From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
Inactivation
of Purified
Cl
By
Lindsey
A.
3-Hydroxypropyl
of
lysis
in
Inhibitor
Miles,
John
acid
Studies
vitro.
of
possible
FIu-HPA
were
undertaken.
The
FIu-HPA
in clot
lysis
was
plasminogen.
influence
a2-antiplasmin
studied.
Plasmin
plates
assays
The
purified
or
the
greater
was
than
1 mM.
in
the
a
dependent
manner.
of
CI inhibitor
purified
M
Urea
ANY
shown
of
amidolytic
on
of Flu-HPA
purified
on the
plasma
the
first
compounds
nonsteroidal
has
agent,
mechanism
of
been elucidated.
in
for
activi-
were
action
They
and
inactivate
concentrations,
purified
also
also
of these
appear
to
fibninolytic
Recently,
compounds.
the occurrence
tor of plasmin,
It reacts
very
plasmin
and
a2-antiplasmin
rapidly
to
is
the
plasma.
6 Prandini
synthetic
fibninolytic
a(isobutyl-4-cyclohexene-
Blood,
Vol.
57,
No.
6 (June),
remove
anti-
to lyse fibrin clots was made.7
It
that blocking
of plasma
inhibi-
tors of urokinase
is effected
and that
a2-macroglobulin,7
Cl-inhibitor’0
are
inactivated
major
by certain
compounds8
anti-thrombin
III,
and
by various
synthetic
plasminogen.2”29
Thus,
has
a
of a fast
been
1:1
inhibitor
et al)7 demonstrated
agents,
orthothymotic
I -yl)
propionic
1981
more
active
From
the
than
Chemistry.
flufenamic
acid
inactivat-
inhibi-
plasmin
that
acid
acid
is inactivated
evidence
by
a
for
of Cl inhibitor
ofCalifornia
in part
by Research
is a recipient
Career
Development
National
Institutes
at San
Congress
Submitted
Address
in
of
Molecular
lion,
©
/0666
I 98/
Health
Training
of
received
this
/980;
requests
Torrey
was
and
and
Calif
HL-18576.
Research
support
to the
from
a
University
of
at
H.
Clinic
VIIth
30,
Griffin,
and
La Jolla,
the
63rd
Societies
London.
January
Scripps
Road.
presented
American
at the
to John
Pines
& Stratton.
of
Haemostasis,
accepted
Immunology,
by Grune
1979,
and
28.
work
Federation
Dallas.
on Thrombosis
North
and
of Health
Grant
Clinic
Biology
La Jolla,
HL-21544
be
lysis
Scripps
of
Diego,
Institutes
LAM.
the
Biology.
reprint
Grants
to
clot
Diego.
of
July
in
Departments
at San
Award.
report
Meeting
shown
Immunology,
the
of a National
of
and
flufenamate
and
by
enhance
fibninolysis
of several
possible
of flufenamic
acid,
3(Flu-HPA),on
both punpurified
Cl
inhibitor
is
of Molecular
University
Supported
Experimental
two
and
(S-
and,
at
It has
kallikrein.’9’22’23
The
reported
to activate
synthesized
Foundation,
J.H.G.
(‘alifornia
descnibed.’3
complex
with
of
that
directly
inactivation
sodium
Department
Research
Annual
form
as
was
indicate
a2-antiplasmin
inactivate
plasmin.
The effect
of a derivative
hydroxypropyl
flufenamide
fled
a2-antiplasmin
and
A preliminary
in plasma
inactivation
by
flufenamic
acid derivatives
could
through
inactivation
of an inhibitor
plasminogen
activators.
of synthetic
activity
and
been suggested
of
macti-
direct
effect of flufenamic
acid on purified
Cl inhibitor
has appeared.
Cl inhibitor
is a plasma
protease
inhibiton that inhibits
Clr, CIs,’8”9 activated
Factor
XII,20’2’
and
plasmin
has also
Cl
Cl inhibitor.
assessed
in this article.
Flu-HPA
has been
to
such
fibrinolysis
or
also
results
compounds
synthetic
profibninolytic
molecules.
It was suggested
that
synthetic
fibninolytic
agents
dissociate
enzymeinhibitor
complexes
such
as plasmin-antiplasmin
or
that they solubilize
and remove
inhibitors
of plasminogen activators
from a fibninolytic
enzyme
complex.6
A positive,
qualitative
correlation
between
the ability
compounds
This
These
activated
Factor
Xl,2#{176}
and plasma
latter
three
enzymes
have been
require
plasma
components,
in addition
to plasminogen,
to induce
profibninolytic
activity.3
There
have
been several
different
hypotheses
for the mechanism
of action
of sodium
flufenamate
and other
related
fibninolytic
acid,
Cl inhibitor.
promot?
plasmin
derivative
also been suggested
that Cl inhibitor
sodium
flufenamate’#{176} although
no
other
including
flufenanthranilate],
antiinflammatory
The
not
may
1623),
higher
studied.3
attempts
were
made
to design
with
certain
chemical
chanactenisitcs
identify
the
structural
requirements
of fibrinolysis.2’4
The fibrinolytic
demonstrated.5
compounds
and
Xlla.
preformed
kallikrein
small
synthetic
compounds
have
been
to enhance
plasma
clot lysis in vitro.”2
were
fibrinolytic
ing a2-antiplasmin
ability
from
water-soluble
reversible.
was
or $-Factor
activity
a2-antiplasmin
and
activity
greater
than
2
did not affect
the
kallikrein,
enzyme
A
H. Griffin
Cl inhibitor
N-flufenamyl-glutamic
be
derivatives
and John
studied.
kallikrein.
a2-antiplasm
synthetic
Human
at concentrations
up to 60 mM
release
acid,
to
also
either
and
J. Kleiss,
of plasmin,
not
of
inhibitor
shown
concentration-
was
Flu-HPA
Flu-HPA
did
complexes
vated
substrate.
Alice
activities
Flu-HPA
flufenamic
125l-fibrin
of a2-antiplasmin
and
inhibit
of
was
concentrations
activity
influence
ability
using
tripeptide
ties of different
N-arylanthranilates,
amic acid
[N (3-tnifluoromethylphenyl)
a
the
on
plasmin
FIu-HPA
inhibitory
to
be dependent
purified
time-dependent
The
derivatives
Subsequently,
molecules
order
to
enhancement
of
activity
spectrophotometric
abolished
Xlla
of action
FIu-HPA
At
fl-Factor
mechanisms
to
clot
Purified
Agents
Goodman,
profibrinolytic
determined
Val-Leu-Lys-paranitroanilide.
and
a series
blood
Murray
abolished
by
mM.
Notably.
inhibit
was
of
enhances
found
of
to
activity
is one
that
and
Fibrinolytic
S. Verlander,
Michael
(Flu-HPA)
derivatives
a2-Antiplasmin
by Synthetic
P. Burnier,
flufenamide
flufenamic
Human
for
International
I 979.
198/.
Ph.D.
,
Research
Calif
Department
Founda92037.
Inc.
0006-497//8//5706-0005$02.00/0
1015
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
MILES
1016
assays.30’3’
In this
directly
inactivates
but does not inactivate
plasmin,
plasma
fl-Factor
Xlla.
Moreover,
such
an
water-soluble
derivative
of flufenamic
antiplasmin
reversible
pooled
study
it is shown
that
Flu-HPA,
a2-antiplasmin
and Cl inhibitor
and on Cl inhibitor
reaction.
kallikrein,
or
effect
of one
acid
on a2-
is shown
and
made
I .6 x 4 cm
to involve
a
Na
mM
phosphate,
washed
with
20 mM
All
grade
chemicals
obtained
available.
Plasminogen
raphy
on
sulfate,
lysine
and
Sephadex
Na
SO ml
Plasminogen
was
equilibration
tion buffer
method
of Wiman
buffer
eluted
Na
with
to
chem)
had
Plasmin
a
and
was
ammonium
10
been
0.1
containing
Tris-CI,
weight
azide.
linear
salt
gradient
and
covalently
and
Plasmin
centrifuged
ratio.
which
activity
4.5
and
titrated
with
benzoate.M
_700C.
site
Aliquots
of
plasmin
Human
to
tripeptide
were
was
in
purified
Ct al.25 Kallikrein
XII
published
stored
to
was
purified
to greater
procedures.35
than
f3-Factor
XIIa
the
was
had
greater
than
of
Moroi
and
Aoki’2
liters of citrated
cm
and
normal
lysine-Sepharose
phosphate,
did
not
bind
the
presence
Na
phosphate,
7.4,
according
to
95%
homogeneity
was
prepared
of 50%
ammonium
sulfate
7.6,
Na
salt
to give
and
used
to elute
a2-antiplasmin.
mm
were
pooled,
lyophilized,
pH
7.6,
cm column
0.02%
Na
containing
that
had
been
Na
azide
and
pylphosphofluoridate.
