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Enzymatic
Activities
of Activated
Hageman
By
Pro-Phe-Arg
chloromethylketone
inactivated
the
man
with
factor
0.75
mm
and
HFf.
. The
-
and
activity
with
factor
reacts
phosphate
with
ty.
Incubation
not
destroy
forms
in native
activated
slowly
with
concomitant
of native
Hageman
with
numbers
Hageman
40
loss
factor,
PPACMK
species.
mM
MM
Hage-
constant
Hageman
These
.
5.26
human
rate
of
Silverberg
at
native
by
mm
its procoagulant
C ONTACT
of
inactivated
0.72
Michael
first-order
detectable
contamination
man
apparent
also
0.82
the
to
activity
activated
were
constants
that
an
Factor
(PPACMK)
amidolytic
The
is due
The
inhibitors
does
and
prekallikrein
cleaved
within
are
Hageman
factor
disulfide
generate
krein],
two-chain
molecules
[HFa
which
are both trypsin-like
active
second
site serines
located
form
of activated
loops
(XlIa)
proteases
on their
Hageman
to
and kalliwith the
light
chains.3
A
factor
is also
formed
that has lost the greater
part of the heavy chain
of HFa.
This
low molecular
weight
form
of active
Hageman
factor
is termed
Hageman
factor
fragment
or HFf(XIIf).4
Studies
performed
have demonstrated
with
purified
the major
that
kallikrein,5
whereas
the major
krein is activated
H F.6 Hageman
vated
in prekallikrein-deficient
reduced
rate,
indicating
that
systems
activator
activator
factor
plasma
the
prekallikrein
evidence
in vitro
of HF is
From
the
Division
State
University
Supported
the National
Submitted
Heart,
of HF
VI1I
in part
Ontario,
Address
Medicine,
Stony
Lung
and
at the 53rd
by
reprint
Health
Stony
requests
to
and
ofthe
N. Y.
2 from
16. 1982.
ofthe
American
1980
and
Sciences
Brook,
Michael
Center,
State
N. Y. I I 794.
Inc.
Silverberg.
Immunology.
University
the
Hageman
a
and
factor
lag
the
reside
proteolysis
is
corn.
effects
and
factor
and
from
amidolytic
Hageman
by limited
of
proteoin
of the
the
native
uncleaved
activate
molecule
prekallikrein
back and cleave
likrein-deficient
cient feedback
krein. However,
onstrated
that
when
exposed
and
at the
Haemostasis,
and that this
to kallikrein,
the HF.
plasma
The
was
autoactivation
tions
autocorrection
explained
of prekalby a less effi-
involving
factor
XIa instead
of kalliin a previous
communication
we dempure
Hageman
factor
autoactivates
to a surface.8
This
autoactivation
is
may
of purified
of the
was able to
could
feed-
account
for
the
zymogen
activity
it has
could
chain3 and
remaining
treatment
Similar
apparent
activity
in Hageman
factor
to ellagic
acid.9
Nevertheless,
of activated
1-lageman
factor
genera-
preparations
the constant
in prepara-
be initiated
by intrin-
of the zymogen.
In support
been reported
that zymogen
slowly
with DFP with
activity.’0
Furthermore,
of bovine
prekallikrein
as
of
is
consequent
loss of
the results
of a
and
Hageman
evidence
cleavage
for a surfaceof prekallikrein
by native
Hageman
factor.
Indeed,
it was proposed
that native
and cleaved
Hageman
factor
were equally
effective
as prekallikrein
activators.
In this
article
we have
source of enzymatic
Division
of
of
initial
at
tion and,
activated
zymogen
York
species
which
accompanied
by cleavage
of the polypeptide
can be initiated
by residual
traces
of activity
in Hageman
factor
preparations
even after
with
diisopropyl
fluorophosphate
(DFP).8
Department
ofNew
component
of factor
XI.
proposed7
that interaction
of Hagea surface
created
a conformational
to expose
an active
site
in the
factor
were
interpreted
dependent,
substrate-induced
USPHS.
November
Thrombosis
Clinical
© 1982 by Grune & Stratton,
/-)06--4971/82/6001-0009$01.00/0
64
Brook.
February
Sessions
Fla..
on
Stony
human
derived
It was originally
man
factor
and
change
sufficient
able to react
its coagulant
recent
study
Center,
1981.
