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Immunoreactive Precipitation of C1 Inhibitor Protein from Plasma of Normal Subjects and of Patients with Hereditary Angioedema after Isoelectric Focusing

Authors:

Abstract

C1-inhibitor is an acid glycoprotein, isoelectric point 3.5-3.6. Plasma of some patients with a variant form of hereditary angioedema contains high levels of functionless C1-inhibitor-albumin complex with an isoelectric point at 4.5-4.6. Therapy with Danazol, which increases C1-inhibitor levels, does not modify the isoelectric focusing pattern of such protein in patients with hereditary angioedema.
Bergamaschini
et
al.:
Isoelectric
focusing
of
C
l-Inhibitor
739
J.
Clin.
Chem.
Clin.
Biochem.
Vol.
24,
1986,
pp.
719-722
©
1986
Walter
de
Gruyter
& Co.
Berlin
· New
York
Immunoreactive
Precipitation
of
Cl
Inhibitor Protein
from
Plasma
of
Normal Subjects
and of
Patients with Hereditary
Angioedema
after
Isoelectric Focusing
By
L.
Bergamaschini,
C.
Valle,
M.
Franzinelli,
M.
Cicardi
and A.
Agostoni
Department
of
Clinical
Medicine,
University
of
Milan,
Milan,
Italy
(Received
March
5/June
2,
1986)
Summary:
Cl-inhibitor
is an
acid
glycoprotein,
isoelectric
point
3.5
3.6. Plasma
of
some patients
with
a
variant
form
of
hereditary
angioedema contains high levels
of
functionless
Cl-inhibitor-albumin
complex
with
an
isoelectric
point
at
4.5-4.6.
Therapy with
Danazol,
which
iricreases
Cl-inhibitor levels, does
not
modify
the
isoelectric focusing pattern
of
such
protein
in
patients with hereditary angioedema.
Immunreaktive
Fällung
von
Ci-Inhibitor-Protein
aus
Plasma
Gesunder
und von
Patienten
mit
hereditärem
Angioödem
nach
isoelektrischer
Fokussierung
Zusammenfassung:
Cl-Inhibitor
ist ein
saures Glykoprotein
mit
einem isoelektrischen Punkt
von
3,5—3,6."
Plasma bestimmter
Patienten
mit
einer Variante
des
hereditären Angioödems enthält hohe Konzentrationen
eines funktionslosen
Cl-Inhibitor-Albumin-Komplexes
mit
einem isoelektrischen Punkt
von
4,5—4,6.
Thera-
pie mit
Danazol,
das die
Cl-Inhibitor-Konzentration
erhöht,
führt
zu
keiner Änderung
des
isoelektrischen
Fokussierungsmüsters
dieses Proteins
bei
Patienten
mit
hereditärem Angioödem.
T _
4-S
strated
that
in
some hereditary angioedema patients
The
inhibitor
of
activated
fir§t
coinponent
(Cl-in-
with
the
variant form, both dysfunctional (94%)
and
hibitor)
is the
major
regidatory
factor
for the
classical
functional proteins
(6%)
are
present,
and
therapy
pathway
of
activation
of
human complement
(l,
2).
with androgen derivatives increases both
the
proteins
It
has
also been
shpwn
to
inhibit
several
enzymes
of
(12),
haemostasis:
activated
Hageman
factor
(Factor
XII),
We
have used isoelectric
focusing
to
study
Cl-in-
Hageman
factor
fragment,
kallikrein,
activated hibitor
in
plasma
from
normal subjects
and in
thromboplastin
antecedent
(Factor
XI) and
plasmin patients with hereditary angioedema before
and
dur-
(3
6).
Plasma from
patients
with"
hereditary angio-
jng
androgen therapy,
to
determine
the
isoelectric
edema
was
found
to
be
deficient
in
Cl-inhibitor
func-
point
of the
protein
and
evaluate
the
effects
of
such
tion
(inherited
äs
an
autosomal
dominant
trait),
lead-
anabolic therapy
on the
characteristics
of
Cl-in-
ing
to the
activation
of
complement.
