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CONCISE
REPORT
Flow
as a Regulator
of the Activation
By C.H.
Gemmell,
V.T.
A novel enzyme
reactor
for phospholipid-dependent
reactions
was
used
to study
the effects
of flow
on tissue
factor=initiated
coagulation.
Microcapillaries
were
coated
with
a phospholipid
bilayer
containing
tissue
factor.
a
transmembrane
protein
that is an essential
cofactor
for a
plasma
procoagulant
enzyme.
factor
VII. We show
that.
in
T
HROMBOSIS
AND
THE
formation
of a hemostatic
plug are related,
highly
dynamic
processes that depend
on factors
in the blood and vessel wall as well as on local flow
conditions.
explored
Mechanisms
and debated;
of these
processes
continue
to be
however,
two basic systems have been
identified:
platelets,
which
of the thrombus
or plug,
which
fibnin, a proteinaceous
tures,
constitute
the cellular
and the coagulation
gel that stabilizes
component
system,
by
these struc-
is produced.’
It
documented
that the deposition
and growth
of
aggregates
on vascular
surfaces
are highly
depen-
is well
platelet
dent
on local
flow
conditions,
specifically
the
shear
rate
at
of Factor
Turitto,
X by Tissue
and Y. Nemerson
contrast
to static,
closed
systems.
the steady-state
lytic activity
is independent
of enzyme
concentration
the time to steady
state
becomes
a function
of the
concentration.
C
by Grune
1988
& Stratton.
Following
the
hydrolyzes
an Arg-Ile
it to factor
VIIa.7
Because
activation
tissue
delivery
of factor
factor
factor
must be an essential
tion. To date, coagulation
been studied
primarily
present
in phospholipid
neously
throughout
investigation,
pholipid
bilayer
on
the dynamic
a great
deal
(factor
X), and wall
formation.
In addition
studies
respect
conducted
to reaction
under static
mechanisms,
the measurement
of
reactant
concentrations.
for
system
studying
formation
as a function
have
therefore
developed
coagulation
continuous-flow
system.
There
is considerable
membrane
protein
not
reactions
evidence
normally
that tissue
in contact
with
thereby
VII,
activates
either
of two
substrates,
initiating
the coagulation
although
a zymogen,
is thought
activity
of factor
that
suggest
the
VIIa5;
zymogen
factor
studies
with
is also
active
human
factor
1% of
factor
in this
ofthe
City
Department
ofMedicine.
University
Supported
in
ofNew
part
by
Mt
Sinai
School
VII
species.6
Institutes
of
Health
Grant
HL29019.
Submitted
April
Address
reprint
Medicine,
York,
Mt
NY
Sinai
payment.
“advertisement”
indicate
C
1988
accepted
to
School
of Medicine,
6. 1988.
MD,
Department
ofthis
This
article
in accordance
article
must
of
1 Gustave
Levy
P1, New
defrayed
in part
by page
& Stratton,
therefore
be
with 18 U.S. C. section
this fact.
by Grune
were
Inc.
hereby
marked
1 734 solely
of
the
the
or Vila),
tubes.
effects
substrate
shear
rate
to measuring
By
of enzyme
concentration
on product
the reaction
the time
AND
present
in a phos-
microcapillary
evaluated
(factor
velocity
to reach
Xa)
in a
steady
METHODS
reactor is a small-diameter
capillary
with phospholipids
containing
tissue
of a stable layer was achieved
of Watts
et al.8 Glass
AL)
were
a single
whose inner
factor. The
by using a modification
of
tubes
obtained
from
Clay
to
prepared
molecule
bovine
brain
phosphatidylserine
from Avanti
Polar Lipids,
(both
as previously
of tissue
factor
described9
and
per vesicle.
and
Pd-
contained,
The
tubes
on
were
then rinsed with buffer at a wall shear rate of 1,300 sec’
for two
minutes (flow rate of 1.35 mL/min
for a 0.56-mm tube). The tubes
were kept filled with buffer until used, and precautions
were taken to
avoid the introduction
of air into the system.
The amounts
of phospholipid
and tissue factor coating
the tubes
were assessed by using vesicles prepared
with tracer concentrations
of ‘4C-dioleoyl
costs
0006-4971/88/7204-0020$3.00/0
1404
June
Y. Nemerson,
10029.