Rheumatology.
Brook.
Institute
Scientific
Clinical
Sciences
1R01HL2371401,
accepted
Beach.
Congress
July
Brook,
Grants
Blood
and
Health
at Stony
1 7, /981;
Miami
International
Allergy,
York
Rheumatology,
Medicine,
by Research
December
Association,
Toronto.
Allergy,
of
ofNew
in part
Presented
Heart
of
Department
activation
that
by
after
factor
is a kinetically
significant
leading
to the activation
tion
of
exposed
generation
of prekallibecomes
actibut at a much
activation
kallikrein
reactions
sic enzymatic
this argument,
Immunology.
of
Hageman
no
Hageman
molecule.
cofactor.2
both
of
provide
forms
occurs
of
inhibits
that
surface-bound
prekallikrein
inhibitor
PPACMK
suggesting
presence
for
is a
activation,
an
activities
activated
of
conditions
by
but
factor.
in uncleaved,
activation
kinetics
lytic
sulphate,
Hageman
is formed
prevented
and prekallikrein
become
activated
in a reaction
which
high molecular
weight
(HMW)
kininogen
During
site
Hage-
PPACMK
dextran
of
initial-rate
ACTIVATION
occurs
when plasma
is
to negatively
charged
surfaces
such as
or dextran
sulphate.’
As a result
of the
the surface,
Hageman
factor
(factor
XII)
exposed
glass,
kaolin,
contact
with
activator
under
activi-
in the
the
of Human
P. Kaplan
factor.
fluoro-
with
of
Allen
Hfa
indicate
factor
even
and
Forms
XII)
active
diisopropyl
factor
(Factor
autoactivation
rate
Uncleaved
Zymogen
of
of its procoagulant
activity,
and
attempted
activity
to determine
in contact
the
activa-
in particular,
to distinguish
between
traces
of
Hageman
factor
and intrinsic
activity
of the
as that source.
These
studies
were facilitated
Blood.
Vol. 60.
No.
1 (July),
1982
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ENZYMATIC
by
ACTIVITIES
the
AND
availability
HAGEMAN
of
inhibitor,
and
Hageman
factor.
FACTOR
a reactive,
65
active
of a polypeptide
site-directed
inhibitor
specific
for
assayed
for coagulant
present,
in units
activity
of
a control
Prekallikrein
AND
METHODS
was
Proteins
before
Prekallikrein6
and Hageman
plasma
by previously
published
-80#{176}C in the
obtained
from
the
prekallikrein
from
IgG
elutes
from
HFa
and
with
kallikrein
HFI
from
Yoshio
Hojima
Lung
and
factor8 were purified
from human
procedures
and stored in plastic
presence
of
benzamidine.
SP-Sephadex
chromatography
fraction
the column
were
before
prepared
corn’2
was
IgG
used
of human
either
from
prekallikrein
Hageman
earlier.8
The
a generous
gift
of the National
Blood
10 mM
the -y-globulin
as described
derived
factor
Institutes
or factor
Xl.
digestion
John
of Health,
Shaw
ries.
H-D-Pro-Phe-Arg-pNA
and Charles
(S2222)
ketone
Kettner
Mannheim.
were
obtained
diisopropyl
from
chemicals
wt
National
I hr at
37#{176}C.The
treatment
prekallikrein
Gel
and
these
was
way
incubated
buffer
contained
did
not
At
system
of
was
the
the end of the
in
fluorophosphate,
BioRad
plasmas,
King Bio-Medical
Inc.
PK)
and
with
incubation
G25
fine
the
(10
9%
factor.
polyacrylamide
gels
Laemmli.’5
RESULTS
of
with
the
we
by the
Drs.
tOO
Laborato-
Greenwich,
was from
obtained
as
Chemical
Co.
Laboratories,
benzamidine
reaction
HF
HFA
HFf
Benzoyl-IIe-Glu-GlyConn.
Boehringer
follows:
St.