I 20
ml
fractions
M
The
equilibration
were
-aminocaproic
step
containing
eluted
buffer
radial
with
in
was
inhibitor
at
acid,
sample
sample
equilibration
containing
Na
taming
gradient
of 350
buffer
containing
a2-antiplasmin
and
step.
Dialysis
2 g/liter
Polybrene
contacting
2-3
=
the
solution
CI
inhibitor
application.
proximal
the
was
NaCI,
(2.5
buffer
high
x
(50
washed
on a
with
140
Cl inhibitor
buffer
in
NaCI
added
chamber.
with
Cl
the
liters
0.3M
chamber
inhibitor
was
was
eluted
M, kininogen.
The
that
fractions
and
applied
had
been
eluted
acid,
buffer
using
proximal
eluted
5.3).
the
gradient
and
a concentration
Cl inhibitor
pH
before
a linear
chamber
to give
conto a SP-
equilibrated
acetate-acetic
ml of starting
was
ml
of 6 liters
6
distal
as
3600
gradient
in the
28 cm)
Na
dia-
10 mM
A-SO
and
dialyzed,
mM
CI
was
Tris-CI,
washed
column.
pooled,
or
human
chromatography
A linear
the
before
were
distal
before
chromatography.
with
to
mM
chamber
O.3M
from
and
C I
analysis,36
Plasma
(40
column
sample
applied.
procedure,
N.J.).
buffer
in
column
in the
the
100 g of DEAE-Sephadex
acid,
starting
isolating
antiserum
mmho)
The
inhibitor
buffer
rabbit
containing
peak
was
for
immunodiffusion
starting
inhibitor
starting
ml
starting
phos-
0.02%
Step
3.
x
13 cm
70
column
then
0.2
were
DEAE-Sephadex
CI
200
starting
then
with
mM
was
ml
inhibitor
column
equilibrated
acid,
diisoproand
benzami-
350
ml
of 0.25M
between
7 and
IS
mmho.
approximately
application
the
(Aldrich),
for each
with
Somerville,
SP-Sephadex
was
2.5
with
Cl
column
to Sepharose
7.6,
or
for
column,
polybrene
chromatography.
8.2,
albumin
the
bound
pH
plasticware
I mM
Throughout
using
after
Sephadex-C50
containing
10 mM
was
DEAE-Sephadex
fresh
double
the
buffer
Cl
to a 2 x 7.5
phosphate.
pH
2.
NaCI
chamber
by
0. 1 2Msuccinic
applied
containing
washed
with
g citric
centrifuged
obtained
contained
scheme
Diagnostics,
buffer
the
The
1 3.6
was
using
before
plasma.
elsewhere.35’38
Step
The
ACD
x 42 cm column
Tris-CI,
azide.
and
acid-
at 4#{176}C
except
prerinsed
is a convenient
of
detected
starting
with
first
H2O
DEAE-Sephadex
after
to a
plasma
buffers
were
20#{176}C
against
starting
been
the
out
prepared
with
syringes
at 20#{176}C.
50 pg/mI
were
rinsed
(Behring
I.
succinic
in
a2-antiplas-
10 mM
I
for
volume
contains
blood
Adams)
the
Na4EDTA,
immunodiffusion,37
of this
buffer
distal
containing
azide,
antecubital
plastic
The
carried
and
the
one-sixth
I liter
Clay
at 20#{176}.
All
containers
was
to elute
buffer
M in the
mdi-
Na
material
from
50-mi
20#{176}Cand
were
plasma
following
inhibitor
had
after
dialyzed
0.02%
starting
with
g for 40 mm
Buffers
I .4 liters
used
75 mM
ml of equilibration
M Na
was
after
Fractions
0.02%
gel
gels.
Cl inhibitor.
The
of
of
the
into
(Siliclad,
andthen
that
applied
of equilibration
pool
buffer
column
buffer
acid.
0.1
this
was
Na
covalently
with
with
against
pH
dissolved
The
Na
precipitated
that
7.6,
I .4
M
portion
and
apparent
by these
were
7.5.
collected
tubes
at
steps
1 mM
described
x 71
fractions
The
sample
of 0.2
plasminogen
washed
The
ml
and
0.1
Sephadex-A50
Fractions
azide.
equilibrated
pooled.
of 900
900
with
The
ml equilibration
a concentration
was
phate,
azide.
gradient
chamber
a 2.5
was dialyzed
phosphate
I 350
azide.
sulfate
DEAE-
with
A linear
added
0.02%
containing
0.1 M Na
proximal
NaC1
were
ammonium
washed
application.
Na
of
over
mm
at 5000
all
was
was
20 g dextrose).
performed
and
lyzed
methods
20
azide.
solution
Step
as
A quantity
passed
equilibrated
0.02%
Sepharose
of the
Cohen.’4
was
column
in 30%
pH
was
and
soluble
with
column
in the
tubing
as described.25
a modification
plasma
containing
5 x 37 cm column
equilibrated
Wiman
human
to lysine
was
using
affinity
pH
pool that
purified
an
70#{176}C.
-
(ACD:
citrate,
glassware
Na
from
95%
volunteers
for
HCI,
10.5
was
sample
containing
had
as judged
pH
at
in centrifuge
purification
dine
been
at
healthy
again
dialysis
5-
volumes
g
was
of
small
3000
centrifuged
which
described.”
a2-Antiplasmin
pure
plasma
anticoagulant
initial
guanidino
prepared
at
0.02%
Sweden)
gels,
was
stored
was
after
buffer
protein
activity
and
Blood
25 g sodium
was
at
all
that
buffer
this
40
column
by SDS-polyacrylamide
90%
phosphate,
human
citrate-dextrose
All
supernatant
paranitrophenyl
purified
Na
inhibitor.
mixed
siliconized
substrate
sample
of normal
rapidly
Na
(Calbio-
remove
by SDS-polyacrylamide
of Bouma
Factor
using
a plasmin
titrant
prekallikrein
as judged
method
Human
with
active
homogeneity,
the
compared
mixture
(Kabi,
the
in
0.02%
the
The
this
with
a2-antiplasmin
citrated
was
acid,
of incubation
the
(H-D-Val-Leu-Lys-paranitroanilide)
determined
hr
and
azide.
of
as judged
at least
40 mM
of Cl
vein
dialyzed
7.6,
The
times
on
ml
of the
urokinase
centrifuged
several
amidolytic
1 20
was
pH
v/v
to
After
was
was
application.
plasminogen
a 25%
coupled.
mixture
column
of
ml
to a
with
of Cl Inhibitor
Freshly
I 20 ml equilibraof 20 mmho
phosphate,
beads
was
Na
eluted
The
containing
against
isolation
10 mM
by a modification
First,
to give
8.2,
The
sample
prepared
35
was
of 70,000,
Purification
DEAE-
pH
after
M Na
(Biorad)
incubation
removed
2251
50%
0.02%
applied
equilibrated
best
with
buffer
Wallen.33
added
Affigel
30#{176}C,the
was
containing
Glycerol
added
beads.
0.02%
equilibration
chamber.
against
was
NaCI,
mM
the
chromatog-
column
40
were
by affinity
buffer
in the proximal
chamber
and
with NaCI
added
to give a conductivity
distal
azide.
a
with
I 3 mM
with
on
equilibrated
7.0,
mM 9-mercaptoethanol,
sources
prepared
precipitation
chromatography
succinate,
the
commercial
was
Sepharose,32
A-SO
washed
from
pH
and
column
a-methylmannoside.
molecular
vidually
METHODS
fl-mercaptoethanol
a2-Antiplasmin
electrophoresis,
AND
with
approximately
application.
Fractions
MATERIALS
1 mM
concanavalin-A-Sepharose
ET AL.
starting
buffer
applied.
the
with
containing
inhibitor
buffer
5.3).
After
(50
the
65 ml starting
in
the
0.4
The
dialyzed,
and
proximal
M
eluted
NaCl,
between
mM
that
Na
sample
buffer.
in the
to
a
been
applied,
A linear
distal
200
chamber,
29
the
gradient
and
16 and
had
acetate-acetic
was
chamber
fractions
applied
DEAE-Sephadex
starting
pH
buffer
Cl
pooled,
containing
NaCI,
washed
chromatography.
were
mmho.
of
ml
of
was
The
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
INACTIVATION
OF
preparation
a2-ANTIPLASMIN
was
AND
approximately
90%
Cl
1017
INH.
pure
as
determined
on
FIu-HPA
SDS
gels.