There
are two
hibitor.
forms
of
this
disease.
In the
majority
of the
patients
(type
l)
there
are
very
low
levels
of
antigen
concentra-
tion
(0.026-^0.052
g/l).
However
15-20%
of the
Materials
and
Methods
patients
have
the
variant
form with normal (type
Ila)
Normal
and
pathological
specimens
or
elevated (type
Ilb)
antigen
concentration
of a
Blood
was
drawn
into
piastic
tubes
containing
0.01
mol/1
dysfunctional
protein
(7—11).
It has
been
demon-
EDTA.
It was
centrifuged
within
l
hour,
at
-h
4°C,
and
sam-
J.
Clin. Chem. Clin. Biochem.
/Vol.
24,1986
/
No.
10
720
Bergamaschini
et
al.:
Isoelectric
focusing
ofCl-Inhibitor
ples
were stored
in
several
aliquots
at
80 °C
until
analysed.
EDTA-plasma
samples
were
taken
from
10
healthy
adult
volun-
teers
and
from
18
hereditary angioedema patients:
10
with
type
I
(Cl-inhibitor
antigen
level:
0.026-0.052
g/l);
5
with
type
Ha
(Cl-inhibitor
antigen level: 0.23
0.29 g/l)
and
3
with
type
Ilb
(Cl-inhibitor antigen level:
0.52—1
g/l).
Cl-inhibitor
functional
activity
was
10—20
U in all 18
patients.
Samples
from
hereditary angioedema patients were obtained
before
and
during
therapy
with
400
mg/day
Danazol
for one
month.
At the
sampling time,
all the
patients
were
asympto-
matic.
Normal
EDTA-plasmas
were
analysed after
2
hours
and
after
storage
for
2-4
month
at
—80
°C.
Chemicals
and
antiserum
Acrylamide,
N'N'-methylenebisacrylamide
and
ampholines
were
obtained
from
SERVA-Feinbiochemica,
Heidelberg (West
Germany).
The
agarose
(low-m
=
0.13
±
0.02)
was a
product
of
-RAD Laboratories, Richmond
Calif.
(USA).
Specific
antisera against Cl-inhibitor
and
albumin
were
obtained
froin
Behringwerke,
Marburg (West Germany).
Isoelectric focusing
(lEF)-lying
on the
immunoelec-
trophoresis
matrix
Thin
layer
IEF was
performed
äs
described
by
Righetti
(13)
with
the
following
modiflcations:
4.4%
polyacrylamide
gel
was
obtained
from
a
stock solution
of 30% T 4% C, and
contained
1.3
mol/1
glycerol, 3.5% Ampholine
3
10 and
2.5%
Ampho-
line
2—4;
gels
were polymerized
on
glass
plates
(U
frame
l mm)
with
2.6
mmol/1
tetramethylethylendiamine
and 1.7
mmol/1
ammonium
persulphate.
The
anolyte
was l .0
mol/1
phosphoric
acid
and the
catholyte
l .0
mol/1
sodium
hydroxide.
The
samples
(18
)
were
applied
to the
cathode
end
side,
in a
well
on the gel
surface.
IEF
were
run on the LKB
Multiphore
apparatus,
with
a
cooling
System
(temperature
on the gel
surface
+
6—8
°C),
for 4
hours,
at 10 W
constant
power
with voltage increasing
from
300 to
1600V.
After
completion
of the
isoelectric
focusing
run,
the pH
gradi-
ent
was
determined
and
Strips
were
cut in the
migfation
area.
They were then
placed
on
0.9%
agarose—4%
polyethylene-
glycol
gel
plates
containing
monospecific
antibody,
and
electro-
phoresis
(3
m
A
for 12
hours)
was
performed.
Tris/barbital
buf-
fer,
ionic strengh 0.1,
pH
8.6,
was
used
for
buffer
vessels
and
gels. Agarose
gel
plates
were
stained with Coomassie Brillant
Blue.