Calbiochem-Behring;
electrophoresis,
MM
dialyzed
pH 7.0. IgG was prepared
for Hageman
on
factor
100
not
of Sephadex
in 0.1 M NaPi,
performed
with
10 mM
prevent
on a column
for use as a diluent
was
by Hageman
mg/mi
prekallikrein
conditions.
desalted
electrophoresis
the
at 0.5-I
therefore
this
0.7 cm) equilibrated
in the same
of its activation
aliquots
under
x
in studies
a preservative;
Heart,
gift
National
Kabi
were
Sigma
500,000),
George
DMF,
inhibitor
Pisano
a generous
and
(Chromozym
Other
(mol
was
of Brookhaven
(S2302)
Benzoyl-Pro-Phe-Arg-pNA
sulphate
10%
Since
activated
Hageman
factor
reacts
kallikrein
substrate
H-D-Pro-Phe-Arg-pNA,8”6
would
expect
it to be susceptible
to inactivation
chloromethyl
Elliot
as
PPACMK
using
Chemicals
Arg-pNA
containing
by treating
for
this
used
cm
The
factor
Drs.
isolate
plasma.
after
Hageman
of
to
was
Institute.
Pro-Phe-Arg
sample,
used
prepared
PPACMK
at
as described
above. The activity
was expressed as a percentage of the
parallel.
MATERIALS
tubes
activity
per milliliter,
dextran
Louis
Mo.;
reagents
Hicksville,
for gel
N.Y.;
Inc., Overland
deficient
Park, Kans.
Methods
Plastic
assays
0.05
pipettes
were
M
Tris-CI
chromogenic
used
and
tubes
conducted
pH
7.8,
substrate.
plastic
used
changes
procedures.
(I
routine
assays
of enzymatic
were
Sarstedt
recorded
equipped
Enzyme
containing
NaCI
(Walter
ing spectrophotometer
in all
cuvettes
0.1 17 M
For
cuvettes
Absorbance
were
in semimicro
0.15)
Inc.,
Ml of
400
and
500
we
Princeton,
on a Gilford
Model
with a cell changer
MM
activity
N.J.).
250
record-
thermostatted
>
to
using
assays
were
activated
factor-Xll-deficient
pooled normal
Protein
Lowry
plasma
human
and
were
calibrated
in 0.001
addition
N
to protein
man
factor
factor
or
phosphate,
by
its
HCI
human
replaced
were
any given
after
were
forms
with
10 Ml of 56.2
of
method
of
for assays
MM
was expressed
a stock
solution
I 0 Ml added
dialyzed
into
0.1
intervals,
diluted
M
of
pH
were
initial
activity
M DFP
5 Ml
and
aliquots
were
Fig. 1 .
with
5.26
of Hageman
saline
that
TIME
activity
of inhibitor.
factor
246
at
in dimethylformamide
Aliquots
in phosphate-buffered
into
remaining
of the original
of inactivation
7.0.
sodium
placed
at 37#{176}C.
Two
the activity
due to the addition
of0.4
Hageman
Five-microliter
times;
to 90 Ml of Hageman
NaPi,
1000-fold
the
PPACMK.
of the kinetics
and
briefly
for
of Hage-
0. 1 M
aliquots
as a percentage
for the dilution
was made
into
dis-
made
as follows.
dialyzed
estimation
was
dilutions
determined
were
at various
the
of inactivation
and incubated
for
For the determination
by DFP,
dilutions
(PPACMK)
appropriate
microliter
tubes
removed
time
with
using
The kinetics
PPACMK
activated
were
correction
factor
and
pH 7.0. One-hundred
removed
times’3
lgG.
ketone
at I mM
solutions.
500 MI microcentrifuge
aliquots
calibrated
determined
with
H-D-Pro-Phe-Arg-chloromethyl
solved
thromboplastin
I-
plasma.
concentrations
et al..’4
partial
10
0
4
37#{176}C.
Clotting
>-
I-
had been
removed
(PBS),
at
and
(MINUTES)
Reaction
of Hageman
factor
and its activated
forms
PPACMK.
Aliquots
were
assayed
for amidolytic
activity
a9ainst
S2302
at the indicated
times as described
under
Methods.
The results
from
several
different
experiments
are
combined
in each panel.
The rate constants
obtained
from the
slopes
are HF: 0.75 min’.
HFa: 0.85 min’,
HFf: 0.72 min’.
MM
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66
SILVERBERG
substrate
analogue
Pro-Phe-Arg-chloromethyl
inhibitor
treated
of kallikrein.’7
with 5.26
M
37#{176}C.