Antibodies
to
injection
of
Serum
from
serum
for
fifth
batch
30
with
mm
M NaCI,
at
Na
Bureau
prep
XXXIV
prepared
by Dr.
John
salt,
measurements
was
meter,
with
pH
CO-NH-(CH2)3-OH
50%
7.5,
0.4
consisted
6.0,
in Fig.
was
U/ml,
N-FIu-GIu
anticoagu-
CF3
by
structures
the
Lowry
2d,
method#{176}
temperature
pH
using
(Radiometer,
at room
CDM
di-
synthe-
as a reference.
room
meter
were
chemical
Chemicals)
at
pH
acid
salt
1.
determined
(Sigma
measured
used
10,350
of plasma
sodium
performed
type
preparation
pH
methods.#{176}’3’ The
were
26
using
sequential
Dr. David
Aron-
N-flufenamyl-glutamic
albumin
model
Conductivity
and
of protein
volunteers.
illustrated
were
conductivity
I hr.
7.7,
N.Y.
pool
chemical
are
serum
Radiometer
The
NaCI,
Albany,
flufenamide,
concentrations
bovine
M
N-flufenamyl-f3-alanine
standard
Protein
mg/mI
pH
Tris-CI,
human
plasma
was a gift from
20 normal
compounds
using
10
precipitated
10 mM
Standards.
plasma
from
and
using
of these
was
gamma
56#{176}C
for
10 g of resin/g
against
Fenton,
human
ACD
at
phosphate,
using
supernatant
dialyzed
in 0.75
3-Hydroxypropyl
sized
cellulose
goat
the
with
treated
CF3
by
period.
unimmunized
3 times
Na
goats
a 4-wk
to isolate
heat
10 mM
of Biological
normal
normal
adsorbed
then
in
over
azide.
thrombin
with
produced
manner
was isolated
from
Human
thrombin
son of the
sodium
and
against
and
0.02%
The
was
22#{176}C.The
Fibrinogen
precipitations.39
lated
bleed
following
DEAE
sulfate
were
plasminogen
at 37#{176}C,and
dialyzed
absorbed
for
week
Serum
was
ammonium
human
in the
20 mm
serum
plasminogen
of
treated
fraction:
kaolin
The
mg
the
were
globulin
human
2.5
CO-NH-CH-CO2Na
a
Copenhagen).
temperature
with
using
cm
a 0.54
(CH2)2
a Radiometer
cell,
type
CDC
I 14.
CO2Na
Assays
ofa2-Antiplasmin
a2-Antiplasmin
or
a
Activity
activity
chromogenic
was
assessed
tripeptide
using
a fibrin
substrate,
5-225
plate
1,
as
assay5
previously
N-Flu--fl-Ala
described.4’
In
a
typical
inactivate
HPA
(0.5
assay
a2-antiplasmin,
dissolved
to
a
antiplasmin,
83 nM
pH
10
x
8.1 1, 0.15
M
The
to the
bottom
cooled
at
TBS-BSA
to give
a final
added
and
sec.
Seventy
pH
7.4,
the
Na
in a narrow
Gilford
model
6050
Chart
cation
of
using
the
unlabeled
tration
2400
of
96
5Miles,
diluted
containing
pipetted
Rothschild
2 x
Angeles,
with
solution
6-mm
was
and
A
with
into
Los
wells
were
et al.42
method.43
was
Bioproducts,
fibrinogen
plates
dried
Griffin,
tube
a
and
and
microliters
culture
of
capacities
of 200
at 37#{176}C
and
the
were
unpublished
results.
plates
that
MI.
kept
0.02%
with
used
purified
human
prothrombint
venom
followed
tMarlar
by
of prothrombin
and
Griffin,
to
Biorex
4/600
clot
was
in
plates
incubated
at
was
removed.
the
were
above
samples
were
in
removed
gamma
was
using
activation
with
chromatography.”
mg/mI
The
wells
were
buffer.
To
assay
triplicate
were
at various
times
prepared
from
counter.
fibrinogen
70
fibrinogen
The
the
at 0.36
manuscript
dried
(I 16 nM)
IOO-Ml
in a Micromedic
the
a-thrombin
isl of
I O-Ml aliquots
thrombin
milliliters
solution
100
activity,
and
use,
human
NaN3.
thrombin
fibrinolytic
counted
The
8.2,
3 times
in wells,
before
of SO ,.Ll purified
I hr. The
plasmin
and
Immediately
pH
washed
placed
Costar
plate
M Tris-CI,
then
to a concen-
used.
by addition
37#{176}C
for
labeled
labeled
clotted
0.1
a modifi-
of a flat-bottom
tissue
Fig. 1 .
Chemical
structures
of 3-hydroxypropyl
flufenamide
(N-Flu-HPA).
N-flufenamyl-glutamic
acid disodium
salt (N-Flu-Glu).
and N-flufenamyI-1-alanine
sodium salt (N-Flu-fl-Ala).
at 4#{176}C
until
a
Gilford
was
Fifty
diameters
The
using
using
of
CO-NH-(CH2)2-CO2Na
placed
length.
Fibrinogen
cpm/ml.
Calif.)
were
and
with
mixture
30
Tris-CI,
recorded,
prepared
in
for
path
was
at
4#{176}C,
were
temperature
the
water
well
mm
diluted
at
deionized
106
each
30
in SO mM
I-cm
nm
of
temperature
equipped
Unkeless
lactoperoxidase
fibrinogen
was
a
at 405
‘25I-fibrin
nM,
from
with
minute
135
at room
spectrophotometer
method
of 56 g/ml
contained
cuvette
per
for
mg/mI
removed
a2-
to bring
plasmin
at room
M NaCl,
were
were
of
centrifuged
of
of
azide)
concentration
incubated
at 0.48
0.012
masked
recorder.
the
solution
(MA.
The
black
in absorbance
tube,
microliters
incubated
mixture
Na
a final
CF3
Flu-
3 zl of buffer
microliters
was
concentration
was
0.02%
to
of
plus
Fifteen
mixture
of S-225l
azide,
I 00 l of the
change
this
mixture
Flu-HPA
concentrations
to give
of the
of
alone,
NaCI,
tube.
0#{176}.
Fifteen
microliters
0.02%
added;
glass
reaction
37#{176}C,and
ability
DMF
in TBA-BSA,
added.
reactants
or
mm
the
various
DMF
75
diluted
were
of
2 zl of
in distilled
Tris-CI,
M
added
all
amidolytic
in 0.05
in preparation.
Echis
carinatus
Five-hundred
M sodium
phos-
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1018
MILES
phate,
pH
buffer
were
7.0,
mixed
and
equilibrated
0.3
with
ofC!
activity
was
assessed
by
cleavage
of
cleavage
a typical
assay
the
Flu-HPA
diluted
DMF
of
alone,
10
various
8. 1 5, 0. 1 5 M NaCI,
0.02%
a final
kallikrein,
which
gave
to a 10 x 75 mm
37#{176}.
Fifty
added.
troanilide
and
0.1
NaCI,
mixture
was
placed
nm/mm.
nmole
In other
DMFor
M Tris-CI,
pH
inhibitor
mixture
was
were
was
incubated
30
nM,
were
20
removed
and
contained
the
at
added
tube.
by recording
Washed
the
clot
described.#{176} Clots
plasma
usually
cified
platelet-poor
were
plasma
(0.1
M calcium
chloride
(0.1
removed,
deposited
ml).
washed
in a 10 x 75
chloride
for lysis
and
was
the
In order
by FIu-I-IPA
plasminogen
±
gamma
antibodies,
presence
clots
did
of Flu-HPA.
required
for the
globulin
shortened
clot lysis
presence
from
74 ± 3 hr
of antiplasminogen
not lyse in
This shows
profibrinolytic
143
that
the
time
to
hr even in the
plasminogen
is
activity
of Flu-H
XIIa,
of 36
for
mixture
path
length
that
pH
determined
as
previously
PA.
Decal-
to a solution
ml),
approximately
30 mm,
M sodium
containing
0.4
were
kept
of 0.1S
and
0.025
the
clots
chloride,
ml of 0.15
and
M
at 37#{176}C
and
recorded.
Dependence
of Clot
Lysis
The
on the
influence
of
Reaction
Flu-HPA
antiplasmin
was studied.
mined
spectrophotometnically
ty, and a2-antiplasmin
Between
on
the
Plasmin
activity
of
Plasmin
activity
was
as lysine amidase
activity
was measured
at higher
concentrations
concentrations,
of Flu-HPA
tration
tested,
70 mM,
the activity
of plasmin
obtained
instead
of
a2-
deteractiviby its
of
activcould
was inhibabolished
as seen
in Fig.
up to the highest
2A.
