Blood-Clotting
Enzymology
Three Basic Reactions
Walt er H. Seegers,
Lowell
McCoy,
and Ewa Marciniak
During the past quarter century perhaps no single phenomenon has attracted so much
attention in biology and medicine as blood clotting. It plays a role in numerous
clinical conditions where it was not taken into account previously. Knowledge of the
basic mechanisms recently passed through a tumulous stage of growth, but is now
delineated in terms of simple enzymology. Most of the components have been obtained in purified form. There are three main events: the formation of autoprothrombin C (F-Xa, thrombokinase),
the formation of thrombin, and the formation of
fibrin. Prothrombin itself is a more marvelous protein than was predicted. It is a
protein aggregate that contains the precursor of thrombin, the precursor of autoprothrombin C, as well as accessory protein. When blood coagulation is initiated by
tissue injury, large amounts of autoprothrombin
C activity arise in the presence of
calcium ions and tissue thromboplastin.
When there is no tissue injury, small
amounts of autoprothrombin
C are produced by platelet cofactor I (F-Vlll,
AHF)
which functions with the lipids of platelets. Autoprothrombin
C is the enzyme which
produces thrombin activity, and being a part of the prothrombin aggregate it is in
close proximity to the specific bond(s) that must be split to get thrombin activity.
Although autoprothrombin
C alone is sufficient to produce thrombin, the reaction is
greatly accelerated by plasma Ac-globulin
(F-V), lipids from platelets, and calcium
ions. The acceleration produced by components functioning in pairs and relays makes
possible the essential integration with the anatomic location of resources. Tissue
materials pair with plasma resources to produce autoprothrombin
C, and then they
pair with another plasma component
(Ac-globulin)
to make autoprothrombin
C
function. In the absence of tissue injury, platelet materials pair with plasma resources
to produce autoprothrombin
C, and then platelets again work with another plasma
component (Ac-globulin)
to support the function of autoprothrombin
C. Platelets are
essential because their lipids are needed for the formation of autoprothrombin
C, as
well as for accelerating its function. Whatever involves platelets can be the stimulus
to accelerate blood clotting;
this includes Hageman factor. Platelets are the true
point of beginning for the first step in blood clotting and are involved in the second
From
the Department
of Physiology
and
of Medicine,
Detroit,
Mich. 48207.
Supported
by Research
Grant
HE-3424-10
Public
Health
Service.
Received
for publication
Oct. 20, 1967.
Phariiiacology,
from
97
the
Wayne
National
State
Institutes
University
of
Health,
School
IT. S.
98
SEEGERS ET AL.
Clinical
Chemislry
and third. Vitamin K deficiency, or counteraction of Vitamin K by Dicumarol reduces
prothrombin synthesis by the liver. As a consequence the precursor of thrombin, the
precursor of autoprothrombin C, and the accessory protein of the molecular aggregate
which constitutes prothrombin is reduced in concentration. It has been found possible
to produce abnormal conditions experimentally
and have relatively more of the
precursor of thrombin released to the blood than is normally associated with the
precursor of autoprothrombin
C. Up to the point of incorporation to form a single
prothrombin
molecule, the synthesis 0f subunits proceeds by separately operating
mecha nisms.
B
LOOD CLOTTING
has popularly
been regarded
as the dominant
influence in hemostasis
and thrombosis.
That would be sufficient
for
special
interest,
but
this abbreviated
appreciation
of its importance
has been enlarged
enormously
during
tile last two decades.
One now
also applies
knowledge
about
these mechanisms
to gain an understanding
of wound healing, fibrinous
inflammation,
metastases
of maligiiant tumors,
fibrinoid
degeneration
in collagen
diseases,
paroxysmal
11oct urnal
hemoglobinuria,
traumatic
shock, hemorrhagic
shock, epidemic hemorrhagic
fever, acute renal failure,
hemorrhagic
pancreatitis,
pituitary
necrosis,
arteriosclerosis,
disseminated
intravascular
coagulation, etc. In the study of protein
synthesis,
prothrombin
is a special
case because
Vitamin
K is needed.
Since its function
can readily
be
antagonized
with Dicumarol,
we have an unusual
opportunity
to study
how such a complex molecule
is produced
and released
to the blood.
Selve developed
the insightful
perspective
that we possess
a multitude of safety
mechanisms
to maintain
our homeostasis
and that we
can survive even if one s stem is overtaxed,
but we may lose our life if
alternative
pathways
that subserve
the same function
are also obstructed.
He goes on to point out that there are pluricausal
thrombohemorrhagic
phenomena
associated
with many clinical
problems
(1).
Disseminated
intravascular
coagulation,
which occurs
frequently,
is
referred
to as an interrne(liate
mechanism
of disease
(2) and as a new
concept
in the etiology of disease (3).
With these diverse
applications,
it is important
to he familiar
with
the basic mechanisms
that are involved
in blood clotting.
This knov,Iedge is largely
the product
of developments
in research
laboratories
during
tile past two decades,
and it cai he asserted
with satisfaction
that the baffling riddle of blood clotting
has been solved.
Ever since the work of Alexander
Schmidt
at tile end of the nineteenth century,
the coau]atioii
of blood has been ascribed
to the enzyme
thromhm.
1)etails
about
how thrombin
functions
to produce
the clot
Vol. 14, No. 2, 1968
BLOOD CLOTTING
99
ENZYMOLO3Y
have beeii documented
regularly
(4, 5). However,
the manner
in which
thrombin
forms was a controversial
question
until recent years. It is
the primary
purpose
of this presentation
to correlate
and present
a
unified concept
of the basic enzymology
related
to the formation
of
thrombin.
Excerpts
from some of our recent experimental
results
are
included.