As shown
in Fig.
rapidly
I , both
forms
lost amidolytic
Hageman
factor
amidolytic
ketone,
which is a potent
(40 g/ml)
were
HFa or HFf
PPACMK
at
of activated
activity
in a
pseudo
first-order
reaction.
The apparent
first-order
rate constants
were 0.85 and 0.72 min
for HFa and
HFf, respectively.
As described
in a previous
communication,8
purified
native
Hageman
factor
always
possesses some
of inactivation
using
amidolytic
of this
a number
activity.
activity
of different
specific
activity
of 2.4
mole
which
),
is not significantly
for
obtained
the
two
different
forms
factor,
both of which
had
800 mole substrate/mm/mole
led to conclude
demonstrable
most
likely
The
due
of
molar
of Hageman
from
activated
of reaction
unchanged
until no more
remains.
We can obtain
any potential
contribution
rate
the
Figure
2
preparation
of
molar
specific
of the initial
activity
could be
site that might
belong
to the
zymogen.
As the initial
activity
the activity
of pure
HFa
or
indicate
that
Hageman
the
factor
less than
that
amidolytic
in the
activity
fluid
of activated
represented
HFf,
these
phase
H F in the
4.6% of
numbers
of
uncleaved
is about
4200-fold
standard
spectro-
0
values
Hageman
activities
of
Thus,
we are
that
the enzymatic
activity
in preparations
of Hageman
with
preparation.
substrate/min/
specific
enzyme.
to contamination
initial
activity
of 36.7 mole substrate/mm/mole.
The initial
reaction
rate falls off to almost
zero, and it is possible
that the apparent
slow rate of inhibition
in the second
mole. In spite of these variations
in specific
activity,
all
of the preparations
reacted
with PPACMK
with the
same apparent
first-order
rate constant
of0.75’
(Fig.
1
of the
obtained
with a single
that
had an initial
estimate
that only 0.5%
due to a slow reacting
factor
with molar specific
activities
of 60-1 20 moles of
substrate
converted
per minute
per mole of protein.
We
also included
a preparation
that had been pretreated
with 10 mM DFP and then dialyzed;
this material
had
a molar
results
factor
KAPLAN
part of the curve represents
the reaction
of a different
kind of active
site with the inhibitor.
By extrapolation
of this second
linear
region
back to the ordinate,
we can
We measured
the rate
by 5.26 .zm PPACMK
preparations
activity
shows the
Hageman
AND
readily
factor
is
HF.
activated
>-
I-
>
I0
with
PPACMK
proceeds
than I % of the initial
activity
from this an upper
limit for
made by the zymogen
to the
4
-J
0
Iz
0
0
LL
100.0
0
I-
50.0
50
z
Ui
0
Ui
ci
_
lo.o-
>-
50-
0
25
>
IU
0
I0-
0.5’-
TIME
A
Fig. 3.
Coagulant
an aliquot
PTT was
0 I
50
2
TIME
Fig.
mg/mi
2.
()
Reaction
with 5 M
of Hageman
PPACMK.
60
70
(MINUTES)
factor
at 1 .4 mg/mI
2
(#{149})
or 0.3
Reaction
3
4
5
6
(HOURS)
of Hageman
factor
zymogen
with
inhibitors.
was measured
at the indicated
times by diluting
1000-fold
and using 50 izl of the dilution
for a PTT. The
calibrated
using dilutions
of normal
plasma
and the
activity
of the aliquots
in U/mI was expressed
as a percentage
of
the activity
of a control
sample
incubated
without
inhibitors.
The
control
had an average
specific
activity
in this assay of 90 U/mg.
Hageman
factor
was 608 mg/mI
and incubated
at 37’C with 40 mM
DFP (0), 200 LMPPACMK
(A).
or 100 MMPPACMK
plus 10 g/ml
dextran
sulphate
(s).
activity
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
ENZYMATIC
ACTIVITIES
AND HAGEMAN
FACTOR
photometric
limit and
assay.
depends
This
figure
on amidolytic
represents
an
measurements
are
distinguishable
barely
med the results
preparations
of
from
of similar
Hageman
zero.