At
concen-
this compound
had no effect on
(Fig. 2B). Similar
results
were
when
Flu-HPA
DMF.
Thus,
was dissolved
in
the small
amounts
methanol
of DMF
used to dissolve
Flu-HPA
had no effect on the
tion of the proteins.
From
these
observations
concluded
that
Flu-HPA
directly
inactivates
interacit is
a2-anti-
plasmin.
The
tration
concenand a2-
influence
on the
antiplasmin
When
of the bovine
serum
interaction
between
in
the
these
experiments
TBS-BSA
was
albumin
Flu-HPA
was
adjusted
investigated.
to a final
concen-
tration
of 4.3 mg/mI
of bovine
serum
albumin,
instead
of the 0.43 mg/ml
concentration
routinely
used, there
was no effect of albumin
on inactivation
of a2-antiplasmm by Flu-l-1PA.
However,
above
this concentration
of albumin,
inactivation
of a2-antiplasmin
by Flu1-IPA was decreased,
and the effect of Flu-HPA
(5.14
mM)
was eliminated
at 21.5 mg/mI
albumin
(0.35
human
plasma.
(0.1
8.0,
was
of citrated
added
an
were
Tris-Ci,
blood-bank
Clots
The
and
incubated
a I -cm
M imidazole
material.
tube.
bottom
of the
by recalcification
tube
concentration
tube
at 405 nm/mm.
performed
of 0.15
(0.5
of fl-Factor
activity
test
in
S Ml of Cl
concentration
Amidolytic
mm
and
glass
in 0.IM
After
cm.
for addition
a final
was
with
2 ml
0.5
concentrations
to the
mixture
0.05
M
2 Ml ofbuffer
a final
was
of
azide),
microliters
ml)
0.05
Effect
ofFlu-HPA
and a2-Antiplasmin
be detected.
The activity
of a2-antiplasmin
ited 50% by 0.4 mM Flu-HPA
and entirely
RESULTS
Plasminogen
by Flu-HPA
for complete
3 hr. In the
at
reaction
of change
linear
siliconized
outdated
8.3,
the
rate
mg/mI
with
test
added
20 mm
substrate.
microliters
(0.1
ml),
were
length
was
Na
giving
in absorbance
assays
were
from
chloride
goat
required
49.5
MI
in
10
at 4#{176}
were
pH
and
to give
The
formed
diluted
for
to various
0.02%
0.125
obtained
M sodium
normal
ability
to inhibit
plasmin
activity.
A concentration
ct2-antiplasmin
that did not fully inhibit
plasmin
ity was chosen
so that small effects
of Flu-HPA
pH
and
70 (41 of TBS
reactants
azide.
change
lysis
nM,
rate
to a microcuvette
Na
of 446
10 Ml TBS-BSA,
37#{176}.
Twenty
0.02%
nM
tripeptide
37#{176}C.Five
80 (LI of 5-2302,
M NaCI,
were
alone,
to bring
to
mm
367
initial
diluted
to 14 Mg/mI,
added
additional
of
or
M Tris-CI,
a path
initial
to the
at
CI
DMF
incubated
as the
in TBS-BSA
mm
distilled
M Tris-CI,
to a 10 x 75 mm
in TBS-BSA
inactivate
microliters
immediately.
with
The
centrifuged
for
diluted
in 0.1
added
measured
diluted
added
and
5-
of benzoyl-Pro-Phe-Arg-parani-
8. 1 5, 0. 1 5 M NaCl,
that
of 26 nM
to
in
at 4#{176}
and
mM)
DMF
or of the
substrate
Two
of
of
substrate
Sweden).
used.
tube
2 ILl of FIu-HPA
in distilled
from
inhibition
Ariz.)45
Flu-HPA
concentration
glass
of kallikrein
assays.
column
eluted
S MI of Cl inhibitor
in a cuvette
at 405
2-48.5
azide),
were
absorbance
time
tripeptide
concentration
(0.14
azide
was
sodium
Fox,
the
of
milliliters
activity
0.05
(Vega
was
of TBS-BSA
Amidolytic
of
Na
one-half
Na
The
its
3 ILl of buffer(0.S
a final
mg/mI
0.02%
over
tripeptide
concentrations
siliconized
microliters
One
ability
zl of TBS-BSA,
give
passed
8.0.
the
of
protocol
TBS-BSA
to
was
was
(Kabi,
the
following
to
then
measuring
(H-D-Pro-Phe-Arg-paranitroanilide)
In
same
were
buffer.
pH
benzoyl-Pro-Phe-Arg-paranitroanilide
fi-Factor-XlIa-dependent
inhibitor,
in the
They
Activity
kallikrein-dependent
2302
same
Thrombin
phosphate,
Inhibitor
70.
mixture
the
buffer.
M sodium
at 20 g/ml
Biorex
I hr. The
equilibration
with
CI inhibitor
the
venom
with
at 37#{176}C
for
with
column
Assays
carinatus
absorbed
70 column
washed
the
Echis
incubated
a Biorex
was
and
separately
ET AL.
Enhancement
mM).
Thus,
the concentrations
of bovine
albumin
used in our experiments
did not appear
to influence
the
effects
of Flu-HPA
and it is noted
that high levels of
albumin
can influence
these
assays,
presumably
by
binding
Flu-l-IPA.
The time dependence
of the reaction
between
FluHPA and a2-antiplasmin
was studied.
Under
the reaction conditions
used,
vated
after
incubation
a2-antiplasmin
with 6.7
was
mM
totally
Flu-I-IPA
mactifor 2
mm.
to test
that
whether
the clot lysis enhancement
had been previously
observed30
was
dependent,
minogen
antibodies
Incorporation
of
the
ability
to inhibit
Flu-HPA
clot
into
of goat
antiplas-
lysis was studied.
a clot
containing
Effect
ofFlu-HPA
on the Ability
to Inhibit
the Cleavage
ofFibrin
The
mm
effect
to
inhibit
of Flu-HPA
the
activity
on the
of
of a2-Antiplasmin
by Plasmin
ability
plasmin
of a2-antiplason
fibnin,
its
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
INACTIVATION
OF
a2-ANTIPLASMIN
AND
Ci
INH.
1019
a
0
0
=
E
0
I.
I’,
,.
II-
vs
-a
0
[Flu#{149}HPA]
mM
b
0
0
.01’
=
2
E
.06’
1#{149}
U,
05’
.04
.03’
c
Fig.
2.
Effect
reaction
of
between
antiplasmin.
Flu-HPA
on
plasmin
The
reaction
the
and
.02’
C’,)
a2-
conditions
-I
are described
in Materials
and Methods. (a) The effect
of Flu-HPA
up to
lOmMand
(b) the effect of Flu-HPA
up
to 70 mM. In both figures
the dashed
line
and
activity
line
activity
open
of
and
circles
closed
chosen
The
circles
of plasmin
natural
substrate,
of a2-antiplasmin
was
indicate
a3-antiplasmin.
represent
.01’
the
I
solid
the
I
‘
0
20
alone.
was tested
that
did
in order
(Fig. 3). A concentration
not fully
inhibit
plasmin
to detect
small
HPA.
In the absence
of Flu-HPA,
observed.
Between
0.5 and I mM
effects
ity of a2-antiplasmin
was abolished.
Thus,
inactivates
the ability
of a2-antiplasmin
plasmin
hydrolysis
of fibnin.
Between
0 and 2 mM Flu-HPA
inhibition
of the fibninolytic
activity
effect
of 20% inhibition
of plasmin
of
60% inhibition
Flu-HPA,
the
Fluwas
activ-
Flu-HPA
to inhibit
there
was a slight
of plasmin.
This
by Flu-HPA
was
very
reproducible.
Flu-I-IPA
activity
had
In
U
U
contrast
no inhibitory
to
effect
1
60
80
this phenomenon,
on the amidolytic
of plasmin.
Effect
ofFlu-HPA
Kallikrein
and
The
inhibitor
I
40
[Flu-HPA] mM
on the
Cl Inhibitor
influence
of
to inhibit
Reaction
Flu-HPA
plasma
Between
on the
kallikrein
ability
was
Plasma
of Ci
studied.
Kallikrein
activity
was determined
spectrophotometnically as amidase
activity,
and Cl inhibitor
activity
was
measured
by its ability
to inhibit
kallikrein.
A concen-
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
MILES
1020
instead
1000’
800-
Flu-HPA
directly
Inactivation
of Cl
incubation
with 0.65
=.