Cl-inhibitor
plasma
levels estimation
The
antigen level
of
Cl-inhibitor
was
determined
by
single
radial
immunodiffusion
on
cellulose
acetate Strips (14);
Cl-
inhibitor
functional activity
was
determined
by its
Inhibition
of
Cl-esterase
activity towards
N-acetyl-L-tyrosine-ethyl
ester
(15).
Cl-inhibitor reference values
Cl-inhibitor antigen level
(mean
± SD)
=
0.26
± 0.8
g/l
Cl-inhibitor
functional activity (mean
± SD) =
0.90
±
0.12
U
of
Cl-inhibitor
(U
of
Cl-inhibitor
=
activity
of l ml of
fresh
normal plasma)
Results
Plasma-EDTA
samples
from
ten
normal
volunteers
were
studied
by
isoelectric
focusing-immunoelectro-
phoresis
after
24
hours
and
after
3
8
months
storage
at
80 °C. In all
samples,
Cl-inhibitor
protein
focüsed
at pH 3.5
3.6,
giving
only
one
peak
in
immunoelectrophoresis
(fig.
1).
3.5
l I
l
8,5
Isoelectric
focusing
6
i
pH
Fig.
1.
Immunoelectrophoretic
pattern
after
isoelectric
focus-
ing
of
Cl-inhibitor
protein
in
EDTA-plasma
from
a
healthy
subject.
Arrow indicates site
of the
sample
application
on the
gel, before
isoelectric
focusing.
Plasma-EDTA
samples
from
ten
üntreäted
patients
with
type
I
hereditary
angioedema
(Cl-inhibitor
anti-
gen
level
less
than
0.052
g/l)
gave
oiily
one
peak
at
pH 3.5
3.6,
äs
observed
for
normal
subjects,
The
same
patients
were
treated
with
Danazol
for one
month
(400
mg/day).
The
antigen
coiicenträtion
of
Cl-inhibitor
increased
to
0.078
0.10
g/l and
func-
tional
activity
to
35
45 U.
There
were
no
differences
between
Cl-inhibitor
in the
plasma-EDTA
before
and
during
anabolic
therapy
(fig.
2).
Of
the 8
patients
with
Type
II
hereditary
angioedema,
five
(Type
Ila)
had
Cl-inhibitor
antigen
levels
in the
ränge
0.21—0.28
g/l and
three
(Type
Ilb)
in the
ränge
0.52—1
g/l. After
one
month
of
therapy
with
Danazol
(400
mg/day),
antigen
levels
had
increased
about
2
times
while
functional
activity
still
ranged
from
35 to
40
U.
Plasma-EDTA
of
Type
Ha
patients
gave
önly
one
peak
at pH
3.5
3,6,
both
before
and
during
Danazol
therapy
(fig.
2).
In
3
üntreäted
patients
with
Type
Ilb
disease,
plasma-
EDTA
exhibited
a
minor
peak
at pH
3.5—3.6,
but
80
-
90%
of
the
reacting
material
focüsed
at pH
4.5
4.6
(fig.
3).
This
major
peak
could
not
be
reproduced
when
crossed
immunoelectrophoresis
was
performed
with
an
intermediate
gel
containing*>anti^albumin
(fig.
4).
Although
the
precisiön
öf
the
quantitative
estirna-
tiön
is
rather
low
with
this
method,
Danazol
therapy
apparently
increased
equally
the
amounts
of
protein
focusing
at
both
isoelectric
points.
J.
Clin.
Chem.
Clin.
Biochem.
/
Vol.
24,1986
/ Np. 10
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Bergamaschini
et
al.:
Isoelectric
focusing
of
Cl-Inhibitor
721
4l
fe
l
o
c
D
ε
ε,
χ
l
l l l l l
3.S
V
1
ι ι
»
ι l ι ι
t
ι ι
4_^
*
ι ι ι ι ι
4.5
5.5 7.5
£Π
2 3.5
Isoeleclric
focusing
ι
ι ι ι t ι ι ι ι ι ι A
4.5
6
8.5
£
PH
Fig.
