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Fibrin
By
Using
Eric
Formation:
P. Brass,
laser
Walter
fluctuation
technique
that
Effect
B. Forman,
Robert
spectroscopy,
measures
a
particle
of fibrin
can
be
studied.
effect
With
this
technique
on
the
three
of clot formation,
fibrinogen,
(2)
formation,
monomer
polymerization,
C
Only
(1)
a small
ALCIUM
monomer
change
flbrin
ions.
that
formation
edly
accelerating
monomer
results
from
interac-
However,
there was a
in the rate
of fibrin
in
These
decreases
fibrin
Lindan
monomer
polymerization
calcium
calcium
investi-
in the
are
IONS
of
(3)
were
period
f’ibrinogen-fibrin
of
monomer
and
induction
Olgierd
tions was observed.
marked
increase
of
proteolysis
Ions
and
Edwards,
the
the
distinguishable
fibrinogen-fibrin
complex
gated.
the
V.
of
size
change
in solution,
the kinetics
clot formation
from
fibrinogen
calcium
phases
of Calcium
the
from
the
data
presence
show
time
fibrinogen
the
that
required
for
by
phase
mark-
of
fibrin
polymerization.
length
known
to accelerate
the
formation
of
a fibrin
clot
from fibrinogen
in the presence
of thrombin.”2
The conversion
of fibrinogen to the fibrin
clot is a multistep
process,
and many studies
have attempted
to
examine
the specific
step influenced
by calcium.
These studies
traditionally
have
divided
fibrin
clot formation
into two steps, (I) the conversion
of fibrinogen
to
fibrin
monomer
by the action
of the proteolytic
enzyme
thrombin
and (2) the
aggregation
of the fibrin
monomer
to form
the visible
fibrin
clot. Examination
of fibrin
conclude
clot
that
formation
from
this
the proteolytic
action
perspective
of thrombin
has led several
on fibrinogen
investigators
is not altered
to
by
calcium
ions and that the accelerated
appearance
of the fibrin
clot results
from
an increased
rate of fibrin
monomer
polymerization.35
Using
laser fluctuation
spectroscopy,
we found
a third
step in the fibrinogenthrombin
system.
During
the early
period
of the reaction,
the fibrin
monomer
that is generated
forms
begins
to accumulate,
a reversible
complex
with fibrinogen.6
the next stage in fibrin
clot formation,
proceeds
rapidly.
This
have dramatic
effects
interaction
between
on the kinetics
of
shown
studies
by the fibrinogen
did not examine
interaction.
To further
From
contain
might
define
the Chemical
A dmi,,istration
the effect
Engineering
Hospital
University,
(‘leveland.
and
of the
systems
Thus
the
the results
of calcium
Department.
Department
in the
Hematology/
of
Medici,,e
and
monomer
system,
of the system.6
fibrinogen-fibrin
used
to
study
Oncologi’
monomer
monomer
comthese results.3’4
fibrinogen-thrombin
system,
Sectiom,,
Pharmacology.
Previous
monomer
fibrin
fibrinogen-fibrin
obtained
from
can
as
Cleveland
Case
Wester,,
we
Vetera,,s
Reserve
Ohio.
Submitted
January
5, 1978;
Supported
by
MDD
Aduzinistratiom,
many
fibrinogen.
be influencing
fibrin
monomer
polymerization,
fibrinogen
and fibrin
the fibrinogen-thrombin
concentration
dependence
the effect of calcium
on the
Furthermore,
polymerization
plexing
reaction
As
N/A
Hospital
accepted
(division
(funded
May
of
bs
26.
N/H)
Project
1978.
Grant
N/A
541-2890-01),
MDD-72-2223,
amid
the
the
Northeast
(‘leveland
Ohio
Veterans
Heart
As-
sociation.
Address
10701
©
654
for
East
1978
reprint
Blvd.,
by
Grumie
requests:
Cleveland,
& Stratton,
Walter
Ohio
Inc.
B.
Formnan,
M.D..
(‘hie/.
Heniatolog
v/Oncology
Sectiomi.
44106.
0006 -4971/78/5204
-0002$01.00/0
Blood,
Vol. 52, No. 4 (October),
1978
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FIBRIN
FORMATION:
monitored
presence
the
OF
EFFECT
CALCIUM
IONS
655
the increase
in particle
size in the system
of calcium
at various
concentrations.
Our
hypothesis
that
the
major
action
of
as a function
results
were
calcium
occurs
of time
consistent
on
fibrin
in the
with
monomer
polymerization.