600-
C.)
after
400
.E
E
us
Co
200-
0
3.
the
4
I
Effect
of FIu-HPA
cleavage
of fibrin
various
dilutions
diluted
in TBS-BSA
of
Flu-HPA
effects
of Flu-HPA
seen in Fig. 4, the
inactivated
mM,
while
‘-:-
.-...
.
plus
of a2-antiplasmin
Thirty
microliters
1 50
to
of
Ml of a2-antiplasmin
concentration
of 0.18
nM
were
that did not completely
chosen
in order
to detect
inhibit
small
on the protein
interactions.
activity
of Cl inhibitor
was
at Flu-HPA
kallikrein
concentrations
activity
was
As
totally
greater
affected
not
than 2
in the
DMF
93%
inactivated.
Reaction
Between
f3-Factor
by
tions.
Figure
was
totally
greater
than
its
ability
to
inhibit
fl-Factor
XIIa.
A
that did not completely
was chosen
in order
to
on the protein
intenac-
5 shows
that the activity
of Cl inhibitor
eliminated
at Flu-HPA
concentrations
2 mM,
while fl-Factor
XIIa
activity
was
not affected
by concentrations
highest
concentration
tested,
were obtained
when Flu-HPA
of Flu-HPA
up to the
160 mM. Similar
results
was dissolved
in metha-
nol, rather
than DMF.
Therefore,
the small quantities
of DMF
used todissolve
Flu-l-IPA
had no effect on the
interaction
of Cl inhibitor
with fl-Factor
XIIa.
These
results,
like those above,
show that Flu-HPA
directly
inactivates
Effect
Cl inhibitor.
ofFlu-HPA
on a Preformed
Plasmin
and
a2-Antiplasmin
The
plasmin
effect
and
of Flu-HPA
a2-antiplasmin
Complex
on a preformed
was studied
of
complex
to determine
of
.01’
.06’
#{149}
#{149}0
.
.
=
.-
,o5.
f--_8--
r-;--
-
I
‘-
i
.04’
.
4.
Effect
and
Materials
and
the activity
indicate
--
0
.01
I
20
U
kallikrein
--
1::
.01-
circles
was
on the ability
by plasmin.
:::1
Fig.
of
.
-
I
Cl inhibitor
measured
of Flu-HPA
concentrations
up to 65 mM, the
concentration
tested.
Similar
results
were
when
Flu-HPA
was dissolved
in methanol
::
.E
E
10 mm,
concentration
of Cl inhibitor
inhibit
fl-Factor
XIIa
activity
detect
small effects
of Flu-HPA
a final
tration
of Cl inhibitor
kallikrein
activity
was
.06-
amounts
inactivates
Cl inhibitor.
inhibitor
was not detected
after
mM
Flu-HPA
for 30 see, but
[FIu-HPAJ mM
in DMF
to give
12
U
added
to a 1 ml polypropylene
snap cap tube and incubated
at
37’C for 30 mm. The tubes were placed on ice for 5 mm and then
1 50 Ml of plasmin.
which
gave a final concentration
of 0.30 nM.
were
added.
One-hundred
microliters
from
each sample
were
placed in each of three fibrin plate wells as described
in Materials
and Methods.
The dashed
line and open
circles
indicate
the
activity
of a2-antiplasmin.
The solid line and closed
circles
indicate
the activity
of plasmin
alone.
When
24 mM Flu-HPA
was tested
(not shown),
the results were the same as for 1 2 mM Flu-HPA.
presence
highest
obtained
small
The effect of Flu-HPA
on the ability
of C! inhibitor
to inhibit
/3-Factor
XIIa
was also studied.
fl-Factor
XlIa activity
was determined
spectrophotometnically
as amidase
activity,
and
Cl inhibitor
activity
was
I.’
Fig.
inhibit
the
Effect
ofFlu-HPA
on the
XIIa
and Cl Inhibitor
-
Q-
Thus,
that
0
C.)
:
DMF.
used to dissolve
Flu-I-IPA
had no effect on the interaction of the proteins.
It is concluded
from these
results
-=
-
of
ET AL.
of
Flu-HPA
Cl inhibitor.
Methods.
of Cl inhibitor
the
‘
activity
The
The
40
FIu-HPA mM
60
‘
on
the
reaction
dashed
line
of kallikrein
reaction
and
The
alone.
I
40
between
are
open
solid
plasma
described
circles
indicate
line and closed
in
I
I
80
FIuHPAi
conditions
plus kallikrein.
80
‘
‘
I
120
U
160
mM
Fig. 5.
Effect
of Flu-HPA
on the reaction
between
fl-Factor
Xlla and Cl inhibitor.
The reaction
conditions
are described
in
Materials
and Methods.
The dashed
line and open circles
indicate
the activity
of Cl inhibitor
plus fl-Factor
Xlla. The solid
closed circles indicate
the activity
of fl-Factor
Xlla alone.
line
and
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
INACTIVATION
Table
OF
a2-ANTIPLASMIN
1 . Effect
of FIu-HPA
AND
Cl
NH.
on a Preformed
1021
Complex
of Plasmin
Table
Preincubated
Plasmin
(A/3
Added
+ a2-antiplasmin
Flu-HPA
0.007
Plasmin
+ a2-antiplasmin
Buffer
0.005
FIu-HPA
+ a2-antiplasmin
Plasmin
0.124
Plasmin
+ buffer
Buffer
0.096
Plasmin
+
Flu-HPA
tration
of
0.02%
mm
added,
were
and
the
added
mixture
1 0 mm.
was
Then
incubated
,
was
at 37
x 75
added
and
Plasmin
0
activity.
The
mm
glass
Flu-HPA
could
control
shows
with Flu-HPA
concena final
tube
and
the
1 0 mm
and
mixture
was
at
incubated
was
370,
at
Methods.
dissociate
did
complex
The
plasma
the
on a Preformed
Cl Inhibitor
determine
whether
complex
complex.
Ci
for
complex
and
of
1).
clot
lysis
assay
concentration
1 .3 mM
mM
assay
tThe
Methods
were
could
and thus liberate
kallikrein
When
Ci inhibitor
was
assays
is
were
for
the
1 25
system
effect
The
performed
concentration
nM,
as described
in
of a2-antiplasmin
assuming
a
value
of
MM
1
for
the
in plasma.
is the same
as that described
in
of FIu-HPA
on the reaction
between
concentration
of plasmin
Materials
in the washed
of a2-antiplasmin
was
134 nM.
plasmin
was
Flufenamic
and
and
Materials
107
acid
nM
and
derivatives
in H2O.
dissolved
inhibitor
with
Flu-HPA
inhibitor
is not
complex
Comparison
Flufenamic
of
to
dissociate
that
preincubation
inactivated
of CI
Cl inhibitor.
capable
of dissociating
to yield free enzyme.
ofthe
Activities
Acid Derivatives
this
Thus,
enzyme-
of Water-Soluble
on the Inactivation
a2-Antiplasmin
and
Clot Lysis Assays
given
in
Flu-Glu)
the
from
shows
Flu-HPA
ied. The concentrations
50% inactivation
of
complex
studied
activity
preincubated
control
on Fibrinolysis
of
in
The influence
of two water-soluble
flufenamic
derivatives
on the activity
of a2-antiplasmin
was
of
on a preformed
inhibitor
was
kallikrein
before
the addition
krein activity
was regenerated
6.3
of a2-antiplasmin
Purified
Washed
plasmin
activity
and a2-antiplas-
Complex
Flu-HPA
+
as amidolytic
When
a2-antiplasmin
before
the addition
was liberated
(Table
not release
of plasmin
effect
of Flu-HPA
kallikrein
and
Assays
Concentration
Required
For 50% Inhibition of
a-Antiplasmint
+
lysis
the concentration
mm.
Effect
ofFlu-HPA
Kallikrien
and
Lysis
+
The final
a2-antiplasmin.
370
incubated
assayed
clot
and
incubated
that preincubation
of a2-antiplasresults
in no loss of plasmin
activi-
ty. Thus,
Flu-HPA
from a preformed
of Purified
Clot
10 mM
N-Flu-fl-Ala
DMF or DMF alone were
mixed
activity
thus
liberate
plasmin
activity.
was preincubated
with plasmin
Flu-HPA,
no plasmin
activity
in Washed
2 mM
N-Flu-Glu
the appropriate
whether
of Water-Soluble
Inactivation
of Washed Clot
Than 36 hr
-
Derivative
to give
an additional
were
on Fibrinolysis
Lysis
8. 1 . 0. 15 M NaCI,
pH
mm
for
buffer
on the
in Less
a final
in TBS-BSA
at 67 mMin
and
plasmin
5 mm
to give
M Tris-CI.
to a 10
Flu-HPA,
an additional
diluted
3 MI of 0.5
37#{176}C.2 i.tl of Flu-HPA
at
Or a2-antiplasmin,
for
and
of the Activities
Derivatives
0. 1 16
in TBS-BSA
1 5 izl of plasmin
nM,
1 35
Na azide
5
for
1 1 8 nM,
of
activity
diluted
Acid
and
Washed
5 ILl of a2-antiplasmin,
C
Comparison
a2-Antiplasmin
Activity
minI
Plasmin
buffer
3.
