Dec 28, 1984 - For personal use only. by guest on July 10, 2011. ..... cellular debris and stoned at -. 20 #{176}C.For the purpose of analysis, the medium.
From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.
1986 67: 616-622
The mutual relationship between the two molecular forms of the major fibrinolysis inhibitor alpha-2-antiplasmin in blood C Kluft, P Los, AF Jie, VW van Hinsbergh, E Vellenga, J Jespersen and CP Henny
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From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.
The
Mutual
Relationship
Between
Fibrinolysis By C. Kluft,
synthesized lar forms: a
less
a
in the
study
ratio
the
constant
for
a
Resynthesis acute-phase
PB
form
after
of the
A
In vitro
was
in blood
after
LPHA-2-ANTIPLASMIN with an apparent
is a
PB form
inhibits
culture
plasma weight
plasmin-activated
of
after
myocar-
that
in the
molecular
in
only
the
medium
PB ±
first
relative
be
a critical
shown
by
role
the
in the
in vivo
hemorrhagic
deficient
diathesis
found
as well as in some
role is attributed form is unknown.
to the
PB form;
the
NPB
Grune
liver
unknown, however, synthesized and the unclear. Circumstantial
cell
line’2
and
form
From
the
Gaubius
The
Netherlands;
versity
Hospital.
Thrombosis
Institute, the
This
of the
appears to be of synthesis
in rat hepatocytes.13
Groningen,
Research,
Denmark;
and
the
Research of
The
It is is forms is that the
South
Jutland
Academic
Division
TNO,
Haematology,
Uni-
the
for
Netherlands; University
Medical
Section
Center,
Centre,
Esbjerg.
Amsterdam,
The
sis and 50: 170,
in part
at the IX
Haemostasis, 1983
Juh’
I 983,
Submitted
Dec 28, 1984; reprint
requests
Division
TNO,
Fund accepted
1986
b; Grune
Sept
No.
Haemostas
Gaubius 2313
Inc.
or inhibitors
did
demonstrate
not that
by the
liver
circulation.
from
the PB form
in vitro’4
and
in
and
mutual
under a
liver
various cell
The
of the
we
PB
conditions
line
study
relationship studied
with
form
in blood
a recently
provides and
data
the
occurrence
and
in culture
devised
that
assay
for
demonstrates
formation
AND
of the two
their
the N PB form
of the
METHODS
Unless otherwise specified, reagents were of analytiand obtained from Merck, Darmstadt, FRG. “Agarose for electrophoresis” (lot No. 33006) and iodoacetamide were obtained from BDH Chemicals Ltd. Poole, England. Carbowax 6000 was from
Fluka
AG,
human
Buchs,
sen. Copenhagen. fraction
Ill
by gel filtration the
of
H-D-Val-Leu-Lys-pNA Stockholm. acid,
was
a gift Dr
prepared
Trasylol
from
E.
was
in rabbits
Dr I. Clemmenfrom
(5880
Bayer
Philipp.
(5-2251) actin
raised from
human
on lysine-agarose,’7
G-l50.
was
courtesy
a gift
chromatography
on Sephadex
units]/mg)
through
Antiserum was
Lys-plasminogen
by affinity
inactivator
Switzerland.
alpha-2-antiplasmin
AG, The
for plasmin
from
Dade
Cohn followed
KIU
[kallikrein
Wuppertal,
FRG,
synthetic
tnipeptide
was from
AB Kabi,
Diagnostics
Inc.
Miami.
Dextran sulphate, sodium salt (mol wt 500,000) was obtained from Pharmacia Ltd. Uppsala, Sweden. Active CIs-esterase was prepared from
outdated
Lepow.’t white
plasma
Trypsin
type
Sigma
lI-T
and
Chemical and
Freehold, granulocytes and
Co.
ovomucoid
Ni.
according
inhibitor
to Vroon
from soybean
et al’8 and
type
diisopropylphosphouluoridate St
Louis. were
Lima from
bean
Haines
and
1-5, from turkey egg (DFP) were from
trypsin
Worthington
inhibitor,
ovoin-
Biochemical
Corp.
Whole extracts of gnanula of polymorphonuclear were prepared as described before.2#{176} Elastatinal, pep-
chymostatin
Microbiological
(see
ref.
Chemistry
21)
were
Research
gifts
from
Dr H. Umeza-
28-443).
wa,
Institute,
Platelet-poor citrated human plasma and pooled plasma were prepared as described by Kluft et a122; serum was prepared by incubation of nonanticoagulated blood at 37 #{176}C for four
10, /985.