MATERIALS
Human
and
tibrinogen
Blomback7
jected
to
from
gel
this
with
was
Fibrinogen
either
et al.9
solutions
Los
in the
All other
chemicals
centrifuged
out
Particle
time
(pH
of the
size
zero
7.4.
centration
by
in all
at
free
was
present.
by
by
of
contaminating
in
method
This
sodium
soluble
Fibrinogen
the
-80’C.
followed
calcium
not
plasma
stored
of
protein.
dodecyl
urea,
poly-
Fibrin
indicating
concentrations
sub-
sulfate
proteins.
5 M
Blombiick
when
formed
that
were
fibrin
determined
by
of
crude
bovine
solutions
were
buflers
used
to
remove
a column
thrombin
stored
in
was
were
as
mixed
Detroit,
Mich.)
by
the
-25’C.
experiments
calcium
or
(Parke-Davis.
at
treated
suggested
with
by
samples,
with
Boyer
allowed
Chelex
100
et
al.4
Chelex
to
equilibrate,
(Calwas
and
solution.
were
of reagent
monitored
addition
ionic
from
Stock
used
was
was
was
of
was
Calif.)
form
outdated
Meniie.8
and
Angeles,
used
and
(SDS-PAGE),
and
METHODS
from
mercaptoethanol
presence
purified
of Prentice
biochem,
and
activity
of Ratnofi
Thrombin
method
urea
in the
(XIII)
AND
prepared
described6
electrophoresis
factor
method
14) was
previously
fibrinogen
stabilizing
the
as
reduction
acrylamide
at
(fraction
by
of
experiments
fluctuation
thrombin
0.15,
strength
grade.
laser
was
pIus
and
any
spectroscopy.6
were
additional
I . II mg/mI:
salts
that
Experiments
at
conducted
23’C
The
specified).
of thrombin
was
in
0.075
were
initiated
barbital-saline
final
bufrer
tibrinogen
con-
NI H units/mI.
RESULTS
Figure
1 shows the relationship
the fibrinogen-thrombin
system
Both
experiments
showed
between
particle
in the absence
and
the expected
rapid
increase
in particle
size.
veniently
characterized
by two
induction
The time
variables,
size and
presence
period
time obtained
of 2.0 mM
followed
from
CaCl2.
by a period
course
of the reactions
can
(1) the length
of the induction
of
be conperiod
U)
9.0
a,
a,
S
8.0
E
0
S
7.0
C-)
E
6.0
a,
5.0
1
(I)
4.0
-
3.0
C.)
t
2.0
0
0..
Fig. 1.
Particle size as function of time in
absence
and presence
of calcium.
Particle
size
in fibrinogen-thrombin
system
was
measured
in the absence
(..)
or presence
(.) of 2.0 mM calcium
chloride.
o
100
300
Time
500
(seconds)
700
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BRASS
656
El
AL.
C.)
a
160
‘
80’
60
.
.
0
40
‘
20
.
g’
Fig.
i
0
.
I
1.0
i
2.0
Calcium
3.0
Concentration
5.0
(mM)
that seen in controls.
When physiologic
small additional
effect on the induction
induction
period
as
a
of
calcium.
Length
of
induction
size
as
experi-
during
the
period
ions decreased
manner.
At
of the induction
calcium
period
concentrations
was noted.
strength,
since the addition
with calcium
chloride
had
of
rapid
particle
the length
physiologic
period
was
This
of the
plasma
80#{176}c
of
were exceeded,
a
effect
was not
of sodium
chloride
to an ionic
no effect
on the length
of the
period.
In contrast
to the minor
particle
size increase
(Fig. 3). The calcium
effect
on the length
was dramatically
concentration
accelerated
dependence
of the induction
was not a result
The separate
of polymerization
of increased
ionic
producing
showed
that
the rate
of
a near-maxithis increased
strength.
measurements
of the length
of the induction
can be combined
to predict
the effect
on
form a visible
clot. Such a prediction
induction
period
the time it would
period,
(400%)
by addition
of calcium
of this effect was similar
to that
seen in Fig. 2, with physiologic
calcium
concentrations
mum acceleration.
Again,
addition
of sodium
chloride
rate
induction
trations
Figure
2 shows
that addition
of calcium
induction
period
in a concentration-dependent
calcium
concentrations
(2.0 mM) the length
in ionic
to that
of
ment.
and (2) the rate of particle
size increase
growth
(fibrin
monomer
polymerization).
due to changes
strength
equal
Length
of calcium
concentration.