The apparent
initial
phase
magnitude.
activities
specific
activities
of rapid
inactivation
We concluded
that
had
tainty
of the
Hageman
become
less
assay
baseline.
factor
that
had
When
experiments
factor,
HFa
found that all the curves
of activity
off, including
those obtained
with
than
67
upper
that
we exam-
with various
and HFf,
we
versus
time
the activated
leveled
forms.
obtained
after
the
varied
by an order of
the true
enzymatic
the
inherent
been treated
uncer-
with
5.26
M
PPACMK
until its amidolytic
activity
was essentially
too low to measure
(Fig. 2) was still able to autoactivate
when
diluted
into
a glass
cuvette
containing
chromogenic
terized
by
substrate.
The autoactivation
long
lag phase
followed
a
by
is characa rapid
acceleration
in the
rate
of amidolysis.
Simulated
autoactivation
curves
using
a simple
kinetic
model8
showed
a similar
pattern
when the initial concentration
. 11
_____
of active sites was equivalent
to less than 0.01 % of the
total number
of protein
molecules.
This value is of the
same
order
react
of
magnitude
as the
activity
of the zymogen
it has been reported
that
enzymatic
Since
with
compared
and with
DFP
with
upper
limit
loss of procoagulant
activity,’0
was
in the uncleaved
Hageman
factor
PPACMK
the
to
we
the reaction
of native
Hageman
with DFP
PPACMK
(Fig. 3). Hageman
factor
at 608
incubated
with 40 mM DFP.
At
aliquots
were removed
and assayed
for their
correct
the PTT of Hageman-factor-deficient
Over several
hours
there
was a progressive
procoagulant
activity
in the presence
of DFP,
ing that the active
site serine is reactive
with
g/ml
for
obtained
above.
native
HF is able
and
zymogen
was also
assayed
form of
incubated
Hageman
with
for procoagulant
factor.
200 iM
activity
in the
same way. There
the procoagulant
was an initial decrease,
but after 5 hr,
activity
remained
at least 70% of that
of
Essentially
the
control.
obtained
from
the reaction
PPACMK
in the presence
phate,
which is an efficient
the
same
results
where
inhibitor,
half-time
it can
be seen
with the latter,
and shown
in Fig.
with those
illustrated
in Fig. 1
that,
activated
Hageman
of approximately
obtained
with
that Hageman
PPACMK
factor
at
/io the
concentration
is inactivated
I mm. Thus,
the
A
“-a..
C
B
Fig.
4.
Autoactivation
of Hageman
factor.
Reduced
SDS polyacrylamide
gels of (A) Hageman
factor;
(B) Hageman
factor
incubated for 7 hr with 10 g/ml
dextran
sulphate;
(C) as B but with
the addition
of 100 MM PPACMK.
were
of Hageman
factor
and
of 10 jtg/ml
dextran
sulpromotor
of contact
activa-
tion)8 There
is thus a clear difference
in the reaction
of
the native
enzyme
with DFP and with an inhibitor
that
is a close analogue
of a substrate
of Hageman
factor.
The results
obtained
3, may be compared
.-,.
intervals,
ability
to
plasma.
decline
of
suggestDFP, even
of
with
a
results
do not support
the hypothesis
zymogen
has appreciable
enzy-
matic
activity
an inhibitor.
expressed
Figure
4 shows
man factor
after
the
presence
toward
SDS-polyacrylamide
incubation
with
or absence
either
a substrate
gels
dextran
of PPACMK
for
or
of Hagesulphate
in
7 hr.
material
(Fig. 4A) shows
a small
forms;
these
arise by autoactivation
factor
is dialyzed
in preparation
experiment.
completion
This
autoactivation
proceeds
almost
to
when
the preparation
is incubated
with
sulphate
(Fig. 4B). However,
when PPACMK
dextran
is present
additional
in the incubation,
cleavage
is seen
only a small
after
7 hr (Fig.
amount
when
for
The
starting
cleaved
Hageman
of
the
the
amount
of
4C). Since
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
SILVERBERG
68
any cleaved
vated
by
consistent
species
produced
the PPACMK,
would
be rapidly
mactigel shown
in Fig. 4C is
of 70% of the initial
the
retention
with
an
equal
volume
of
12-16
AND
ng/ml
HF
KAPLAN
in
3.8
procoagulant
Comparison
occurs
only
activity
measured
at this time (Fig.
3).
of gels B and C shows that autoactivation
slowly,
when
the concentration
of active
The Hageman
factor
stock
solution
had
with
lOO M
PPACMK
before
dilution.