Sd,
by
plasmin-
cal grade
Foundation,
Tokyo.
Plasma.
AD
Leiden,
Health The
hours & Stratton,
0006-497l/86/67O3-0O1O$03.OO/O
616
(project
to Dr C Kluft, Herenstraat
Netherlands. (#{149}i
on Thrombo-
(Thromb
Stockholm
and
is produced
MATERIALS
statin,
by the Prevention
Address
Congress
fabstr526/).
Supported
Research
International
uninfluenced
fibrin.
at was
Inc.
the origin
behavior
hibitor,
Netherlands. Presented
and
an
Materials.
as
NPB
Health
conversion
in the
to
with
The
is formed
be obtained
more and is
relevance
Department
form,
in vitro.
of alpha-2-antiplasmin,
Ellagic
Leiden,
0.55
±
spontaneous demonstrated
& Stratton.
can
synthesis
congenitally
which form of alpha-2-antiplasmin mutual relationship of the two evidence has been provided
2.86
NPB
results
in
demon-
vivo.’5
against
in an established
form
blood,
was
These
of alpha-2-antiplasmin
by
to
of enzymes
process.
that
the
PB
by
decrease
days.
fibrinogen.
PB form
and
heterozygotes.9”#{176}
The site of synthesis of alpha-2-antiplasmin the liver, as recently supported by demonstration
in
appeared
synthesis
from
dependent
and
by rapid
stabilizing
in
serum
of a variety the
To determine
of fibninolysis,
regulation
and
with
in the circulation.
plays
the eight
the
forms
factor (factor XIII).5’6 The NPB form reacts much slowly with plasmin,3 does not bind to plasminogen, not significantly cross-linked to fibrin.7 Alpha-2-antiplasmin
form
An
from
of about
Additions
interfere
coagulation
fibnin
NPB
components
forms.’6
the
the
temperature
both
of
rapid
apparently
of
action
a more ratio
media
the
revealed PB-NPB
conversion
form of
SD).
of a stoichiometnic plasmin complex.2’4 It further interacts with fibninolysis by forming a reversible complex with plasminogen and by covalent binding to fibnin during by
NPB
of 6.
to
a 1986
the inhibition
in the
fibrinolytic
NPB of
formation
caused
while
Henny
after to
(mean
in plasma
ogen.
Ch.P.
0.24
half-life
found
G2.
therapy
order ‘C
Hep
and
by a change
apparent
plasmin(NPB).3
fibninolysis
line
Clearance
form
strated
Major
of
glycoprotein (mol wt)
forms: binding
cell
L-asparaginaSe the
of the
J. Jespersen,
1 1 days.
the
of the
discontinuation
demonstrated day
and 0.34
synthesis
and
human
37
±
increase
It occurs in blood in two molecular binding (PB) and nonplasminogen
68,000.12
ogen The
after therapy
one
the in the
2.41
=
the
1 .74
heterozygotes
a specific
studies
present
of
variation
to be.
or increased
showed
or streptokinase
infarction.
This
to 138%).
found
PB/NPB
depletion
molecule
and
alpha-2-antiplasmin.
cirrhosis:
reaction
1-asparaginase PB form
of
liver
wide
volunteers,
deficiency
form
form.
(16% was
Forms in Blood
E. Vellenga,
after
relationship
Despite
inhibitor
is
molecu-
(PB)
Molecular
Hinsbergh,
fibrinolysis.
mutual
in vivo
healthy
a stable
the
dial
forms
among
with
and in vitro.
of the
two
congenital
patients (SD).
and
van
in two
(NPB)
origin
in vivo
concentration
between
main.
of
in blood
plasminogen-binding
the
forms
in plasma
inhibitor
occurs
Two
Alpha-2-Antiplasmin
Jie, V.W.M.
nonplasminogen-binding
investigates two
and
active
active
these
major
liver
a very
Inhibitor
P. Los, A.F.H.
Alpha-2-antiplasmin,
the
in plastic
tubes
before
Xlll-deficient
plasma
were
Inc.
Park,
Kansas.
Overland
centnifugation. obtained Plasma
Blood, Vol 67,
from
Factor George
XIIKing
depleted
in plasma
No 3 (March),
1986:
and
factor
Bio-Medical urokinase
pp 6 16-622
From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.