Flufenamic
and a,-Antiplasmin
the
with
of Flu-HPA,
no kalli(Table
2). In this table,
.08
of these
a2-antiplasmin
derivatives
was
Table
3. N-flufenamyl-glutamic
and N-flufenamyl-f3-alanine
acid
stud-
at which
observed
is
acid
(N-Flu-fl-Ala)
(N-
-
.
.O
-
..-.
U,
.5-
Table
2.
Effect
of Flu-HPA
of Kallikrein
on_a Preformed
:1
Complex
a-
and Cl Inhibitor
aC-,
Preincubated
C!
Inhibitor
+ kallikrein
C 1 Inhibitor
Flu-HPA
+_
+ Cl
FIu-HPA
<0.001
kallikrein
Buffer
<0.00
Inhibitor
Kallikrein
0.020
Kallikrein
0.028
Buffer
Buffer
Kallikrein
Activity
(A ,,,/min)
Added
+ Flu-HPA
Kallikrein
1
0.025
20
25 MI of Ci
of
Na
39 nM,
azide).
3
mm
at
of
,
in DMF
370
preincubated
(0.5
in TBS-BSA
M Tris-CI,
5 Ml of kallikrein
glass
FIu-HPA
diluted
buffer
.Ll of
and
concentration
siliconized
inhibitor
.
for
40
nM
tube
and
10
mixture
further
assayed
as amidolytic
mm
added
in TBS-BSA
in
incubated
at 37
alonewere
added
as indicated,
Cl
at 3 7
incubated
activity
for
inhibitor,
0 ,
5
and
mm
a final
order
to
C for 5 mm.
at
as described
kallikrein
a final
75
incubated
and
Tris
was
37#{176}.Kallikrein
in Materials
x
mm
2 izl of 1 . 14 M
further
Flu-HPA,
then
10
#{176}
and
0.02%
to give
a
IFIu#{149}Na1’
] mM
concentration
pH 8. 1 5, 0. 1 5 M NaCI.
diluted
were
or DMF
to give
for
buffer
added
and
activity
and Methods.
10
were
the
was
Fig. 6.
Effect
of sodium
flufenamate
on the reaction
between
plasmin
and a,-antiplasmin.
The assay procedure
is the same as
that described
in Materials
and Methods
for the effect of Flu-HPA
on the reaction
between
plasmin
and a,-antiplasmin.
The concentration
of plasmin
was
1 34 nM.
and the concentration
of a,antiplasmin
was 1 07 nM
Sodium
flufenamate
was dissolved
in
H,O.
The dashed
line and open
circles
show
the activity
of
a,-antiplasmin
indicate
plus
the activity
plasmin.
of plasmin
The
alone.
solid
line
and
closed
circles
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1022
MILES
did
not
inactivate
compound,
sodium
plasmin,
Table
although
the
parent
above
5 mM
totally
flufenamate,
that
the
to inactivate
corresponded
to
ability
of N-Flu-fl-Ala
and
directly
purified
a2-antiplasmin
their
activity
in
washed
oflnactivation
ofcx2-Aniiplasmin
lysis
by
activity
was lost.
mixture
containing
a2-antiplasmin
to
remove
the
N-Flu-Glu
restored
antiplasmin
to inhibit
plasmin.
of a2-antiplasmin
by N- Flu-Glu
Reversibility
of Inactivation
ability
of
by
The
reversibility
of the direct
inhibitor
by N-Flu-Glu
was also
When
Cl inhibitor
was preincubated
inactivation
of Ci
studied
(Table
5).
with N-Flu-Glu,
it lost its ability
of the reaction
However,
N-Flu-Glu
inhibitor
inhibitor
restored
to inhibit
most of the observed
kallikrein.
Thus,
the
Cl inhibitor by N-Flu-Glu
Table
4.
Reversibility
dialysis
and CI
of a,-Antiplasmin
0.003
buffer
sO.0O1
0.004
a2-antiplasmin
0.050
N-Flu-Glu
a2-Antiplasmin
+
Control
without
Ml of a2-antiplasmin
in H 20 or H 20 alone
at 3 7
from
each tube
7.0.
0.02%
also kept
0.02%
at 0.48
against
Na
mg/mI
were
black-masked
absorbance
cuvette
at 405
after
43
then
with
nm/mm
hr.
a
was
at 450
tube
and
removed
assayed
1 -cm
mixture
path
30
at
sec.
was
length
The
27
70
and
in
g/ml
and
zl of S-225
placed
values
activity
pH 8. 1 , 0. 1 5 M
plasmin
for
recorded.
MI of each
for a,-antiplasmin
M Tris-CI,
1 5 121 of
The
pH
At 1 9, 43, and 68 hr. the
3 ILl 0.5
temperature
added.
iLl were
at 4#{176}C.200
dialysis.
were
adding
glass
1 liter of 40 mM Na phosphate,
with
at room
mm
1 00
of
1 mMfl-mercaptoethanol,
azide,
mxiture
75
C 1 inhibitor
200
ILl of Cl
inhibitor
in H2O
or H2O alone
80
of
each
reaction
1 3 1 nM
incubated
and
for
mixture
were
separate
flasks
containing
50 mM
pH
5.3,
1 mM
Na4EDTA,
1 mM
50
g/ml
polybrene.
After
as described
1 4 M’ of N-Flu-GIu
30
mm
and
dialyzed
Na acetate-acetic
24 hr of dialysis,
in Materials
and
acid,
HCI,
reaction
at 450
at 37#{176}C.Two
removed
benzamidine
40 Ml of each
dialysis.
0.045
0.040
at
were
12
mixture
Cl
at
of
4#{176}Cin
70 mM
0.02%
mM
aliquots
NaCI.
Na azide,
were
inhibitor
also kept
activity
and
at
was
Methods.
acid,
of action
of Fluas a fibninolytic
enhance
clot
through
hypothesis
a
plasmin,
the
lysis
by
direct
disruption
plasminogen-independent
that Flu-HPA
directly
major
inhibitor
of
fibnin
or
pathway.
The
inactivates
a2-anti-
of plasmin
in plasma
was
purified
proteins.
Plasmin
activity
was
using
‘25I-fibrin
plates
or the spectrophoD-Val- Leuthat Flu-HPA
a2-antiplasmin.
ability
of Flu-HPA
a protease
inhibitor
to
Lys paranitroanilide.
directly
inactivates
inactivate
of several
directly
plasminogen
Cl
krein,
or fl-Factor
XIIa
as assayed
with spectnophotometnic
tnipeptide
substrates.
This
finding
differs
from the results
obtained
for orthothymotic
acid and
5- 1623,
a(isobutyl-4-cyclohexene-I
-yl)
propionic
-
14 Ml of N-Flu-Glu
aliquots
at 4#{176}C
without
and aliquots
the
and
to a 1 0 x
Four
1 5 ILl of each sample
incubating
nM
added
mm.
and dialyzed
was changed
Ned.
at 140
were
C for 30
Na azide,
were
by mixing
obtained
0
Dialyzed
0.045
+
0.0
0.001
inactivates
the
inhibitory
activity
of CI inhibitor
against
plasma
kallikrein
and purified
fl-Factor
XlIa.
Flu-HPA
by itself does not inhibit
plasmin,
kalli-
Activity
(.A,/min)
a2-Antiplasmin
N-FIu-GIu
activators,
was also studied.
Cl inhibitor
was isolated
using a new procedure
that employs
DEAE-Sephadex
and SP-Sephadex
chromatography.
Using
purified
Cl
inhibitor,
it was demonstrated
that Flu-HPA
directly
Not Dialyzed
buffer
without
ILl
Dialyzed
<0.001
Control
purified
The
inhibitor,
ability
of Cl
inactivation
of
Plasmin
sample
+
tometnic
tnipeptide,
The results
showed
by N-Flu-Glu
incubated
inhibitor
tested
using
determined
is reversible.
of Inactivation
Activity
agent
were undertaken.
The profibninolytic
activity
of
Flu-HPA
in washed
clot lysis assays
was found
to be
dependent
on plasminogen.