2.
Effects
of
Danazol
therapy
on
Cl-inhibitor
in
EDTA-plasma
frora
patients
with
hereditary
angioedema:
Type
I = a
Type
Ha = c, d. '
a
=
pretreatment sample, Cl-inhibitor antigen
level
=
0.04
g/l
b
= one
month
of
therapy, Cl-inhibitor antigen
level
=
0.09
g/l
c =
pretreatment sample, Cl-inhibitor antigen
level
=
0.24
g/l
d
=
one
month therapy, Cl-inhibitor antigen
level
=
0.46
g/l
PH
\
i.
t.
i
.r?V-j-lj
i
ι-u
r
ι
ι
ι...
U.JL.
3.5
4.5 6 8.5
Isgelectfic
focusing
Figf
3.
Effects
of
Danazol therapy
on
Cl-inhibitor
in
EDTA-
plasma
from
a
patient with
Type
Ilb
hereditary angio-
edema
with
a
high
Cl^injubitpr
antigen level.
a
=
pretreatment
s
mples, Cl-inhibitor antigen level
=
0.62
g/l
(sample dilution
l:
2)
b
= one
month treatment, Cl-inhibitor antigen level
= 1.2 g/l
(sample dilution
l:
4)
angioedema
had
normal Cl-inhibitor
at pH
3.5
3.6.
In
type
Ha,
with normal antigenic levels
of Cl-
inhibitor,
we
could
not
differentiate
functionless
pro-
tein
from
normal Cl-inhibitor.
In 3
patients (type
Ilb)
with Cl-inhibitor antigen
levels
2—4
times
the
normal
value,
the
majority
of
antigenic
material
was
focused
at pH
4.5—4.6
and
imm
noprecipitated
both
with
anti-Cl-inhibitor
and
anti-albumin,
suggesting
the
presence
in the
plasma
,of
a
functionless complex
Cl-inhibitor-albumin
s
previously
reported
(17).
In all 3
patients,
the Cl-
inhibitor
in
pretreatment plasma
was
indistinguish-
able
from
the
Cl-inhibitor
obtained
during
Danazol
treatment,
which
increased
the
synthesis
of
both
the
dysfunctional
Cl-inhibitor
and
normal Cl-inhibitor
gene
products.
The
fact
that
in
type
Ha
disease both normal
and
functionless
protein
have
the
sanie
Charge
confirms
that
the
chemical
difference
responsible
for the
dys-
funetion
must
be
very
small
(probably
a few
amino
acid
substitutions),
since
it
does
not
cause
any
detect-
able
change
in
size
or
antigenicity.
Discussion
From
this study
we
conclude
that
Cl-inhibitor
is a
very
acidic
protein,
with
an
isoelectric
point
of 3.5
3.6, which
is
intermediate
between
those
previously
reported
by
Haupt
(16)
and
Curd
(12).
The
Charge
on
the
protein
did not
change during
storage
at
-80
°C for 8
moaths.
All
our
patients
with hereditary
Acknowledgement
We
thank
Dr.
P. G.
Righetti
for his
helpful
comments
and
assistance.
This
work
was
supported
by a
grant
of the
"Special
Project
on
Genetical
Engineering
and
Molecular
Basis
of
Hereditary
Diseases"
of
C.N.
R./Rome,
Contract
No.
83.00700.51.
J.
Clin.
Chem.
Clin.
Biochem.
/
Vol.
24,1986
/ No. 10
722
Bergamaschini
et
al.:
Isoelectric
focusing
of
Cl-inhibitor
l
l l l
.1
l l l ' l 1 Ι Ι Ι Ι 1 l A
2
a5
45 6
8
Isoelectric
focusing
PH
Fig.
4.
Disappearance
of the
major
peak focused
at pH
4.5—4.6
in
plasma
from
a
patient with type
Ilb
hereditary
angioedema
in
an
anti-albumin
intermediate
gel.
a: gel
containing anti-albumin antibody
b: gel
containing
anti-Cl-inhibitor
antibody
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ResearchGate has not been able to resolve any citations for this publication.