It is consistent
with all experimental
observations
to state that there
are three main events involved
in the coagulation
of the blood, and all
other
related
facets
of molecular
biology
subserve
these
essential
reactions.
Tile following
occurs in sequence:
1. Formation
of autoprothrombin
C
2. Formation
of thrombin
3. Formation
of fibrin
The resources
for supplying
the materials
needed
for these essential
steps in blood coagulation
are found primarily
in tissues,
plasma,
and
platelets.
Any condition
which serves as an adequate
stimulus
such as
injury,
foreign
surface
contact,
and tile numerous
conditions
that alter
platelets
may release
the necessary
molecular
components.
The last
steps in blood coagulation
are fibrin stabilization
and clot retraction.
Proth rom bin
Transformation to Autoprothrombin
rfhe
observation
which
C and Thrombin
served
as a basis
for
demonstratuig
clearly
and unequivocally
the minimum
requirements
for obtaining
thrombin
was made 19 years ago by Seegers
(6). He had succeeded
in obtaining
purified
prothrombin
from bovine plasma
and found that it converted
to thrombin
in strong salt solutions.
The experiment
was the subject of
continuous
study ever since it was first recorded,
and recently
was
again performed
in two ways for presentation
in this paper. Thrombin
activity began to form spontaneously
after 8 hr. (Fig. 1), and eventually
the yield was 100%, i.e., the yield was equal to the maximum
which was
obtained
from the same prothromhin
by two-stage
analysis.
Purified
thrombin,
added at zero time, primed the reaction
so that the full yield
was obtained
in 6 hr. The same result
was also obtained
by adding
*The
term
autoprothroinhin
C is used
in this
paper.
It
very
likely
corresponds
to
active
thrombokinase
(Milstone)
or active F-X. The latter
two terms were used from a perspective
which regarded
this enzyme as being separate
from the precursor
of thromhin.
‘When the terms
autoprothrombin
C and F-X were introduced,
no attempt
was made
to rule out possible
similarity
to autoprothrombin
I, which was earlier
described
in this laboratory.
The latter
is
a variant
of the basic structure
found in autoprothroinbin
C. Autoprothromhin
C is an enzyme
which occurs as part of the prothromhin
molecule
and is found there in precursor
form called
autoprothromhin
ITT.
100
SEEGERS fT AL.
Clinical
purified autoprothrombin
C at zero time. Either
thrombin
formation,
but to obtain
the same
more thrombin
was needed than autoprothrombin
Chemistry
enzyme thus accelerated
result,
about 100 times
C.
The same experiment
was repeated
by using purified
from the same lot. This time, however,
the generation
thrombin
C was rcorded
rather
than that of thrombin.
It
dent (Fig. 2) that this enzyme activity
developed
under the
prothrombin
of autoprois quite evisame condi-
4
0
Fig.
1. Spontaneous
tion of thrombi,,
prothrombin
in
z
citrate
creased
thronibin
tlirombin
O
i
TIME
IN
genera.
by
25%
purified
sodium
solution.
Rate
is inby
adding
purified
or purified
autoproC at zero time.
HOURS
-J
Fig.
0
2. Spontaneous
generation
of autoprothrombin
protliroinbin
in 25%
C by purified
sodium citrate
solution.
inci’eased
Rate
adding purified
autoprotlirombin
is
by
thrombin
or purified
C at zero time.
0ID
4
TIME
IN
HOURS
tioris and at the same rate as thrombin.
It generated
niore rapidly
when either thrombin
or autoprothromhin
zero time.
Subunit Activation:
Prethrombin and Autoprothrombin
spontaneously
and
C was added at
Ill
Although
it was known that either
soybean
trypsin
inhibitor
(7),
3,4,4’-triamiuodiphenyl
sulfone
(6),
diisopropylfiuorophosphatel)FP-(8)
would block the generation
of thrombin
from prothrombin,
it
was not precisely
clear what was happening.
An attempt
was then made
to separate
the precursors
of the two enzymes,
for which purpose
prothrombin
was
dissociated
with the use of thrombin.
Thereupon
we
.
Vol. 14, No. 2. 1968
BLOOD CLOTTING
101
ENZYMOLOGY
obtained
prethrombin,
autoprothrombin
III, and, in addition,
an inhibitor.
Properties
of the two purified
enzyme
precursors
were then
studied
(9). Ti#{236}rombin
activity
was readily
obtained
from mixtures
of
prethrombin
and autoprothrombin
III in 25% sodium citrate
solution,
but thrombin
did not accelerate
this reaction.
Evidently
the autoprothrombin
Ill transformed
to autoprothrombin
C and the latter
functioned as the enzynie for attacking
the bond(s)
in prethrombin
which
are broken in association
with the emergence
of thrombin
activity.
The
curve for thrombin
generation
was a sigmoid,
and it was thus evident
that an activator
was building
up in concentration
until no more of the
respective
precursor
remained.
When purified
autoprothrombin
C was
added to the prethrombin,
the generation
of thrombin
began at once
and followed
a straight
line course (Fig. 3).
0
0
‘C
-J
Fig. 3. Conversion
of prethrombin
(5 mg./
tiil.) to thrombin
in 25% sodium
citrate
solsition at 370, and at pH indicated
on each curve.
Purified
autoprothrombin
C (200 U./ml.)
was
used for activation.
z
0
I
I-
o
20
40
60
80
100
TIME IN MINUTES
In place of strong
salt solutions
physiologic
saline was also found
to be a suital)le
solution,
but then much more autoprothrombin
C was
required
and a time period of 5 hr. instead
of 1 hr. was needed.
The
conclusion
is that autoprothrombin
C alone is sufficient
for the formation of thrombiii.
Thus we have reached
a milestone
in the history
of
blood coagulation.