Thus,
Flu-HPA
did not
a2-
N-Flu-Glu
to inhibit
kallikrein.
mixture
containing
by N-FIu-G1u
0.033
Studies
of possible
mechanisms
HPA,
a derivative
of flufenamic
Thus,
the inactivation
is reversible.
of Cl Inhibitor
Inhibitor
DISCUSSION
Glu,
its ability
to inhibit
plasmin
However,
dialysis
of the reaction
the
fibrinolytic
compound
and
600
inhibitor
Cl
assayed
The
reversibility
of the direct
inactivation
of a2antiplasmin
by N-Flu-Glu
was studied
(Table
4).
When
a2-antiplasmin
was preincubated
with N-Flu-
mM
c..
4#{176}C
without
the
of Cl
Not Dialyzed
assays.
Reversibility
N-Flu-Glu
of Inactivation
AL.
UA,/m,n)
N-
clot
Reversibility
Kallikre,n
inactivates
plasmin
(Fig. 6). In Table
3 it can also be
seen that N-Flu-fl-Ala
was effective
in the washed
clot
lysis assay
in 36 hr at concentrations
above
2 mM,
while
N-Flu-Glu
was readily
effective
at 10 mM.
It
appeared
Flu-Glu
5.
ET
1
in a narrow
the
change
the
table
in
were
acid, which
inhibit
both plasmin
and a2-antiplasmin,’7
and also for other
fibninolytic
compounds
studied
which
fibrinolysis
is inhibited
at high concentrations
in
of
the synthetic
derivatives.7
Thus,
FIu-HPA
may
be
more
specific
in its mechanism
of action
than
these
other compounds.
It was found that Flu-HPA
does not
liberate
enzyme
activity
from preformed
complexes
of
either
a2-antiplasmin
kallikrein.
These
plus plasmin
results
do not
on Cl inhibitor
plus
support
a previously
proposed
mechanism
for the enhancement
sis by synthetic
compounds
that involved
of fibninolyeither
disso-
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
INACTIVATION
OF
a2-ANTIPLASMIN
AND
ciation
ofa plasmin-antiplasmin
of plasminogen
activator
inhibitor
been
reported
albumin.46
In our
to a concentration
However,
the
was completely
1023
complex
from
activity
Kaulla’s
synthetic
that
or liberation
an activator-
These
dialysis.’7
Thus,
do not interact
binds
to
Flu-HPA
at albumin
on a2-antiplasmin
concentrations
are
that
were
consistent
higher
required
of
with
concentrations
to lyse
von
of
plasma
to
the
binding
of
the
It was
also
found
up
I8
that
activities
of these
compounds
assays
correspond
to their
ability
the
in washed
to macti-
activity
N-Flu-Glu
that has been abolished
is restored
after
dialysis
Thus,
appears
differ
from those
which
directly
dissociated
from
the
effect
to be
by
to
inhibitor
inhibits
of plasminogen
activated
kallikrein,
all
plasminogen.24
since
Cl
activated
of which
29 The
have
rela-
Derivatives
vitro assays
of flufenamic
acid
or other
procedures
could
be utilized
in in
in which
removal
of
the
protease
is desirable.
plasma
inhibitors
Moreover,
Flu-HPA
is the first synthetic
fibninolytic
agent
tested
that does not appear
to inactivate
plasmin.
Hence,
this
property
of the compound
and related
derivatives
may
make
them
plasminogen
especially
activator
of this compound
reversible.
These
useful
assays.
for in vitro
fibninolytic
or
ACKNOWLEDGMENT
for the fibninolytic
compound,
inactivates
a2-antiplasmin
but
and
antibodies
a2-antiplasmin
Dr.
Charles
after
activators,
Factor
X I I,
‘
tive importance
of these different
activities
of Ilufenamic
acid derivatives
in different
fibninolytic
assays
will depend
on the design
of the particular
assay itself.
remove
N-Flu-Glu.
on a2-antiplasmin
is not
that these fibrinolytic
agents
can mactimajor
inhibitor
of plasmin
in plasma”
the
acid,
pun-
due
a2-antiplasmin.
results
5-1623,
demonstrates
vate both
to mactiinhibitor
Factor
XI, and plasma
been reported
to activate
was
substrate.
a2-Antiplasmin
treatment
with
restored
the inhibitory
activity
of Cl inhibitor.
The ability
of flufenamic
acid derivatives
vate
directly
both
a2-antiplasmin
and
Cl
such
tnipeptide
vate
the same mechanism.
The
interaction
of N-Flu-Glu
with
CI inhibitor
appears
to be reversible,
since dialysis
of a mixture
of
N-Flu-Glu
and Cl inhibitor
to remove
the compound
this
fled a2-antiplasmin.
They do not inactivate
plasmin
to the highest
concentrations
tested,
40 mM and
mM, respectively,
as assayed
with
the spectrophotofibninolytic
clot lysis
S-l623
presumably
through
exactly
and an
inhibitor
fibrinolytic
compounds
to inert
plasma
proteins
as albumin
during
clot formation.7
Two
water-soluble
derivatives
of flufenamic
N-FIu-Glu
and N-Flu-fl-Ala,
directly
inactivate
metric
N-FIu-Glu
and
with a2-antiplasmin
synthetic
compounds
were
incorpolyse
preformed
hanging
clots.
He
into which
than
to
that
acid
no effect of albumin
up
mg/ml
was
observed.
results
observation
compounds
suggested
flufenamic
experiments,
of 4.3
effect
of
abolished
mg/mI.
clots
rated
INH.
complex.6
It has
21 .5
Ci
extensive
The
authors
thank
Gregory
Beretta
to plasminogen.
G. Cochrane
for preparing
The
are
support
gratefully
and
a-Factor
XlIa
encouragement
of
acknowledged.
REFERENCES
I.
von
Kaulla
KN:
Suppl
32:63,
Haemorrh
in
2. von Kaulla
vitro
studies
Haemorrh
3.
von
4.
certain
agents.
I 975,
von
von
7.
von
von
mechanism
plasma
Biol
KM:
derivatives
as
106:530,
1961
Med
fibrinolysis
Kaulla
KD,
Agents.
model
Davidson
TF
Springfield,
TA:
KN:
Fibrinolysis
KN:
agents,
Kluft
for
On
New
LB:
Blocking
Fed
E,
von
for
unmasking
by certain
synthetic
the
96:4,
(eds):
in
JM
Moroi
York,
mechanism
of
Wiley-lnterscience,
of urokinase
Proc
34:290,
Kaulla
KN:
Factor
fibrinolytic
14.
of
1970,
binding
to plasma
Eur
p 1
VIII
activity
agents?
removal
in
in von
the
hemophilic
Kaulla
Raven,
of
flufenamate
1975,
and
Agents.
on
in Davidson
in Chemical
euglobulin
JF.
Samama
Fibrinolysis
and
p 375
some
in human
KN,
Aoki
I 5.
properties
plasma.
Wiman
Biochem
Eur
of a new
J Biochem
fast-
69:209,
78:19,
17.
Chem
primary
of
25 1 :5956,
inhibitor
a21976
of plasmin
1976
the
kinetics
of the
a low-molecular-weight
reaction
form
between
of
plasmin.
1978
Collen
D:
the
fast-acting
antiplasmin,
of a2-plasmin
Med
B,
characterization
J Biol
I: The
D: On
and
87: 143,
and
plasma.
J 159:545,
B, Collen
Wiman
Harpel
Isolation
human
S. Clemmenson
plasma.
J Biochem
N:
from
Mullertz
human
inhibi-
M,
antiplasmin
16.
inhibitor
Effect
Progress
York,
Thrombolytic
p 176
inactivator,
(eds):
inhibitor
J Biochem
Fibri-
1975
Is
I 3.
aromatic
synthetic
Control
P:
D: Identification
inhibitor
human
Chemical
I 975,
ofCl
PC
New
in human
by
Brakman
plasmin
12.
of
1962
vitro
(ed):
activity
Fibrinolytic
I 976
C
1966
in vitro
C,
Collen
.
reacting
Synthetic
C Thomas,
Involvement
Thrombolysis.
Charles
fibrinolytic
15:1 171,
induction
Biophys
in Schor
The
Pharmacol
(eds):
Charles
Desnoyers
I I
A structure-activity
in von
Biochem
Kaulla
Kaulla
Urea
Gryglewska
by diiodo-salicylate.
9.
Exp
Biochem
8. Nanninga
10.
plasmin
nolysis-Thrombolysis.
tor
RL:
Thrombolytic
RJ,
Arch
TF
fibrinolysis:
p 227
Kaulla
fibrinolytic
Soc
agents,
5. Gryglewski
6.
Smith
Kaulla
N-arylanthranilates.
derivatives.