Article
Full-text available
Highly purified inhibitor of the first component of complement (CāINH) was shown to inhibit the capacity of active Hageman factor fragments to initiate kinin generation, fibrinolysis, and coagulation. The inhibition of prealbumin Hageman factor fragments observed was dependent upon the time of interaction of the fragments with CāINH and not to an effect upon kallikrein or plasmin generated. The inhibition of the coagulant activity of the intermediate sized Hageman factor fragment by CāINH was not due to an effect on PTA or other clotting factors. The inhibition by CāINH of both the prealbumin and intermediate sized Hageman factor fragments occurred in a dose response fashion. The CāINH did not appear to be consumed when the activity of the Hageman factor fragments was blocked, although the fragments themselves could no longer be recovered functionally or as a protein on alkaline disc gel electrophoretic analysis. These results suggest that the CāINH may have an enzymatic effect on the fragments or that an additional site on CāINH is involved in Cā inactivation.
Article
C1(-)-inhibitor (C1(-)-INH) proteins from normal persons and members of eight different kindred with dysfunctional C1(-)-INH proteins associated with hereditary angioneurotic edema (HANE) were compared with respect to their inhibitory activity against purified preparations of C1s-, plasma kallikrein, activated forms of Hageman factor, and plasmin. Each dysfunctional C1(-)-INH protein showed a unique spectrum of inhibitory activity against these enzymes. Although none of the dysfunctional C1(-)-INH proteins significantly impaired amidolysis by plasmin, all but one inhibited activated Hageman factor. One purified dysfunctional C1(-)-INH (Ta) inhibited purified C1s- to a normal degree. Another C1(-)-INH (Za) had almost seven times as much inhibitory activity as normal C1(-)-INH against activated Hageman factor, but had decreased activity against C1s- and no activity against plasmin. Analyses of mixtures of plasmin and C1(-)-INH proteins in SDS gel electrophoresis revealed variability in the patterns of complex formation and cleavage of dysfunctional proteins after exposure to C1s- and plasmin. Some bound to plasmin and were cleaved, even though none significantly impaired the amidolytic activity of plasmin. Two were cleaved by C1s-, whereas neither normal or other dysfunctional C1(-)-INH were cleaved. Dysfunctional C1(-)-INH proteins from patients with HANE are thus heterogeneous in their inhibitory properties and there must be different structural requirements for the inhibition of the various plasma enzymes that can be regulated by normal C1(-)-INH. The data suggest that in addition to common sites of interactions between these proteases and C1(-)-INH, there are also points of contact that are specific for each protease. Genetic mutations leading to structural changes at some of these sites may have differing effects on the interaction between individual proteases and abnormal C1(-)-INH proteins. These alterations may allow these proteins to serve as probes for structural requirements for inhibitory actions of normal C1(-)-INH.
Article
Normal human serum contains 18 +/-5 mg/100 ml of C1 esterase inhibitor (alpha-2 neuraminoglycoprotein) as estimated by immunochemical means. Of 118 patients with hereditary angioneurotic edema, the sera of 80, from 42 kindred, contained a mean concentration of 3.15 mg/100 ml or 17.5% of normal. The mean serum concentration in 35 patients in 7 other kindred was 20 mg/100 ml or 111% of normal, and 3 patients in another kindred contained over 80 mg/100 ml or greater than 400% of normal. The nonfunctional inhibitors in patients' sera of these eight kindred were identical with normal C1 esterase inhibitor by Ouchterlony analysis, but they were different from normal and from each other with respect to their electrophoretic mobility, their capacity to bind C1 esterase, and their ability to inhibit esterolysis of N-acetyl-tyrosine-ethylester.
Article
The fraction of human serum designated as C'1 esterase inhibitor is known to inhibit the action of C'1 esterase, a plasma kallikrein, and PF/Dil, an enzyme in plasma enhancing cutaneous vascular permeability. In the present study, C'1 esterase inhibitor has been found to block the actions of plasmin and the C'1r subcomponent of the first component of complement, and to retard the generation of PF/Dil. No inhibition of blood clotting or of the generation of plasmin was demonstrable.