MOLECULAR
was
FORMS
prepared
urokinase
OF
ALPHA-2-ANTIPLASMIN
by chromatography
as described
column
on
by Kluft
617
coupled
et al.23 Plasma
antibodies
depleted
to
in plasmin-
ogen was prepared by chromatography on lysine-agarose (cf ref. I 7); the depleted plasma showed no response on immunochemical assay for plasminogen (detection limit about 5%) and a residual activity of 1.6%
in the streptokinase
method
of Fniberger
et al.24
Modified crossed immunoelectrophoresis with added lys-plasminogen was carried out as described by Kluft and Los.’6 Briefly, the 1% agarose gel for the first dimension in 0.03 mol/L buffer, pH 8.6, contained 1,000 KIU/mL Trasylol and 0.04 mg lys-plasminogen/per milliliter added to the agarose solution just before casting the gel. Before electrophoresis, 5 zL of plasma or serum, 2 zL of lys-plasminogen solution (2 mg/mL), and I ML of 10,000 KIU/mL Trasylol were sequentially and rapidly introduced into the punched well. The gel for the second dimension contained antiserum against alpha-2-antiplasmin. The immunoprecipitation peak surface at -mobiIity represents the concentration of the PB Assays.
form
of alpha-2-antiplasmin,
form.
The
antiserum
alpha-2-antiplasmin
at a-mobility,
that
of the
NPB
both forms of of the PB and
discontinuation of 1-aspanaginase intravenous therapy. dosage varied from 5,500 to 7,496 U/m2. The total duration of the therapy varied from seven to I 7 days and resulted in reduction in the immediate plasmin inhibition test to 5% to 30% of normal, reverting to normal again after discontinuation.29 Informed consent was obtained. Cell
culture.
human Institute produce
liver
25-cm2
flasks
The
established
assay of alpha-2-antiplasmin,
test,
was
crossed
immediate
performed
as
the immediate
described
by
immuno-electrophoresis
plasmin
a known
with
titrated
inhibition
plasmin.26
plasmin
Kluft
et
a19 and
simultaneous
by
runs
using
modified
with
the
a standard
result
of
containing
0.1
supplied
(pooled
on day
crossed
pooled
Alpha-2-antiplasmin of
plasma
immunoelectrophoresis
normal
plasma
determined
Laurell27 (1.07
with
in
known
amounts
was
in the conversion
of alpha-2-antiplasmin
I 8 days
for
immunochemically
expressed
by the
in percentage
of
pooled
tech-
normal
j.smol/L).
Changes
rate
were
at 37 #{176}C and
of plasma
assessed
assay
immunoelectrophoresis.
of the
Depleted
in vitro
of the two
by incubation pattern
or
of the
of the
deficient
modified
plasmas
forms
plasmas crossed
were
com-
or pooled normal plasma. Effects of inhibitors (25 zL added to 250 L plasma) were compared with buffer controls. The effects of CIs-esterase, granulocyte enzymes, dextran sulphate, and ellagic acid on the antiplasmin activity in the pared
with
starting
plasma
immediate
plasmin
incubation
at 37 #{176}C in I : 1 mixtures.
Volunteers institute.
8. The
inhibition
and
test
were
studied
with
after
antiplasmin
media
volunteers
congenital
units
and
a maintenance
was increased
dosage
stopped
after
therapy
were analyzed
sis assay
in a special
97 hours.
to
Samples way.
intermediate
agarose
minogen’6
was
necessary.
Three
admitted
dose
gel
introduced. patients
to hospital
of 5,000 U/h
obtained
were
from
a
mL/cm2
(Dulbecco’s
modified
were
centrifuged
to remove of
cellular
analysis,
the
concentrator
B IS
The
steady
data
individuals,
from
antiplasmin
deficiency
in Fig
on was
the
state obtained
heterozygotes
plasma concentration, with an apparently
with
1 , the ratio synthesis
in
congenital
and from patients impaired synthesis
half
with of the
by Launell immunoassay. (Fig I) was 2.41 ± 0.34
The
ratio
(SD),
well
stable normal
alpha-2-
approximately
normal
liver cirrhosis inhibitor in a
stable period. (It was recently demonstrated enhanced catabolism occurs in such patients.30) amount of alpha-2-antiplasmin (PB + NPB) mined group
PB is not
of alpha-2-
conditions, eg, from apparently for
an
PB-N rate
that no The total was deter-
PB-NPB in
experience in a larger group of 29 apparently teens showing 2.33 ± 0.29 (SD).
in this
accord
healthy
with
volun-
ratio
the defi-
layer
during
and
shortly presence
samples,
the
against
precaution
study28 of patients
a diagnosis
of myocardial
an plas-
with
acute
studied
lymphocytic before
the
leukemia start,
at the
proved infarction,
described end,
and
0
0
____
00
50
--- ____
100
ANTlP1ASMIN(immun/,)
with chest
by electrocardiography and enzyme studies (AMI