Aliquots
of the mixture
were periodically
assayed
for
kallikrein
activity
using
the chromogenic
assay.
The
results
are shown
in Fig. 5, plotted
according
to a
sites is kept
is consistent
below
with
first-order
differing
which
remain.
with
the level of detectability.
This result
the hypothesis
of a steady
state
in
very
low concentrations
Alternatively,
zymogen
enzymatic
activity,
is no more
site(s)
against
We
the
but
readily
the
gels
of
may
4 show
against
the
of the 1-lageman
factor
polypeptide
oligopeptide
substrate
or inhibitor.
next examined
the possibility
that
that
this
the same
cleavage
chain
than
activation
dextran
prekallikrein
could
induce
enzymatic
activity
in surface-bound
Hageman
factor
so that
prekallikrein
might
be activated
to kallikrein
by Hageman
factor
zymogen.
In
these experiments,
vate prekallikrein
of the
Hageman
factor
was used to actiat very low ratios so that the kinetics
reaction
conditions.
was treated
removed
could
The
with
be
by gel-filtration
experiments,
100-200
.tg/ml
rate
in Materials
prepared
and was
to minimize
losses
of the highly
adsorbtion
initial
diluted
prekallikrein
protein.
was
and
used
due
the effect
concentrations.
of
after
very
from
molar
concentration
of prekallikrein
sulphate
occurred
(Fig.
5). In contrast,
in the presence
rapidly
from
the
of 10 sg/ml
the beginning
a very short lag phase.
Although
this lag was not
pronounced,
it was seen in the results
obtained
three
separate
experiments.
We repeated
the
experiment
using an inhibitor
derived
from corn, which
for Hageman
factor
and does not inhibit
kallikrein.’2
This inhibitor
forms
a complex
with HFf
that is stable
on alkaline
disc gel electrophoresis
but is
is specific
these
experiments
which
was then
as described
Methods.
lgG was similarly
dilute
the Hageman
factor
surface
under
observed
prekallikrein
for
100 1tM PPACMK,
rate
equation’9
to eliminate
substrate
(i.e., prekallikrein)
In the fluid phase, prekallikrein
became
activated
in an
accelerating
reaction,
which after 3 hr, approached
the
rate of prekallikrein
activation
by preformed
1-IFf of
active
enzyme
have very weak
in Fig.
expressed
mg/ml
IgG.
been treated
to
to
In our
mixed
not stable
inhibitor
Hageman
in the presence
did not decrease
factor
zymogen
those
performed
that a complex
of SDS.
We found
that
the procoagulant
activity
in an experiment
DFP or
was not formed
with
man
factor.
In the presence
inhibitor,
the activation
of
this
of
similar
to
PPACMK,
suggesting
with uncleaved
Hageof 0.05
prekallikrein
mg/ml
was
corn
com-
go
0.
..-.
0
aC
TIME
(minutes)
Fig. 5.
Activation
of prekallikrein
by Hageman
factor
under initial rate conditions.
At indicated
times.
5 Ml aliquots
of the reaction
mixtures
were assayed for amidolytic
activity
against chromozym-PK.
The activity
obtained
at time t. in i A/mm.
is used as the value of K,.
and the value of PK is calculated
as the activity
that would
be expressed
upon total conversion
of prekallikrein
to kallikrein.
This figure was
checked
by the addition
to some samples
of 5 ILl HFf (76 Mg/mI) and assay of the kallikrein
formed
after 1 -2 hr incubation.
The composition
of the reaction
mixtures
was as follows:
(#{149})
36.3 g/ml
prekallikrein.
6.1 ng/ml
Hageman
factor.
10 g/ml
dextran
sulphate;
(A)
58.5
pg/mI
PK. 6.1 g/ml
HF. 10 pg/mI
DS. 0.05 mg/mI
corn inhibitor;
(U) 107.5 g/ml
PK. 8 ng/ml
HF; (0) 58.5 pg/mI
PK. 6.1 ng/ml
HF. 10
g/ml
DS. Note the different
time scales
in the left and right panels.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
ENZYMATIC
ACTIVITIES
AND
HAGEMAN
pletely
prevented
(Fig.
5A),
Hageman
factor
was required
tion. Thus,
it would
appear
dextran
verted
FACTOR
indicating
that activated
for prekallikrein
activathat
in the presence
of
sulphate,
Hageman
factor
to H Fa by traces
of kallikrein
prekallikrein.