Particle
fibrinogen-thrombin
system
was
in the presence
of varying
concen-
period was determined
from particle
a function
of time
plots
from
each
I
4.0
2.
function
size
in
measured
can be made by adding
take to reach a given
period
and
the overall
the rate
time to
to the length
of the
size at the observed
4,
U)
0
4)
(,)
‘V
0.06
-o
0.05
.
U)
u,
Fig. 3.
Rate of particle
size increase
as a function
of calcium
concentration.
Rate of particle
size increase
following
induction
period
was
determined
by
comparing
increase
in particle
size and
time from a series of fibrinogen-thrombin reactions.
0.04
‘Z0.03
.
o
#{176}
0
0.02
01
E
,
Calcium
Concentration
(mM)
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FIBRIN
FORMATION:
EFFECT
OF
CALCIUM
IONS
1. Prediction of Visual
Table
Acceleration
Clotting
(mM)
Time
The visual
size was
chosen
then
can
Time
Visual
(Percent
71
2.0
53
55
2.5
49
53
3.5
45
52
5.0
49
46
time
to
is predicted
reach
because
by adding
a 4-nm
of
the
particle
onset
of
to the visual
rate.
Thus
acceleration
to
size
gel
this
the control
at the
length
observed
formation
clotting
rate
observed
time
using
the
predicted
overall
clotting
time that is spent
during
which
no polymerization
predictions
are made for calcium’s
It
Clotting
100
polymerization
I
Actual
of Control)
70
be compared
observed
on
0.5
take
time
Based
100
clotting
it would
Time
Reactions
Visual
(Percent
0
time
Clotting
of Polymerization
Predicted
Ca2’
657
of polymerization
accounted
induction
in
period
size
growth.
this
region.6
will
for by its acceleration
sec)
This
compare
the
the
particle
calculated
portion
polymerization
as opposed
i.e., the induction
period.
on the visual
clotting
time
by laser
(150
A 4.pm
reagents.
fluctuation
also presents
the actual
observed
effect of calcium
can be seen that the effect of calcium
in shortening
be completely
the
of particle
previously
same
time
during
occurs,
effect
of
of Control)
of the
to that time
In Table
I
based on its
spectroscopy.
Table
on the visual
clotting
time.
the visual
clotting
time can
of polymerization.
DISCUSSION
The
dividual
ability
to divide
the
reactions
or groups
by which
clot
clot
fibrinogen
from
formation
conversion
of reactions
is regulated
and
of fibrinogen
has provided
or
thrombin
perturbed.
includes
the
to the
details
The
fibrin
clot into inof the mechanism
formation
proteolytic
of the
action
and the subsequent
polymerization
of the generated
fibrin
monomer.
tion, the importance
of an intermediate
series of steps, the formation
versible
complex
between
fibrinogen
and fibrin
monomer,
was recently
Two
dysfibrinogens
(Cleveland
abnormal
complex
suggested
by studies
reaction.
In earlier
work
I’#{176}
and DetroitTM)
formation
that
rather
did
not
we monitored
would
than
abnormal
include
methods
the
increase
appear
to evaluate
size,
In addiof a reshown.6
to be examples
polymerization,
in particle
fibrin
of thrombin
of
as initially
this
step
a direct
of the
measure
of
the growth
of the fibrin clot, as a function
of time in the fibrinogen-fibrin
system.
Such monitoring
permits
the kinetic
effects
of the fibrinogen-fibrin
monomer
interaction
to be evaluated
and visualized
in the form
of an induction
period
prior
thrombin
changes
to
the
onset
system
of
predict
rapid
that
in the proteolytic
polymerization.
the
action
length
The
of
of thrombin
the
kinetics
induction
and
to changes
fibrin monomer
interaction.
In contrast,
the rate of size
tion period
is a measure
of the polymerization
reaction.
The
period
particle
present
study
shows
that
with
the addition
is decreased
only slightly,
while there
size increase.
This increase
in the
of
the
period
in the
increase
of calcium
fibrinogen-
is sensitive
after
ions
is a dramatic
increase
rate of polymerization
the
to
fibrinogenthe
induc-
induction
in the rate
is similar
of
to
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BRASS
658
that
observed
by previous
tion of purified
tored by optical
that
calcium
ions
fibrinogen,3’4
the
do
not
the small
possibility
complex.
investigators
fibrin monomer
density
changes
alter
of a major
be expected
when
the
rate
in the
effect
of
studying
thrombin’s
length
of
of calcium
a small
decrease
the polymerization
of polymerization
fibrinogen-fibrin
by ourselves
the
on
the
are enhanced.
by visual
time.
magnitude
observed
during
Kanaide
clotting
Thus
is sufficient
fibrin
an increase
account
reported
compared
that
the reversible
to our studies.
irreversibly
removes
would
be lengthened
effects
rate
of the
observed
of the induction
system
as
rate
of the
the acceleration
by
formation.
and Shainoff’2
is long
for
can
increased
in polymerization
to quantitatively
can be converted
to an irreversible
factor
(FSF,
factor
XIII).