The publication
charge
1 1, 1988;
requests
walls
technique
average,
of Medicine
York.
National
factor
factor
is
homoge-
Parsippany,
NJ, or Rochester
Scientific,
Rochester,
NY
to 1 . 1 mm internal
diameter,
75 to 150 mm in length),
were
boiled in dilute detergent
(0.2% wt/vol Sparkleen,
Fisher Scientific
Co. Pittsburgh)
for 30 minutes and rinsed with several changes
of
distilled,
deionized
water in an ultrasonic
bath for 1 5 minutes.
The
tubes were dried at 120#{176}C
for 30 minutes.
The capillaries
were filled with a 2 mmol/L
phospholipid
vesicle
suspension
containing
20 nmol/L
tissue factor in HEPES/albumin
buffer (0.01 mol/L
HEPES,
0.14 mol/L
NaCl,
1 mg/mL
bovine
serum albumin,
pH 7.5) and incubated
for 20 minutes at room
temperature.
The phospholipid
vesicles (100 to 150 nm in diameter)
ham,
the
In
tissue
VII
containing
30% (mole/mole)
70% egg phosphatidylcholine
From
of coagulapathway
has
medium.
have
the
to tissue
(0.34
IX or X,
cascade.4
Bovine
to possess
about
formation
stream
reaction
MATERIALS
the
blood
Adams,
injury
to a vessel,
may be a primary
in the hemostatic
and thrombotic
processes.
Tissue
functions
by forming
a proteolytically
active
complex
factors
VII or VIIa,
a circulating
blood protein.
This
complex
the
factor,
a transwith blood,
is a
rapidly
converting
in membranes,
the moving
true steady state, a new parameter,
state, has been defined.
The dynamic
wall is coated
Xa
thereby
immobilized
the
we
(factor
of
a
in a well-defined
major
initiator
of coagulation.4
Following
the contact
of blood
with tissue
factor
event
factor
concentration
be evaluated.
generated
from
conditions,
particularly
with
these studies
are limited
to
product
We
system
only
factor
step in the initiation
via the tissue
factor
the
where
While
this
X,
VII,
in systems
in which
tissue
vesicles
that are dispersed
we have
using
factor
in factor
is active
from
cataand
enzyme
Inc.
of
bond
VII
the vessel wall.2’3 However,
most studies
of blood coagulation
have been conducted
in test tubes
(static,
closed
systems)
aspects
of flow cannot
of information
has been
Factor
phosphatidylsenine,’4C-dioleoyl
phosphatidylcholine
(both from Amersham
Corp. Arlington
Heights,
IL), or 3H-tissue
factor.9 The capillaries
were drained, broken into 1.5-cm segments,
and vigorously
shaken with 3 mL of scintillant.
The mass of each
component
was calculated
from its associated radioactivity.
A reaction
mixture
consisting
of bovine factors X and VII (or
VIla)
and 5 mmol/L
CaCl2 in HEPES/albumin
buffer was perfused
Blood,
Vol 72, No 4 (October),
1988:
pp 1404-1406
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
ACTIVATION
through
OF FACTOR
the
X BY TISSUE
capillaries
by using
FACTOR
a syringe
1405
pump
(model
351,
Table
Sage
Instruments,
Cambridge,
MA). The coagulation
factors were punfled and quantified
as previously
described7”0”;
factor VIla was
prepared
by cleaving
factor VII with factor Xa.7 All reactions
were
of Factor
X as a Function
of Flow
Diameter
Length
Wall Shea
Hyotysis
Hy&olysis
(mm)
(mm)
(sec’)
(Observed)
(Expected)
0.56
75
26
9.2
-
1.10
75
4
9.3
9.2
1.01
0.56
150
11
25.0
25.9
0.97
0.56
150
26
14.1
14.6
0.97
0.34
125
120
15.6
13.1
1.19
0.34
125
430
4.0
5.4
O.74
run at 22#{176}C.
A radiometnic
emerging
from the capillary.
centrifuge
The
acetic
factor
was collected
sialic
acid
into
EDTA
X
micro-
per 25-tL
intervals
residues
for up to
of factor
X
via reduction
a heavily
activation,
separated
effluent
peniodate-oxidized
radiolabeled
During
The
the activated
containing
Fractions
35 minutes.
to measure
75 iL of 50 mmol/L
were collected
at the indicated
tubes
sample.
were
was used
assay’2
from
with tnitiated sodium borohydnide.
labeled
peptide
is released
that
is
components
by extraction
into 5% tnichlono-
other
acid and counted.