Such
Hageman
factor
total prekallikrein.
traces
if they
69
was
rapidly
present
would
be equimolar
comprised
only
conin the
with
0.01%
the
of the
procoagulant
centrations
by the
tant,
activity
after
of the inhibitor.
presence
of an activating
that
experiments
it
described
results
of earlier
prekallikrein
and
in
this
paper
demonstrate
factor
become
21
Hageman
and
the
that purified
activated
in
the
presence
of certain
negatively
charged
substances.
described
in the introduction,
Hageman
factor
is
the probable
location
of the first enzymatically
active
sites. We have considered
four hypotheses
that might
As
account
for the initiation
ofcontact
( I ) Hageman
factor
zymogen
has
activity.20
matic
(2)
activity
proteolysis.9
the
Hageman
when
(3)
factor
bound
An active
activation
intrinsic
zymogen
in vitro:
enzymatic
acquires
to a surface
conformation
enzyprior
to any
is induced
by
substrate
zymogen.”
vation
in surface-bound
Hageman
factor
(4) Autoactivation
and the reciprocal
actiby kallikrein
are initiated
by traces
of active
the Hageman
although
substrate
if at all,
is barely,
considered
substrate
that
which
kallikrein
enzyme,8
to Hageman
factor
to traces
of activated
belonged
it seems
likely
that
formed
in the experiments.
The cleavage
factor by HFa was demonstrated
earlier8
treated
in detail
in another
publication.2’
In our studies we have made extensive
tors to eliminate,
or at least
minimize,
activated
forms
of Hageman
factor.
have used Pro-Phe-Arg-chloromethyl
an active-site-directed
irreversible
the
zymogen
derivatives
activities
of Hageman
and has been
use of inhibithe effects
of
In particular,
ketone,
inhibitor
we
which
is
of Hage-
man factor
and has a close structural
similarity
to a
synthetic
Hageman
factor substrate.
Our results
with
PPACMK
and chromogenic
substrates
indicate
that
Hageman
factor
zymogen
can have only very weak
reactivity
with these oligopeptides
and we estimated
an
upper
limit
for the hydrolysis
of 500 sM substrate
as
4200-fold
less
than
that
of HFa
or HFf.
Attempts
estimate
a Km for this reaction
were not successful
our best estimate
would
be in the mM range.
The reactivity
also
very
low
of the zymogen
as demonstrated
with
PPACMK
by the retention
to
and
is
of
chain
is
Hageman
factor,
the zymogen.
We
possibility
that
the
other
be cleaved
contact
a positive
by
Hageman
activation
feedback
enzymatically
factor
of normal
reaction
in
activates
Hageman
generation
is normal
in factor(suggesting
that factor
Xl is not
factor
activation).
We, there-
concentrated
on the interactions
prekallikrein.
The interaction
of prekallikrein
with
of
Hageman
factor and
possesses
the
is
therefore
property
subject
to
of
rapid
rate
of
prekallikrein
fac-
feedback
acceleration.
of Hageman
to prekallikrein.
measure
kallikrein
these experiments,
Hageman
positive
becomes
difficult
to investigate
the
We attempted
to avoid
this difficulty
the
actually
that were
polypeptide
fore,
concentration
fold relative
ascribed
might
factor
and bradykinin
Xl-deficient
plasma
required
for Hageman
remove
all traces
factor autoactivates
activated
the
zymogen.22
However,
plasma
depends
on
tor
HFf.
Since
Hageman
catalyzed
by traces of
factor
for activated
cleaved
by
natural
substrates
of Hageman
factor,
namely
factor
XI and prekallikrein,
could
be activated
efficiently
by
the zymogen.
Since
it is clear
that contact
activation
can proceed
in prekallikrein-deficient
plasma,2
one
would have to postulate
the factor
XI would
be the
cleaved
Hageman
factor
that are always
present.8
The first and second
hypotheses
arose
from experiments
in which
the preparations
of Hageman
factor
used had not been treated
with effective
inhibitors
to
of HFa
and
in a reaction
It is impor-
to consider
the possibility
that a macromolecular
substrate might be able to induce
significant
activity
in a manner
not possible
for low molecular
weight
compounds.