The equation
follows:
autoprothrombin
Prethroinbin
C
thrombin
&
peptide(s)
The 25% sodium citrate
solution
proved
to be an adequate
medium
for the spontaneous
conversion
of purified
autoprothrombin
III to
autprothrombin
C. The addition
of autoprothrombin
C at zero time
*Certain
preparations
of autoprothromhin
TTT do not
convert
to autoprothromhin
C in 25%
sodium
citrate
solutions.
Under
certain
conditions
thrombin
produces
a modified
autojirothronibin
ITT which remains
stable
in 25e/
sodium
citrate
solution
but is activated
by
stypven
or tissue
thromboplastin.
All other
enzymes
tested
leave the protein
iii a condition
which yields autoprothrnmbin
C in strong
salt solutions.
102
SEEGERS fT AL.
Clinical
Chemistry
accelerated
the reaction
(10). Soybean
trypsin
inhibitor
blocked it because it neutralizes
autoprothrombin
C; 3,4,4’-triaminodiphenyl
sulf one
also retarded
the reaction
because it is a competitive
inhibitor.
The whole series of developments,
starting
with the study of purified
prothrombin,
extended
over a period of two decades.
One can prove the
first two main events in blood clotting
by having
only purified
prothrombin,
two of its derivatives,
and sodium citrate.
Furthermore,
the
same prothrombin
yields the thrombin
which is needed
for the third
main event-the
formation
of fibrin. In the course of presenting
the evidence and carrying
out the technical
manipulations,
nothing
was required from plasma,
platelets,
tissues,
or aiy natural
source. This approach
ruled
out the consideration
that
a contaminant
might
be
associated
with one or another
substance
that might have been isolated
from plasma,
platelets,
or tissues. Only the purified prothrombin
needed
to be studied
critically.
It was pointed
out by Kline (ii)
that it is impossible
to rule out the existence
of hypothetical
material
in association
with anything,
and especially
with a protein.
The sodium citrate
was
of analytic
grade.
The prothrombin
was studied
(12, 13)
by electrophoresis,
with the ultracentrifuge,
with the electron
microscope,
by
N-terminal
amino acid analysis,
immunochemistry,
etc. We can thus
outline two main events in blood clotting
by a simple diagram
(Fig. 4).
Autoprothrombin C
Accessory Factors for Activity
The evidence
for the production
of thrombin
by means of autoprothrombin
C alone is clear. Autoprothrombin
C is the enzyme, and any
PROTHROMBIN
J
Fig.
pH 7.0
I
PRETHROMBIN
4
INHIBITOR
I
of
prothrombin.
Prethrombin,
autoprothrombin
III,
and
inhibitor were produced. In 25% sodium
AUTOPROTHROMBIN
AUTOPROTHROMBIN
+ (Peplide(s))
4. Degradation
C
citrate
solution to convert
autoprothrombin
formed sufficient
autoprothroinbin
C which
alone
prethrombin
throinbin.
IIIto
was
THROMBIN
#{247}
(Peptlde(s))
acceleration
is properly
considered
to be in direct
support
of that
enzyme’s
function.
This support
is needed because
there is neither
a
sufficient
amount
of the enzyme
nor enough
time, under physiologic
conditions,
for the reaction
to occur with sufficient
promptness.
Ac-
Vol. 14. No. 2, 1968
BLOOD CLOTTING
103
ENZYMOLOGY
globulin,
lipids, and calcium ions make the enzyme function
efficiently
(14).
To show how autoprothronibin
C, Ac-globulin,
and lipids are
interrelated,
each one was made tile single variable,
and prethrombin
was the substrate
(is).
The composition
of tile reaction
mixtures
was
as follows:
Prethronibin
Autoprothrombin
Ac-globulin
Lipid
Ca ions
C
Prethrornbin
Autothrombin
Acglobulin*
Lipid
Ca ions
Varied
in concentration
from zero and upward.
3 projections.
The physiologic
saline solutions
were
C
Prethrombin
Autothrombin
Ac-globulin
Lipid*
Ca ions
C
Everything
else was kept constant
buffered
with imidazole
at pH 7.2.
for
all
Without
autoprothrombin
C no thrombin
formed
(Fig. 5). With autoprothrombin
C the rate at which thrombin
was generated
and the
amount
of thronlbin
obtained
increased
in direct
proportion
to tile
amount
of purified
autoprothrombin
C which was added.
Without
purified
Ac-globulin
a little thrombin
activity
developed
which
is due to autoprothrombin
C alone.
The lipid,
without
Acglobulin,
did not make the enzyme more effective;
as Ac-globulin
was
added and as the concentration
was increased,
the yield of thrombin
went up proportionately
(Fig. 5). The same relationship
held with lipid
as the variable
(Fig. 5). The Ac-globulin
did not help the enzyme
without
the presence
of lipid. Lipids
are surface-active
agents
while
Ac-globulin
is concerned
with the substrate
specificity
of autoprothrombin
C.
To determine
the Michealis
constant
for the reaction
under
consideration,
all conditions
for calcium
ions, Ac-globulins,
lipids,
and
autoprothrombin
C were fixed at the optimum
and the substrate
concentration
was varied.
The K5 was found to be 3.14 X 10#{176}.
For the
experiments
described
above,
prethrombin
was preferred
to prothrombin
because
the use of prothrombin
altered
conditions
considerably owing to the development
of autoprothrombin
C activity
from
the autoprothrombin
III portion
of the protein
aggregate
(16).
Activity Development
As soon as it was clear that autoprothrombin
C is the enzyme needed
for generating
thrombin
activity,
one could say that the appearance
of thrombin
implies the presence
of autoprothrombin
C because there
is no substitute
for it in blood. The potency
of tissue extracts
would he
due to the autoprothrombhi
C generated
and the lipid which is needed
after autoprothrombin
C is formed.