Davidson
Springfield,
MM,
Proc
C,
Fibrinolytic
Thomas,
Diath
Chemical
structure
and fibrinolysis
induction
126 synthetic
compounds.
Thromb
Diath
KN,
fibrinolytic
Synthetic
Thromb
1962
Kaulla
I-Iansch
Fibrinolytika.
1969
KN:
with
7:404,
promoting
Synthetischc
Purification
and
plasmin
characterization
inhibitor
of
in plasma.
Eur
1977
PC:
Plasmin
inhibitor
146:1033,
1977
Prandini
MH,
inhibitor
in the
Wiman
interactions.
presence
B, Samama
The
of a2-macroglobulin.
M,
Collen
effectiveness
J Exp
D: Effects
of
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
MILES
1024
the
synthetic
the
reaction
13:165,
fibrinolytic
agents
between
human
PC.
vator-enzyme
and
19.
Cooper
trypsin.
of the
inhibitor.
first
CG,
Pensky
factor
and
by purified
serum
Cl inactivator.
Schreiber
factor
kinin
plasma
I, Mason
Kagen
human
J Clin
Bouma
plasma
of
Biochemistry
kallikrein.
28.
a2-plasmin
Factor
Burnier
derivatives.
Ph.D.
DG,
by
by
Hage-
XII
and
Meth
(Hageman
for
immunological
Progress
Heremans
JH:
in
Allergy.
JF:
Immunochemical
immunodiffusion.
Human
and
blood
Immuno-
coagulation
mechanism
by
acti-
1977
D,
Schwartz
SA:
Amino
fibrinopeptides.
cape
Factor
of activation
252:6432,
artiodactyl
deer,
OH,
Rosebrough
with
the
buffalo.
NJ,
folin
I
Arch
Farr
phenol
interaction
Blood
and
to the
Taylor
FB
54:850,
relation
of
acid
Dromedary
.
Biochem
AL,
Biophys
Randall
reagent.
of
J Biol
(eds):
The
XI
plas-
Regulation
1980,
M,
properties
of some
of
p 555
Doolittle
R,
Good-
flufenamate
University
activity
of California,
by avian
43.
McConahey
Ri:
Protein
Chem
193:265,
44.
tion
Diego,
ET:
Plasminogen:
Purification
from
RNA
Morita
of
diates.
T,
bovine
carinatus
viruses.
I. Chick
Iwanaga
and
by
E, Svendsen
in:
in
DHEW
Publication
Chignell
albumin.
with
and
and
Disease.
DF:
Mol
(NIH)
Optical
of flufenamic
of
1973
29:1 85,
mechanism
the
of
trans-
iodination
new
of
1966
of activa-
isolated
L, Vennerod
kallikrein
No
I 37:85,
Allergy
activator
DB,
from
active
Echis
interme-
1976
Chemistry
Health
Rifkin
of trace
T: The
an
JP,
Invest
fibroblasts
Med
Arch
characterization
79:1089,
plasma
Int
of a
Lab
transformation
embryo
J Exp
means
Clin
with
A method
5, Suzuki
J Biochem
System
46.
FJ:
studies.
Amundsen
of
viruses.
Dixon
by
J
L, Quigley
associated
prothrombin
venom
mination
serum
A, Ossowski
tumor
PJ,
E: Determination
blood
Scand
function
for immunologic
derivative,
of flufenamic
San
by oncogenic
in human
substrate.
Tobia
enzymatic
formed
45.
1978
fibrinolytic
fibroblasts
P. Gyznader
inhibitor
tripeptide
JC,
E: An
proteins
Factor
contact-mediated
Verlander
and
1977
Unkeless
Friberger
plasmin
specific
42.
1979
phenomena:
AC,
rapid
37:403,
fibri-
fibri-
the
as coupled
I 2:707,
Mertz
a
1967
Interaction
Deutsch
Factor
(eds):
radial
Schubert
on
Teger-Nilsson
plasmin
substrates.
I 979
32.
AO,
Chem
RF,
Lowry
41.
plasma
factor-dependent
coagulation
thrombolytic
Thesis,
plasmin,
HCI.
methods
BH
and
J Biol
studies
of a new
factor
plasminogen.
M,
Synthesis
human
of activation
in man
KG,
Res
trypsin,
Human
by single
Griffin
XII.
measurement
1969
Elsevier/North-Holland,
and
of
of
1973
p 20
properties,
mule
40.
Factor
36:25,
1965
Doolittle
Reich
antecedent)
JP:
by
35:273,
activity
deficiency
Thromb
39.
I 18:456,
Human
Hageman-factor-dependent
and
York,
M: Synthesis
derivatives.
acid
AP:
Sakarellos-Daitsiotis
.
JH:
1977
XI and
in Mann
New
Factor
demonstration
1976
G, Carbonara
BN,
vated
CG:
Waksman
1962,
Bouma
Purification,
by an
of plasminogen
p’-guanidinobenzoate
Diffusion-in-gel
Kallos,
2:235,
XI.
and
J Biochem
Titration
45:56,
ofantigens
chemistry
E:
plasminogen
changes
plasmin
Eur
Cochrane
0:
in
Mancini
sequence
phosphofluoridate.
Hageman
fibrinolytic
H: Fibrinolysis
generation,
Commun
AP:
Kaplan
inhibitor
Coagulation.
Res
252:6097,
of
S Karger,
of human
I 91
mechanism
R Jr.
Basel,
of
factor
by activated
of plasminogen
Kaplan
thromboplastin
30.
activation
Hageman
Griffin
diisopropyl
in Hageman
activated
Saito
(plasma
The
and
G,
participation
Generation
29.
31
Jr.
Chem
Mandle
human
by
II.
camel,
Beretta
prekallikrein’s
and
J Biol
nolysis:
1964
170:1095,
1970
Ouchterlony
38.
of the
Shaw
Enzymol
analysis
1970
45:604,
0:
XII)
of its activation
Biophys
R
plasma
factor
nolysis.
Ratnoff
of
1978
LA,
Activation
Biochem
Human
62:54,
inactivation
RW:
Mandle
man
properties
(Factor
Meth
37.
I 9: 1 15 1 , I 980
Colman
27.
H,
Studies
its
104:574,
physical
Pathol
factor).
to
p-nitrophenyl
JH,
quantitation
Interaction
Jr.
I 9:20,
Griffin
fibrinolysis,
KF:
Science
Structural
compound.
T
35.
P: Activation
activation
with
36.
by Cl INH
J Immunol
Br J Exp
Miles
Enzymol
Inhibition
Austen
and
factor
C’l
1973
RW,
Saito
Invest
BN,
prekallikrein.
and
man
Jr.
by Hageman
fragments.
mm
G
KF:
inhibitor.
globulins.
Goldsmith
25.
Cl
by serum
1970
of
Chase
thrombin
76:809,
chromatography.
of urokinase.
intermediate
34.
of
B, Wallen
course
Cl’r
of activated
affinity
derivative
the
the
of coagulation,
biochemical
in
antecedent
Med
52:1402,
Colman
with
Cls,
by
Wiman
insoluble
and
thromboplastin
activation
the
Some
permeability
24.
plasma
AL.
1970
Cl macti-
inhibition
Inhibition
Clin
Austen
Invest
JW,
with
KJ:
plasminogen
AP,
J Clin
kallikrein
23.
OD:
J Lab
fragment
generation.
Gigli
Ratnoff
plasma
Kaplan
The
factor,
of complement
1969
activated
AD,
of Hageman
26.
human
possible
D, NaffGB:
129:315,
J.
Hageman
XII
on
Res
1975
permeability
component
Med
plasma
of interaction
55:593,
plasma
J Exp
Forbes
22.
5-1623
Thromb
on human
J, Ogston
kallikrein,
subcomponent
esterase
Invest
Pensky
plasma
Studies
I . Mechanisms
J Clin
RatnoffOD,
plasmin,
20.
NR:
interactions.
plasmin
and
and
antiplasmin.
33.
Harpel
.
acid
and
1978
18.
21
ortho-thymotic
plasmin
ET
the
Intl
K: Detertripeptide
Kallikrein-Kinin
Ctr
Proc
No
27,
1974
studies
Pharmacol
of
Fogarty
and
Laake
chromogenic
Biology
76,
acid
AM,
a new
of drug-protein
other
5:455,
complexes.
N-arylanthranilates
1969
III.
with
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1981 57: 1015-1024
Inactivation of purified human alpha 2-antiplasmin and purified human C1
inhibitor by synthetic fibrinolytic agents
LA Miles, JP Burnier, MS Verlander, M Goodman, AJ Kleiss and JH Griffin
Updated information and services can be found at:
http://www.bloodjournal.org/content/57/6/1015.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.