However,
the data of Fig. 4 show
therefore
The
surface.
however,
a satisfactory
DISCUSSION
treatment
with high conThis result
is unchanged
and
It
thus
initiating
events.
by reducing
the
factor
several
This sufficiently
activation
to
thousand
reduced
allow
us
to
activity
as a function
of time.
In
both proteins
were pretreated
with
PPACMK
to reduce
the concentrations
of active
sites
to below
the level of detectability
with synthetic
substrates.
Nevertheless,
prekallikrein
became
activated
indicating
that enzymatic
activity
must still be present
in some form.
Significantly,
the initial
rate of activation of prekallikrein
by Hageman
factor
in the fluid
phase
approach
indicating
fraction
was
negligible
that
and
of activated
that
the zymogen
of the prekallikrein
took
a period
Hageman
of
factor
has at
activating
hours
(Fig.
to
5B),
most
a small
activity
of the
activated
enzyme.
In the presence
of a surface,
the
activation
of prekallikrein
proceeded
rapidly,
but the
effect of the surface
was to shorten
the lag phase, not
eliminate
it. This is consistent
with the hypothesis
that
the surface
induces
a conformation
more
susceptible
to proteolytic
in Hageman
cleavage.5
All
factor
of our
experiments
showed
evidence
of lag phases
at early
times,
but even at these dilutions
of Hageman
factor,
the acceleration
of the activation
of prekallikrein
was
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
SILVERBERG
70
too great
time.
It
to permit
estimation
of the true
is this rate
that
would
reflect
activity
enzyme
that
of the zymogen
or the
present.
In either
case,
activation
of Hageman
can
express
maximal
prekallikrein.
We
attempted
to
enzyme
and
of
activation
factor
must
in
occur
the
it
of
between
activity
activated
by repeating
the
experi-
of an inhibitor
of Hageman
factor
In the presence
of this inhibitor,
prekallikrein
occurred
for
over
an
such activity.
If Hageman
to consider
factor
how
are
nevertheless
able
and contact
man factor
steady
zymogen
is inactive,
PPACMK-treated
to
it is imporpreparations
both
initiate
autoactivation
activation.
It seems likely that
is able to autoactivate
there
since Hagewill arise
a
state where
the rate of generation
of new active
will equal
their rate of removal
through
reaction
PPACMK.
This amount
of activated
Hageman
sites
with
x 10
M, two
for prekallikrein
tor
is effective
However,
reversible
hour, even in the presence
of dextran
sulphate.
The
most probable
explanation
for this result
is that activated
Hageman
factor
is the
species
required
for
prekallikrein
activation
and that the zymogen
has no
tant
Km
KAPLAN
subenough
the value
of
than the values
Corn
factor
inhibitor,
on
with a K, of
orders
of magnitude
lower than the
(5 x 10
M). Thus,
corn inhibi-
in preventing
it should
inhibitor,
prekallikrein
be noted
that
and therefore,
that
even in its
activated
Hageman
able prekallikrein
These
synthetic
rapidly
because
higher
found
with synthetic
substrates.’6
the other
hand,
inhibits
Hageman
2.5
distinguish
is undetectable
using
to activate
prekallikrein
contact
activation
for this reaction
is much
kca,/Km
before
activation
which
is able
initiate
to
concentration
of active
we are led to conclude
activity
zymogen
ment in the presence
derived
from corn.’2
no
factor,
strates,
rate at zero
the possible
AND
presence
would
activation.
considerations
activation.
corn
we
inhibitor
is a
would
expect
the equilibrium
eventually
produce
also
apply
to the
level
of
detect-
macromolec-
ular inhibitors
of plasma
which,
with the exception
of
a2-macroglobulin,
are reversible
and permit
low equilibrium
concentrations
of active
enzyme.23
Our results
suggest
that the residue
of active
enzymes
that escape
inhibition
in vivo can have
a significant
physiologic
role. Although
it is not possible
to rigorously
exclude
enzymatic
activity
in the zymogen,
our conclusions
are
that unactivated
Hageman
factor
has, at best, several
thousand
fold less enzymatic
activity
than proteolytically cleaved
forms of the protein.
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From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1982 60: 64-70
Enzymatic activities of activated and zymogen forms of human Hageman
factor (factor XII)
M Silverberg and AP Kaplan
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