1 . Hydrolysis
Calcul ated assuming
fHydrolysis is
ratio is the observed
was
obtained
from
9.2
The
quantity
lary
was
6)
of tissue
found
and
concentration
of 2 mmol/L
only five to
required
to saturate
the capillary
surface.
factor
to be 4.58
5.97
0.87
±
respectively.
If one
and
phospholipid
0.53
±
(SD)
(SD)
x
assumes
10_b
the
on the
x l0
mol/cm2
formation
capil-
mol/cm2
(n
(n
=
6),
=
of a phospholipid
control
deviation
toward
noted
kinetic
at 430
diffusive
processes
are
where
L
factor
X concentration.’6”7
molecule
as the
44%#{149}13
These
data
coat
phospholipid
single
bilayer.8
toward
The
weight
nm2
fraction
for
of water
varied
from
activation
remained
constant
IA). In addition,
production
unknown.
stable:
system:
4,000
microcapillary
of wall shear
20 to 60 sec’,
sec’
for large
technique
rates found
250
veins,
a
factor
X
and
at a wall
permits
sec
‘,
and
2,000
but
100 nmol/L
factor X,
CaCl2
was perfused
at 27 sL/min
(wall
shear
rate of 26
level (9.2 ± 0.6 nmol/L,
n = 10) was
after
in the
a delay
shear
low
of >20
rate
minutes.
experiments;
This
however,
lag
was
when
the
shear
rate was increased
to 430 sec’
the lag was significantly
reduced
(Fig lB). In addition,
the use of factor
VII
instead of factor
VIIa did not alter the time to reach steady
state
nor
the
rapid
cleavage
the
steady-state
tissue
of
VII.’5
only),
of factor
level
(not
shown),
factor-dependent,
factor
(phospholipid
activation
In
factor
X
which
factor
a departure
was
‘
from diffusion
and
steady
state
Enzymic
extensively
dynamic
sions),
to
diffusion
flow
the
static
Km
and
can
be rate
average
Q is
the
limiting.
product
is proportional
to
flow
x [X],
(L/Q)2”3
rate,
and
[X]
is the
length,
tube
that our system
rates of <120 sec’
was diffu(Table
1).
showed
shear
conditions,
the
In
concentra-
in tube
Variations
rate
is delivery
catalysis
treated.’6”7
and
the
length,
these
and
but
outlet
tube
at wall
under
important
rate-limiting
step
in the
of substrate
to the catalytic
complex.
in immobilized
flow systems
has been
The V,,.., has the same
meaning
in
systems
(albeit
with
in flow conditions
convection.
At
different
dimen-
contains
terms
relating
the
limit
when
flow
approaches
oo, Km (flow)
approaches
Km (static).
Because
amount
of substrate
required
to achieve
0.5 V,,
will
the
vary
the
VII
occurred
absence
or VIla,
(not
The formation
on the catalytic
of factor
rate
Xa within
constants
c2TT
:o253s
reflects
Xa-catalyzed
of
tissue
or calcium
factor
ions,
no
0
6
8
10
shown).
DISCUSSION
only
suggests
to the
hydrolysis
to
attained
within
five minutes
(Fig 1A). Decreasing
the factor
VIIa
levels
to 0.075
nmol/L
resulted
in similar
product
marked
K refers
in which
10
mixture
containing
VIIa,
and 5 nmol/L
a capillary
a steady-state
formation,
where
experiment
the microcirculation,
‘4
When a reaction
10 nmol/L
factor
through
sec’),
sec
regimes
reactor
is the
diameter,
Thus,
to investigate
the
in the cardiovascu-
to 1,200
arteries,
at the
sion-controlled
for at least 35 minutes
(Fig
offactor
Xa was not diminished
by pre-rinsing
the tube with buffer
for ten minutes
shear
rate of 3,000
sec’
(not shown).
This finding
lar
1 5% to
also agree
with Watts
et al who found
on glass
coverslips
to be a continuous,
Whether
all the tissue
factor
is oriented
the lumen
of the tube is presently
coated capillaries
were remarkably
the use of the
complete
range
tion
a phosphatidylcholine
K(L/Q)213,
total
substrate.