It has been known since 1948 that
104
calciuni
slowly
SEEGERS fT AL.
and
thromboplastin
are
from
purified
prothrombin
sullicient
Clinical
for
)roduci1lg
thrombin
the formation
of
This implied
was, therefore,
(17).
Chemistry
autoprothrombill
C. Throniboplastin
the
to produce
autoprothrombin
C from purified
prothrombin
prothrombin
C was first purified
in Illis laboratory
(18).
selected
agent
when
auto-
-J
-J
LiJ
uJ
0
z
z
:1
z
2
I
I-
I
20
I-
40 60 80 100
TIME IN MINUTES
TIME
IN MINUTES
-J
LLi
0
z
2
I
I.-
0
20 40 60 80 100 120
TIME IN MINUTES
Fig. 5. Interrelationships
of autoprothrombin
C, Ac-globulin,
and lipid. Top left, Ac-globulin
concentration
progressively
increased.
Top right, lipid as variable;
lower left, autoprothrombiii
C as variable.
Any one of the variables
determined
the yield and rate of formation
of thrombin.
#{149}When
purified
III was also obtained,
it was possiAs expected,
there
was rapid
activation
in the presence
of calcium
ions and sedimentable
brain
thromboplastin
made by the procedure
of Hecht
ei al. (19). Notiling
else was needed and evidently
no hypothetical
substance
such as factor
VTI was needed.
The pH optimum
for the reaction
was 7.2, and a restricted
amount
of the thromboplastin
reduced
the rate of the reaction
ble to test
its
autoprothrombin
reaction
to thromboplastin.
Vol. 14. No. 2, 1968
BLOOD CLOTtING
105
ENZYMOLOGY
and
the quantity
of autoprothrombin
C obtained.
The question
which
was to determine
how autoprothrombin
C forms
when no
tissue
extracts
are involved.
It had frequently
been postulated
that
there might be an extrinsic
and an intrinsic
prothronibinase.
However,
the enzyme
is evidently
always
autoprothrombin
C and does not share
this function
with any other
enzyme.
It forms
slowly and in small
quantity
if tissue extracts
are excluded.
rFhe slow clotting
of plateletiich plasma
is due to small quantities
of autoprothrombin
C, and its
accelerated
clotting
by tissue extracts
is due to relatively
more autoprothronibin
C. The near failure
of platelet-poor
recalcihed
plasma
to
clot is associated
with very limited
formation
and function
of auto-
remained
C. [nder
those conditions
there
are no platelet
lipids foi’
the production
of autoprothrombin
C or to function
with tile little autoprothronibni
C that is produced.
To contribute
to a fuller understanding
of the way in which autoprotllromnbin
C originates,
we again prepared
bovine autoprothrombill
UI and also platelet
cofactor
I. This was done by making a combination
of technics described
by Bidwell
(20) and by Hurt et al. (21). The preparatioli
represented
a thousand-fold
purification
but was not a single
component.
With this preparation
and calcium
ions, some autoprothrombin
III converted
to autoprothrombin
C (Fig. 6). The rate of the
plotill’ombin
60
50
“
z
Fig. 6. (‘onversioll
of purified
bovine
autoprothrombin
III to autoprothrombin
C by adding
a
plasma
fraction
(F-vTIfl
and calcium
ions, 01’ l)y
also adding
lipid activator
(crude
ceplialin).
Reaction mixture
was used nsa substitute
for thromboplastin
in the standard
one-stage
prothroinbin
time test. Note that much niore autoprothrombin
C
(short
clotting
time)
generates
when lipids
are
present.
40
i.ij
30
o
Z
,..-N0
b
i0
I
0
I
I
1020304050
TIME
reactioti
and the yield of autoprothromhin
accelerated
l)y adding
a crude
“cephalin”
brain tissile. It was possible
to use platelet
lipids,
or purified
platelet
factor
3. The
substance
associated
with the preparation,
LIPIDS
H 20
IN MINUTES
C was, however,
greatly
preparation
made from
homogenates
as a source of
antihemophilic
factor,
or a
produces
autoprothrombin
106
SEEGERS fT AL.
Clinical
Chemistry
C from autoprothrombin
III. Let us discount
the possibility
of a hidden
substance
in the preparation
and go on to say that platelet
cofactor
1
ordinarily
functions
with the lipid of platelets
to generate
amitoprothrombin
C activity.
The conclusion
reached
is supported
by the prothronlbin
activations
described
in many studies
(12) from this laboratory
where it was reported
that platelet
cofactor
I was found to function
with platelets,
the main active
component
being platelet
factor
3. The designation
platelet
cofactor
I was intended
to imply something
which works with
platelets
and from that point of view is a more suitable
term than
AHF or F-VIII.
It is not found in serum.
There
one finds platelet
cofactor
IT (autoprothrombin
TI) which
some regard
as factor
IX
(which is not strictly
accurate
l)ecause factor IX is supposed
to be only
in plasma).
Autoprothrombin
IT is derived
from prothi’ombin
and thus
occurs only in serum. Substance
P. found in urine, also functions
as a
platelet
cofactor
(22). Tn recent experiments
the P fraction
from urine
was found
to convert
autoprothromhin
ITT to autoprothrombin
C.
Calcium
ions, but not lipids,
were required
for this, and there was
secondary
inactivation
of the autoprothrombin
C.
In the case of our bovine
preparation
of platelet
cofactor
I, the
lipids were important
but not essential.
‘We then received
some material of human origin.* We found that it alone, or with calcium ions,
would not generate
autoprothrombin
C from autoprothrombin
ITI. It
was, however,
very effective
when purified platelet
factor 3 was added.