Article
Purified Cl inactivator has been shown to inhibit activated plasma thromboplastin antecedent (PTA) and activated Hageman factor (HF). It has no action on activated Stuart factor. Evidence is presented that this inhibitor represents only part of the inhibitory activity in plasma against activated PTA.
Article
A minority of patients with hereditary angioedema (HAE) have normal concentrations of a dysfunctional C1 inhibitor protein (C1INH) in their plasmas. We purified C1INH from the plasmas of one such patient before and during treatment with the anabolic steroid stanozolol. Both the pretreatment plasma and plasma obtained during stanozolol treatment contained varying amounts of two extremely similar C1INH proteins that were functionally distinct. The pretreatment plasma contained primarily (94%) dysfunctional C1INH that did not inactivate or complex with either purified C1s, activated Hageman factor, or kallikrein and small amounts (6%) of functionally normal C1INH. Stanozolol treatment increased the plasma concentrations of both of these proteins as well as the proportion (23%) of functional C1INH in the plasma. The purified dysfunctional and functional C1INHs had identical or nearly identical molecular sizes, charges, amino acid compositions, and amino sugar contents, and could not be distinguished physicochemically from each other or from normal C1INH. From these studies of purified C1INH proteins we concluded that HAE associated with dysfunctional C1INH is due to a defect at the structural locus for one C1INH gene and that both the dysfunctional C1INH gene and the normal C1INH gene products are present in the plasma of the affected subject. Treatment with stanozolol comparably increased the synthesis of both C1INH proteins. The disproportionate rise in the level of the normal C1INH protein is consistent with the view that it is more rapidly catabolized as a consequence of its interaction with the proteases it inactivates.
Article
One hundred and four patients affected by hereditary angioedema belonging to 31 families have been studied. Twenty-two percent had the variant form related to the deficiency of the functional activity of serum C1 esterase inhibitor. The remaining 78% of patients had the predominant form, characterized by low antigenic levels and low functional activity of serum C1 esterase inhibitor. Attacks of swelling affected the subcutaneous tissue in 86% of patients; the upper airways in 76% of patients, and the bowel mucose in 75% of patients. Before treatment was available the mortality rate was 56%. One or more attacks a month were present in 46% of cases. The infusion of C1 inhibitor concentrate promptly reversed 14 severe attacks without any side effect. Twenty-nine patients were given long term prophylactic treatment with androgen derivatives with full success. Tranexamic acid reduced the frequency of swelling of 70% of the patients.
Article
Serums of patients with hereditary angioneurotic edema lack inhibitory activity against the esterase derived from the first component of complement. In one group of patients this lack appears to result from failure to synthesize the esterase inhibitor of the first component of complement, whereas in another group of patients an abnormal, nonfunctional protein is synthesized.
Article
An assay is described for measurement of a serum inhibitor of an esterase derived from preparations of the first component of complement (C'1-esterase). Esterolysis of N-acetyl-L-tyrosine ethyl ester by C'1-esterase is inhibited instantaneously and stoichiometrically by fresh human serum. Units of C'1-esterase and of serum inhibitor are defined. The inhibitor in human serum is stable at 45 for at least 4 months, at 37° for at least 24 hr. and at 48° for at least 30 min. It is completely inactivated during 30 min. incubation at 60° and is labile at 0° at pH values below 6. The inhibitor is inactivated at —5 in the presence of methanol concentrations of 15° or greater but is recovered quantitatively in a 40° ammonium sulfate supernatant fraction. These data are being applied to purification of the inhibitor from human serum. Purification procedures and properties of the purified inhibitor will be reported elsewhere.
  • F S Rosen
  • P Charache
  • J Pensky
  • V H Donaldson
Rosen, F. S., Charache, P., Pensky, J. & Donaldson, V. H. (1965) Science 148, 957-958.