The
predicted
hydrolysis
the reactants
to the capillary
wall. Since blood flow is usually
laminar
and the diffusion
of blood proteins
slow, the rate at
which
reactants
arrive
at the surface
via convective
and
figure
to 0.72
=
of the
The
control.
diffusion-controlled
nm2
(%)
of the first
bilayer,
then a single
phospholipid
molecule
would
occupy
0.56 nm2, a value in reasonable
agreement
with the published
of 0.59
-
0.6 (SD, n = 10).
±
The
At a phospholipid
ten minutes
were
Ratio
flow.
as a percentage
by the expected.
hydrolysis
constant
proportionality
RESULTS
Poiseuille
expressed
divided
Parameters
the reactor
depends
but also on the delivery
not
of
Fig 1 .
Effect
of enzyme
concentration
on factor
Xa levels at
the tube outlet at two wall shear rates. 26 sec’
(A). and 430 sec
(B). The factor X concentration
was 100 nmol/L.
and factor
VIla
was 10 nmol/L
(#{149}).
0.1 nmol/L
(s). and 0.075 nmol/L (i).
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
GEMMELL,
1406
with
the
flow
conditions
vasculature,
the Km
systems
without
(flow)
on
to what
made.
The
present
shear
might
introduction
more
intravascularly.
its
in
realistically
thereby
coagulation
pathologic
interesting
rates,
occur
that
our
reactor
of
the conditions
that
hope
understand
to better
in the arrest
with
is
present
this
levels
light,
of factor
actual
or
after
the
Thus,
one
may be
steady-state
it is
cannot
reactor
VII
coagulation.
a long
arterial
seen with
lag phase
reduced.
bleeding
the
in an undiminshed
Xa, albeit
typical
of
prolonged
of bleeding
In
varying
are
result
factor
rates
is an attempt
AND
NEMERSON
may suggest
why patients
with moderate
decreases
in factor
VII levels do not bleed: As seen in Fig 1, levels as low as 0.075
nmol/L
for studying
reactions
thrombosis.
results
of the
dependence
no extrapolation
from static
in the vasculatune
can be
factors
correlate,
segment
inherent
coagulation
We
of various
role
the
of a continuous-flow
phospholipid-dependent
to simulate
in a given
considering
TURIT1’O.
the
critical
steady-state
production
delay.
Indeed,
at wall
circulation,
430
sec’,
low factor
VII
parameter
time to steady
state
production
of product.
be extracted
adds an entirely
of
shear
the
concentrations
with
respect
rather
This
than
the
parameter
from
static
systems;
thus
new dimension
to the study
to
the flow
of blood
REFERENCES
I . Sixma
vessel wall
Haemostasis
283
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ii:
Role
in
haemostasis,
platelets,
in
and Thrombosis,
Bloom
plasma
AL,
ed 2. Churchill
proteins
Thomas
and the
DP
Livingstone
(eds):
1981,
p
Baumgantnen
HR: Shear dependent
fibnin formation
on blood vessels:
Studies
utilizing
patients
with quantitative
and qualitative
platelet
defects. i Clin Invest 78:1072,
1986
3. Badimon
L, Badimon
ii, Galvez A, Chesebro
JH, Fuster V:
Influence
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6:3 1 2, 1986
4.
Hi,
of platelets
Nemenson
Tunitto
VT,
in promoting
Y: Tissue
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Blood
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1988
5. Zur M, Radcliffe RD. Oberdick J, Nemerson
Y: The dual role
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Initiation
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proteolytic
system by a zymogen.
J Biol Chem 257:5623, 1982
6. Broze
GJ in, Majerus
PW: Purification
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human
coagulation
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R, Nemenson
Y: Mechanism
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Xa. J Biol Chem
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2. Weiss
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9. Bach R, Gentry
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8. Watts TH, Brian AA, Kappler
JW, Marrack
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IAd: Proc Natl Acad Sci USA 8 1:7564,
1984
R, Nemerson
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R,
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W:
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J Biol
1986
Purification
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256:8324,
1981
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Y: The activation
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Y: Radiometnic
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VT: Rheological
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Koyayashi
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Biochem Cell Biol 64: 1 39, 1986
16.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1988 72: 1404-1406
Flow as a regulator of the activation of factor X by tissue factor
CH Gemmell, VT Turitto and Y Nemerson
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