The fraction
of human origin was more dependent
on lipids than the
bovine material.
Organization of Data
The study with the 25% sodium citrate
solution
made it possible
to
assert that autoprothrombiri
C arises solely from its precursor
(autoprothrombin
ITT), and that this enzyme
alone produces
thrombin
activity from its precursor.
This is summarized
in Fig. 2, which outlines
two main events in blood clotting.
Now we know which of these reactions
is supported
by accessory
materials
located
in plasma,
platelets,
and
tissues.
The basic diagram
can then be enlarged
(Fig. 7) to include an
assignment
of “clotting
factors”
to their exact place in support
of
the two main reactions
in blood clotting.
Ac-globulin
is exclusively
concerned
with
thromhin
formation
by determining
the reaction
specificity
of autoprothrombiri
C. Tissue
thromboplastin
and platelet
cofactor
I are colicei’lled with tile formation
of aiitoprothrombin
C. The
lipids of thromboplastin
are coucerlied
with thrombin
and autoproKindly
supplied
by Dr.
Allen
Johnson
of New
York.
Vol. 14. No. 2. 1968
BLOOD CLOTTING
107
ENZYMOLOGY
thrombin
C formation.
When tliromboplastin
is excluded,
the lipids
must come froni platelets,
since those ordinarily
found in plasma
are
not effective.
The lipids of I)latelets
are concerned
with autoprothrombin
C formation
and its function
in tile production
of thrombiri.
PROTHROMBIN
Fig.
7.
Degradation
Autoprothrombin
prothrombin
C
of
protlirombin.
III
converts
spontaneously
to
in
auto25%
sodium
citrate
solution;
in physiologic
saline
solution,
calcium
ions and tissue
extracts
accelerate
tile
reactions,
with
tissue
extracts
excluded,
calcium
ions,
platelet
cofactor
I, amid lipids contribute
to formation
of autoprothim’omnbin
C. Note
that lipids
are iinportaimt
at two places
and originate
frommi platelets
when tissue
extracts
are excluded.
IThbdh1
‘t p
I
I
PRETHROMBIN
Co iOnS’
c-gIobuIn’
INHIBITOR
AUTOPROTHROMBIN
Sse
Extract
Ca ions
Platelet
Cof actor I
Lipi s
AUTOPROTHROMBIN
+ Peptide(s) 7 C
THROMBIN+
Peptide (s)
These
accelerate;
Autoprothrombin
C alone is sufficient.
In this design for the proper
functioning
of an important
biologic
system,
there is the underlying
theme of functioning
in pairs.
This
starts
with the combination
of tissue materials
with plasma
following
injury,
or platelet
substances
with plasma.
The adequate
stimulus
for
clotting
is the combination
of materials
from two anatomic
compartments.
This translates
to thromboplastin
+ autoprothrombin
III, or
platelet
factor 3 + platelet
cofactor
I + autoprothrombin
III. Then the
platelet
lipids
or thromboplastin
pair with the plasma
protein
(Acglobulin)
and support
the work of autoprothrombin
C, the latter being
a product
of a previously
paired
function.
A paired
function
must he
created
in producing
the adequate
stimulus.
Functioning
in pairs also
serves as a safety device. Note, for example,
that autoprothrombin
C
can be injected
into the circulation
without
lethal effects because
there
are no suitable
lipids with which it can function.
By contrast,
thromboplastin
is lethal because
autoprothrombin
C forms and can function
with the lipids of thromboplastin.
Another
interesting
fact is the close association
of prethrombin
with
autoprothrombin
III in the form of prothrombin.
This arrangement
contributes
to obtaining
maximum
efficiency
in molecular
collisions.
The autoprothrombin
C does not have to traverse
space in plasma;
if
that were so, it would collide with an undetermined
number
of albumii
or globulin
molecules
and the like before
getting
to its prethrombin
substrate.
The distance
from autoprotllromhill
C to prethrombm
corre.
sponds to molecular
length and width. Furthermore
we can anticipate
108
SEEGERSfT AL.
iin(iing spatial
relationships
thrombin
C and prethronibin
that are advantageous
ititeractions.
Clinical
for
the
Chemistry
aiitopro-
Prethrombin Compared with Prothrombin
Pretllroml)in
was derived
from purified
prothronibin
by first digesting
with thrombin
at pH 7.0. This resulted
in a degradation
product
whicil,
utilizing
chromatography,
yielded
i)rethrombin
in more than one fraction. One of tllese was further
purified
to obtain a single component
(23).
The physicochemical
properties
of the prethrombill
are niore
nearly
like those of thronlbin
than prothromhi1l.
Prethrombin
cannot
be determined
quantitatively
by using the two-stage
analytic
reagents
commonly
applied
to prothrombin.
it is necessary
to increase
the procoagulant
strength
of tile reagents
1)y adding
purified
Ac-globulin
and
autoprothronibin
C. It is well known that plasma
mixed with lipid of
the crude cephahn
type, and tilell recalcified,
will have all of tile prothrombill
utilized.
Furthermore,
we found that all purified
prothrombin
added to plasma
under the same conditions
was also utilized
in the
prOtllrombin
consumption
test (Fig. 8). Tile native
prothromhin
and
-I
600
400
4
t’
z
PLASMA *
PRETHROMBIN
-,
Fig.
8. Prothromhimi
consumption
es.
PLASMA
200
/PROTHROMBIN
PLASMA
ALONE
ia
0
0
10 20 30 40 50
TIME IN MINUTES
the added prothrombin
were both completely
utilized. tTpon studying
the
serum,
very little thrombin
was generated
even when purified
autoprothrombin
C and purified
Ac-globulin
were used in attempts
to
convert
precursor
material
to thrombin.
