and by
Lapchak et al38 using microplasmin in experimental embolic
stroke. These studies used systemic (intravenous) rather than
regional (intra-arterial) infusion with the objective of depleting,
rather than preserving, antiplasmin activity. The authors conclude
that neurologic improvement with microplasmin was achieved
without increased risk for parenchymal hemorrhage,38 but because
the rate of hemorrhage with placebo was much higher than in
historical controls,39 the safety of this approach requires
further study.
Our results indicate that the threshold pattern of plasmininduced bleeding occurred only on complete depletion of clotting
factors, thus providing a unique circumstance for laboratory
monitoring of treatment. In this scenario, plasmin treatment could
be monitored and judged as safe from excessive bleeding so long as
significant amounts of clotting factors are present. Such monitoring
is not feasible for TPA administration, because changes in clotting
factor concentration do not correlate with bleeding complications.26
In conclusion, we have compared plasmin with TPA in a
randomized and blinded study in an established rabbit model of
fibrinolytic hemorrhage. The data provide insights into rational
dose selection of plasmin and the role of plasma coagulation for
hemostatic safety during plasmin infusion.
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