The time
mation
period
will
remove
fibrin
monomer,
favoring
dissociation
monomer
complex
by mass action.
The major
change
measured
complex
stabilizing
monomer
The
with a minor
change
in the length
the behavior
of the fibrinogen-thrombin
of
eliminates
of the induction
in the rate of polymerization
period
accurately
predicts
calcium
attack
period
fibrinogen-fibrin
in the length
reactions
as monipreviously
proteolytic
induction
AL.
polymeriza-
in the absence
and presence
of calcium
(data not shown).
Since it was shown
change
Furthermore,
and
El
fibrinogen-fibrin
complex
for this
Since
the
action
fibrin
monomer
from
the
or the rate of polymerization
are consistent
with
calcium’s
effect
monomer
by the action
of
irreversible
complex
of
FSF
system,
the
retarded.
of accelerating
on the complex
induction
Neither
fibrin
fibrin
for-
of
period
these
polymerization.
Thus the experiments
presented
here provide
strong
support
for the conclusion
that the major effect of calcium
in the fibrinogen-fibrin
system
is to increase
the
rate of fibrin
monomer
polymerization,
thereby
decreasing
the time required
to
form
a visible
mechanism
tion.
clot.
Further
studies
by which
calcium
interacts
are
required
with
to
fibrin
delineate
monomer
the
during
molecular
polymeriza-
REFERENCES
I.
Rosenfeld,
fibrin
2.
G.
effect
celerating
Potts
of calcium
version
of
33:206.
1954
to
Lab
Invest
266.
Blomback
415,
accelerating
I
fibrin
the
Clin
RatnoffOD,
Ratnoff
1972
Endres
GF,
Scherago
monomer.
HA:
Equilibria
IX.
reversible
Arch
Ac-
Fibrin
fibrinogen-fibrin
Haemostasis
Forman
Effects
polymerization
Biochem
36:37,
1976
RatnofT
on
OD.
the
for
samples
of
Breckenridge
nature
of
principle:
by
method
37:3 16. 1951
Clin Med
CR,
New
in small
thrombin.
Br
I
the
pro-
Alteration
of
Haematol
13:
Forman
WB,
Brass
EP,
Edwards
RV,
in
of
script
of
Biophys
WB,
formation:
monomer
10:
0: Fibrinogen
Cleveland.
I. Abnormal
fibrinogen-fibrin
complex
formation.
(Manu-
153:
in preparation)
II.
M:
RV,
Edwards
The
complex.
role
of
Thromb
the
Kudrky
Fibrinogen
with
gen
EP,
Kemi
1967
10.
1972
0:
I Lab
bin-converting
898.
C:
Menzie
Experiments
throm
calcium
Purification
Ark
Lindan
conversion.
the
OD:
by
polymerization
39:382,
on
RT:
i Clin
M:
fibrinogen.
of fibrinogen
proaccelerin
ShainoffiR,
fibrin
ions
Scand
Blomb#{228}ck
bovine
determination
plasma.
Invest
B,
and
1957
8.
con-
polymerization
ions.
Blood
6. Brass
Lindan
7.
of human
1952
on
fibrin.
ac-
1969
MH,
of
calcium
The
cations
calcium
of
fibrinogen-fibrin
fibrin
AM:
Delayed
24:29,
Boyer
5.
The
fibrinogen-
116:36,
other
to
the
9. Prentice
HC:
removal
celeration
ions.
and
fibrinogen
3. Godal
due
B:
on
Science
RatnoffOD,
4.
ianszky
calcium
transformation.
elTect
the
of
merization
12.
Detroit.
An
non-functional
domain.
Kanaide
fibrinogen
B, Blomb#{228}ck B, and
and
Blomb#{228}ck
abnormal
fibrino-
NH2-terminal
Thromb
H. ShainofTlR:
poly-
Res 9:25. 1976
Cross-linking
of
fibrin by fibrin-stabilizing factor
(factor XIIIa). I Lab
Clin Med
85:574,
1975
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1978 52: 654-658
Fibrin formation: effect of calcium ions
EP Brass, WB Forman, RV Edwards and O Lindan
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