Likewise,
wilen blood was
added to a test tube containing
a small amount
of prethromhin,
serum
formed
and all the prothrombin
was coiisiimed
l)ut tile I)l’etilrombill
remained.
Tile procoagulallt
power of blood was not sufficient to convert
prethrombin
to thrombin.
Prethrombin
was Ilot utilized
in the prothrombin
consumption
test (Fig. 8). it was also not neutralized
by antithrombin,
and thus was like neither
protilrornbin
nor t.ilrombin.
It is a
true degradation
product
of prothrombin
and quite different
from
prothrombin
or thrombin.
Vol. 14 No. 2. 1968
BLOOD CLOTTING
109
ENZYMOLOGY
Prothrombin Structural Suppositions
Most of what is known about the structure
of prothrombin
is by inference
from the store of general
inforniation
about proteins.
Nevertheless,
we can venture
in the directioll
of constructing
a working
hypothesis.
Even a two-dimensional
projection
is helpful
(Fig. 9). From
AUTOPROTHROMBIN
-
C
HROMBINIII
AUTOPROTHROMBIN
Fig. 9. Prothrombin
structural
suppositions.
Autoprothrombin
III portion
of protlirombin
probably
yields 1 or more peptidcs
whemi autoprothromhiii
C is formed
and a peptide(s)
may
be removed
when autoprothrombin
C produces
thrombin
activity.
Accessory
protein
can be
available
in association
with thrommibimi or autoprothronmbin
C to modify
structure
and activity.
with proper conditions,
prothrombin
can form other derivatives
called autoprothrombin
I and
autoprothxrombin
II which are modified
forms of autoprothromhin
C and thrombin,
respectively.
These derivatives
are important
in modifying
the rate of thrombiu
formation.
the molecular
weight of 68,900 for bovine prothrombin,
25,000 is needed
for thrombin
and about the same is required
for autoprothrombin
C.
A substantial
amount
of material
remains
and can be designated
as
accessory
protein.
The thrombin
and autoprothrombin
C precursors
probably
lose one or more peptides
when each enzyme activity
develops.
It will then be necessary
to determine
the nature
of the bond(s)
broken
during
each activation.
It was found
autoprothrombin
basic thrombin
in this laboratory
that
II (24). Tilis component
unit. It could conceivably
prothrombin
can also yield
in some way is related
to the
form by cleavage
of pro-
110
SEE6ERS fT AL.
Clinical
Chemistry
thrombin
at some place that would leave accessory
protein
with the
thrombin
portion.
Autoprothrombimi
II is not inactivated
by antithrombin
and it functions
as a platelet
cofactor;
it is found
in serum.
Prothrombin
can be activated
under conditions
quite differeiit
fi’om
those which yield autoprothrombiim
II. Conditions
which yield autoprothrombin
I have been studied
most extelisively
(24).
This latter
derivative
is closely related
to autoprotilrombin
C amid, like autoprotilrOmbin
C, is inactivated
by antithi’ombin.
This component
could conceivably
forum by degradation
of the pI’otllronlbin
in a manner
that
would leave accessory
protein
with the autoprothrombin
C portion
of
the original
molecule.
Commonly,
serum contains
autoprothronibin
II, a small amount
of
prethrombin,
and appreciable
quantities
of autoprothrombin
III. In
fact, autoprothrombin
111 was found in the serum
of all 10 species
tested (25). The derivatives
of prothrombin
found in serum are not in
plasma in the same form. Failure
to recognize
this fact has frequently
been the basis of confusion.
Effect of Dicumarol
In past years the literature
carried
many reports
on the effects of
Dicumarol.
The concentration
of factors
II, VII, IX, and X in the
plasma
was supposed
to be lowered,
and each one at a different
rate.
In this laboratory,
the hypothesis
for factors
VII and IX was not
accepted.
It can still be asserted
that factors
VII and IX are hypothetical
plasma
factors.
No one has succeeded
in isolating
such substances from plasma. In this laboratory
it was found that the properties
of the prothrombin
molecule
can be used to account for all phenomena
ascribed
to factors
VII and IX. It is thus not possible
to discuss
the
effect of Dicumarol
on factors
Vii and IX. With factor
X (autoprothrombin
III portion
of prothrombin),
we are concerned
with a plasma
component
associated
with the precursor
of thrombin.
A prime question
was whether
any experimental
facts could be produced
to indicate
that
autoprothrombin
III can appear
in the blood in a form which is not
prothrombin,
even though it is generally
in tile prothromhin
aggregate.
Likewise,
it had to be asked whether
prethromhin
can occur in blood as
an entity separate
from prothromhin.
Many authors
used “factor
VII deficient,”
“factor
1X deficient,”
or
“factor
X deficient”
plasma
as a test substrate.
Whatever
served as a
corrective
procoagulant
with the respective
plasma
was then said to
contain,
respectively,
factor VII, factor IX, or factor X. This carried
with it the error of not ruling out the effect of prothrombin.
In factor
Vol. 14, No. 2, 1968
VII
deficiency,
BLOOD CLOTTING
sensitive
to the thromboplastin
of sensitivity
can easily be produced in normal prothrombin
by a variety
of conditions.
In factor IX
deficiency,
the prothrombin
does not readily
form autoprothrombin
II
(12, 13). In addition
to using abnormal
piasmas
we worked with three
other tests: (1) time well-known
two-stage
assay which develops
a certain
thronibill
titer
ull(ler
standard
conditions;
(2) the two-stage
assay
nlo(lihed
l)y a(iding more procoagulallt
power in the form of purified
autoprothromhiii
C and purified
Ac-globulin
(this was called the prethrombin
assay);
(3) a two-stage
assay in which the maximum
autoprotilrombill
C activity
is developed
with Russell’s
viper venom and
in the
tilen
prothrombin
Ill
ENZYMOLOGY
two-stage
measured
reagents.
is not very
Such
loss
quantitatively.
When
prothrombin
synthesis
was stopped
ill the dog by giving
I)icumarol,
the residual
prothronlbin
disappeared
and the rate was the
same whether
measured
as the thrombill
titer that developed
or as the
autoprothrombin
C titer
(Fig. 10). Tn another
experiment
the drug
-J
...J200
5D
r)
I60
4
zrnl2O
30
0
80
21-
m
I
040
0
2
DAYS
412345
HOURS
Fig. 10. Prothromnbin
plasma
during
body weight).
time, 2-stage
prothrombin
assay, amid autopiotlimoimibimi
( titer
iii (log
administration
of Dicumarol
and after
injection
of Vit-iniimi K (L5 iiig/kg.
Dicumarol
dose was 100, 75, and 50 mg. on successive
days.
was continued
after the prothrombin
concentration
ilad been lowered;
then purified
bovine
prothrombin
was infused
to restore
the prothrombin
concentration
to normal.
This foreign
prothrombin
temporarily
restored
the
bleeding
time
to normal.
The
prothrombin
concentration
diminished
quite like that of the original
residual
dog
prothrombiri.
Whether
measuring
maximum
thrombin
titer or maximum autoprothrombin
C titer, the result was the same. At the height of
112
EEGERS
fT AL.
Clinical Ckemis+ry
the Dicumarol
effect,
it was always
possible
to obtain
much more
thrombin
by using the prethrombin
assay than with the prothrombin
assay
(26).
One is left with the impression
that prethrombin
is the
major precursor
of thrombin
in plasma
under the extreme
conditions
of prolonged
and excessive
use of Dicumarol.
In normal
plasma
about
20% of the total thrombin
potential
might be in the form of prethrombin
(26).
When Vitamin
K1 was given to dogs being treated
with Dicumarol,
the thrombin
and autoprothrombin
C titers
began to rise promptly.
First,
relatively
more of the thrombin
precursor
was found
(25, 26).
Then, after 3 hr. the proportion
of thrombhi
to autoprothrombin
C
titer was the same as in the average
sample
of plasma
from dogs.
Evidently
the thrombin
and the autoprothrombin
C precursors
are
synthesized
independently
by tile liver and then are combined
in the
form of prothrombin.
Under conditions
of metabolic
turmoil,
they may
also appear
in the blood witilout
being combined.
The combination
is
evidently
a loose one and, besides
thrombin
and autoprothrombin
C,
includes accessory
protein which is added at an undetermined
time. The
combination
of the proenzymes
and accessory
protein
is so stable that
the aggregate
can be isolated
as a single component
if proper
methods
are used. In this connection
it is also of interest
that an antibody
to
purified
autoprothrombin
C forms
a precipitin
reaction
with plasma
prothrombin
and thereby
removes
most of the thrombin
precursor
of
plasma
(27).
Role of Platelets in Blood Clotting
Accurate
observations
frequently
reasserted
in the older, as well as
recent, literature
are that bleeding
is associated
with tllrombocytopenia,
that prothrombin
consumption
is poor in patelet-free
plasma,
and that
platelets
contain tile kind of lipids needed in blood coagulation.
Nevertheless,
Epstein
and Quick (28) found that platelet
materials
could be
infused
intravenously
with no resultant
gross disseminated
intravascular clotting
and with survival
of tile animals.
This is in contrast
to the
effect of tissue thromboplastin
which can be lethal because
it produces
autoprothrombin
C and supplies
the lipids needed
for the function
of
the enzyme. Purified
autoprothromhin
C was infused
intravenously
by
Marciniak
et a!. (29) and, again, no disseminated
intravascular
coagulation occurred,
and the animals
survived.
This is a most spectacular
experiment,
and it is also impressive
that no one seems to have taken
special notice of it.
Just reflect n the fact that the niost powerful
procoagulant
knowii
to date is not lethal when put into the blood stream.
Why noti It is
Vol. 14, No. 2, 1968
BLOOD CLOTTING
I1
ENZYMOLOGY
because
the lipids ordinarily
in the plasma
are not the right kind to
function
with autoprothrombin
C. We can eat whatever
we like and the
lipids,
as they ultimately
appear
in the blood, do not function
with
autoprothrombin
C. Those found in platelets
are special. However,
the
platelets
must first undergo
viscous metamorphosis
in order to create
conditions
for paired
functioning.
Platelet
homogenates,
purified
platelet
factor 3, or the lipids from platelets
were lethal when injected
intravenously
with autoprothrombin
C. It was the combination
which
was effective.
By reconsidering
the data given ill Fig. 5 and 7, we can see that tile
platelet
lipids are important
in at least two places. They function
with
platelet
cofactor
I in the production
of autoprothrombin
C, and Witil
autoprothromhin
C in the production
of thrombin.
However,
unless
the concentration
of platelet
materials
and platelet
cofactor
I is
sufficient,
the procoagulant
power does not gain
ascendelley
over
the
anticoagulant
resources
of blood. Areas of slow flow are conducive
to
platelet
clumping
amid to the release
of materials.
These are also the
natural
conditions
for platelet
lipids to operate
effectively
after platelet
viscous metamorphosis.
Here it is well to recall
that purified
prothrombin
converts
to a
derivative
and some thrombin
(hence also autoprothrombin
C) in the
PLATELET
#{149}
ADHESION
#{149}
COHESION
#{149}
ACGREGATION
Fig. 11. Sensitivity
of platelets
to stimuli.
#{149}
VISCOUS
METAMORPHOSIS
From
this
of accelerated
blood coagulation.
Platelet
lipids are needed
amid for enzyme function
of autoprothrombin
C.
point
of view
in formnatiomi
they
are
the
beginning
of autoprothrombis
C
14
EEGERS
fT AL.
Clinical Chemistry
presence
of calcium
ions and platelet
homogenates.
In other words,
platelets
alone are sufficient
to produce
a small amount
of thrombin
from purified
prothrombin.
This experimemit,
first recorded
by Seegers
(30) has been repeated
many times. Furthermore,
tile same result was
also obtained
with 1)Urified platelet
factor 3. All this points to the fact
that the platelets
are the point of beginning
for accelerated
blood
coagulation
when tissue thrombloplastin
is excluded.
There is nothing
in plasma that can substitute
for them. For this reason we do iiot believe
that anything
else can be nominated
and selected
as tile substance
which
initiates
blood coagulation.
Any such substance
would have to alter the
platelets
and ill that way be the prime mover by ili(hirect means.
Multiple
ways to alter platelets
is a dominant
theme that has appeared ifl tile literature
of the last decade;
Fig. 11 highlights
this fact.
The accuracy
of its implications
can he verified
by reviewing
the
presentations
found in the symposium
edited by Johnson
and Seegers
(31). Even Hageman
factor is effective
via platelets.
It does not produce disseminated
intravascular
coagulation
when infused
intravenously (32). In two widely publicized
hypotheses,
Hageman
factor was
assigned
the top and only position
in beginning
a cascade
of reactions
leading
to fibrin formation
(33, 34). It cannot
accurately
be assigned
this role. It is one of those
substances
that can influence
platelet
responses
and, as is well known, is not essential
for blood clotting.
The
widespread
interest
in platelets
seems to be a most fortunate
development. Their
participation
is critical
at two places
in blood clotting
which is one of the main reasons
for their importance
in hemostasis,
thrombosis,
and blood clotting
irregularities.
References
1.
2.
3.
4.
5.
6.
Selye, H., Throinbohemorrhagic
Phenomena,
Thomas,
Springfield,
Ill., 1966.
Hardaway,
R. M., Syndromes
of Disseminated
Intravascular
Coagulation,
Thomas,
Springfield, Ill., 1966.
McKay,
D. 0., Disseminated
Intravascular
Coagulation,
Hoeber,
New York, 1965.
Blomb#{228}ck,
B., “Fibrinogen
to Fibrin
Transformation.”
In Blood
Clotting
Enzyrnology,
Seegers,
W. H., Ed. Acad. Press, New York, 1967, Chapt.
4.
Lorand,
L., Physiological
roles of fibrinogen
and fibrin. Federation
Proc. 24, 784 (19651.
Seegers,
W. H., Activation
of purified
prothrombin.
Proc. Soc. Exptl.
Biol. Med. 72, 677
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7.
Glendening,
M. B., and Page, E. W., The site of inhibition
of blood clotting
by soy bean
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30, 1298 (1951).
8. Miller,
K. D., and Van Vunakis,
H., The effect of diisopropyl
fluorophosphate
on the
proteinase
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activities
of thrombin
and on prothrombin
and its activators.
J. Biol. Chem. 223, 227 (1956).
9. Seegers,
W. H., and Marcinialc,
E., Some activation
characteristics
of the prethrombin
subunit
of prothrombin.
Life Sci. 4, 1721 (1965).
10. Kipfer,
R., and Seegers,
W. H., Transformation
of autoprothromhin
III
to autoprothrombin
C in sodium citrate
solution.
Thromnb. Diath. Hasmorrhag.
In press.
11. Kline, D. L., Blood coagulation:
Reactions
leading
to prothrombin
activation.
Ann. Rev.
Physiol.
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12.
13.
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20.
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CLOTTING
ENZYMOLOGY
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Scegers,
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Cambridge,
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Harmuisomi, C. R., and Mammemm, E. F., “Molecular
Characteristics
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Active in
Blood Coagulation.”
In Blood Clotting
Enzymologjj,
Seegers,
w. H., Ed. Acad. Press,
New York, 1967, Chap. 2.
Marciniak,
E., and Secgers,
W. Ii., Prethrombimi
as a new subunit
of prothronmbin.
Nature
209, 621 (1966).
Baker,
W. J., and Seegers,
W. H., The conversion
of prethrombin
to thrombin.
Throinb.
Diath.
Haemorrhag.
17, 205 (1967).
Seegers,
W. H., Cole, E. R., and Aoki, N., Function
of Ac-globulin
and lipid in blood
clotting.
Can. J. Biochem.
Physiol.
41, 2441 (1963).
Ware, A. 0., and Seegers,
W. H., Studies
on prothrombin:
Purification,
inactivation
with
thrombimi,
and activation
with thrombopiastimi
and calcium.
J. Biol.
Chenm. 174, 565
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Seegers,
W. H., Cole, E. R., Harniisomm, C. R., and Marciniak,
E., Purification
amid some
properties
of autoprothromnbin
C. Can. J. Biochem.
Physiol.
41, 1047 (1963).
Hecht, E. R., Cho, M. H., amid Seegers,
W. H., Thromnboplastimm:
Nomenclature
and preparation of protein-free
niaterial
different
from platelet
factor
3 or lipid activator.
Am. J.
Physiol.
193, 584 (1958.
Bidweli,
E., Time purification
of bovine nmitihemnoplmilic globulin.
Brit. J. Haematol.
1, 35
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21.
22.
2.
24.
25.
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33.
34.
Hurt, J. P., Wagner,
R. H., and Brimmkhous, K. M., Humnan nntihemophilic
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(AHF)
purificatiomi:
A comparison
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