Nature of the Blood Coagulation Mechanisms
in SPCA* Plasma
By WALTER H. SEEGEHS, P H J ) . AND NORMA ALKJAERSIG,
MS.
Derivatives of purified prothrombin and substances required for the formation of a derivative
have been studied in connection with SPCA deficient plasma. The deficiency can be corrected in
vitro with prothrombin derivatives. It is concluded that the SPCA patient cannot convert prothrombin to autoprothrombin and thrombin in the same way as a normal individual, and that
the precursor of SPCA is prothrombin.
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ALEXANDER and associates1 described
/ % a hitherto unrecognized hemorrhagic
-ZTTm. disease which involved the blood coagulation mechanisms in an unknown manner. The
hemorrhagic diathesis could be corrected with
the use of serum and serum fractions. They
ascribed the therapeutic value of serum to a
substance called serum prothrombin conversion
accelerator or SPCA. The bleeding tendency
has also been diagnosed by others2 and properties of the serum factor have been extensively
described.1"7 It has not been obtained in pure
form, and its origin has not been adequately
clarified. It has been postulated that it arises
from a precursor ordinarily found in plasma,
and that this precursor is a substance distinctly
different from but with many properties quite
like prothrombin. Thromboplastin and platelets and calcium ions are believed to play an
important role in the activation of the
precursor.
prothrombin, is prepared from prothrombin by
adding calcium ions, a purified preparation of
platelet factor 3, and a small amount of Acglobulin to the purified prothrombin. In such a
mixture the prothrombin activity disappears
rapidly. The preparation is then inert for it
cannot be converted to thrombin with any of
the known biologic materials commonly associated with the blood coagulation mechanisms.
Although this protein, derived from prothrombin, is inert with respect to its capacity to
transform to thrombin it possesses powerful
accelerator activity.11 Autoprothrombin accelerates the interaction of prothrombin, calcium ions, thromboplastin and serum Acglobulin, the latter substance being derived
from plasma Ac-globulin by means of thrombin.
Thus we may represent a small segment of the
blood coagulation mechanisms by the following
equation:
Plasma Ac-globulin
This laboratory has conducted an extensive
study on the purification of prothrombin, 8 ' •
and the prothrombin products obtained are uniformly found to be devoid of the serum prothrombin conversion accelerator.10 Recently it
has been possible to prepare derivatives of
prothrombin that possess powerful accelerator
activity."' 1 2 One of these derivatives, auto-
Thrombin
Prothrombin
Ca++
Thromboplastin
Serum Ac-globulin
Thrombin
Autoprothrombin
Ac-globulin
Platelet factor 3
From the Depurtment of Physiology and Pharmacology, Wayne University College of Medicine,
Detroit, Mich.
This investigation was supported by a research
grant H-2026 from the National Heart Institute,
National Institutes of Health, Public Health Service,
Bethescla, Md.
Received for publication May 9, 1955.
* SPCA-aerum prothrombin conversion accelerator.
In this paper we show that the slow activation of prothrombin associated with SPCA
plasma can be accelerated by autoprothrombin
and also by platelet factor 3. This suggests that
SPCA, the serum accelerator, is derived from
514
Circulation Restarck, Volume III, Srplember IMS
WALTER H. SEEGERS AND NORMA ALKJAERSIG
prothrombin. In the normal individual prothrombin Incomes partly converted to autoprothrombin and partly to thrombin. In the
SPCA patient prothrombin is probably not
readily converted to SPCA (autoprothrombin)
as in the normal individual.
PROCEDURES
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The materials employed in this work were all
prepared by methods previously described; namely,
bovine platelet concentrate,13 purified platelet factor
3,14 purified prothrombin,8'g autoprothrombin,11
citrate-autoprothrombin,11 and barium carbonate
eluate.13 The analytical methods used for the quantitative determination of prothrombin16 and Acglobulin14 have also been described previously. The
plasma from the SPCA patient was kindly supplied
by Benjamin Alexander and Robert Goldstein of
the Beth Israel Hospital, Boston. The plasma was
citrated, dried from the frozen state, and reconstituted to its original volume with distilled water just
prior to use in our experiments.
RESULTS
In the quantitative determination of Acglobulin concentration18 purified prothrombin is
used as a substrate. The quantity of thrombin
that forms and the rate at which it forms is
measured. Without Ac-globulin activation of
prothrombin is negligible under the conditions
of assay and a unit of Ac-globulin is denned in
terms of rigid specifications." In the work described below we made use of an observation
recorded by Johnson and Seegere.10 They found
that proconvertin deficient plasma (same as
SPCA plasma) gave an apparent Ac-globulin
concentration lower than 15 per cent of normal.
If a concentrate of the serum factor (BaCO3
eluate) was supplied in the analytical procedure
the Ac-globulin concentration was found to be
normal. Their interpretation of the data was
that the test indicates, in a semiquantitative
manner, whether SPCA is present or not, provided Ac-globulin is not lacking. Whether
SPCA is required to overcome an inhibitor
under the conditions of the analysis is not
known. In our work the observations of Johnson
and Seegers were confirmed by analysis of
plasma supplied by Alexander and Goldstein.
In the analytical procedure the apparent Acglobulin concentration was 1.8 units per ml.
(table 1), which is a much lower concentration
515
TABLE 1.—Ac-Olobulin Concentration in SPCA
Plasma under Varying Conditions of Analysis
Reagent Added
None
BaCOj ehmte
Autoprothrombin
Citrate-Autoprothrombiil
Platelet suspension
Purified platelet factor 3
UniU Ac-G/ml.
l.S
3
S.9
S.6
9.S
9.0*
9.7
* Small correction made for platelet accelerator
(platelet factor 1) activity.
than ever found for plasma from healthy
individuals.
BaCOj eluate is a preparation obtained from
serum and known to be rich in SPCA. It contains a small amount of Ac-globulin, but in the
concentrations used in our experiments this involved only a small correction factor. This
reagent was added to the SPCA plasma and the
analysis for Ac-globulin concentration was then
8.9 U/ml., which is a low value but often found
with normal individuals.17
Autoprothrombin was prepared from purified
prothrombin by adding calcium ions and a purified preparation of platelet factor 3. After autoprothrombin had formed to fullest extent the
platelet factor 3 was removed by sedimentation
in an ultracentrifuge. When the autoprothrombin was supplied in the analytical procedure for Ac-globulin the SPCA plasma was
found to contain 8.6 units of Ac-globulin. The
autoprothrombin is thus equivalent to BaCOj
eluate in this test. Both preparations fully develop the total Ac-globulin titre of the plasma.
Since citrate-autoprothrombin is closely related
to autoprothrombin in its accelerator properties
it was of importance to determine its effect on
SPCA plasma.
Citrate-autoprothrombin was prepared from
purified prothrombin by dissolving the latter in
25 per cent (w/v) sodium citrate solution. In a
short time the prothrombin molecule is degraded in this solution so that multiple components appear upon examination in the electro
phoresis apparatus or the ultracentrifuge.18-'0
Prothrombin itself is not soluble in 7 per cent
(w/v) trichloroacetic acid, but after a short
while in 25 per cent sodium citrate solution
large quantities of nitrogen containing material
516
COAGULATION MECHANISMS IX SPCA PLASMA
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become soluble. In due course, the solution
acquires thrombin activity, however, the development of this thrombin activity can be
inhibited by adding 3,4,4'-triaminodiphenyl
sulfone or purified soybean trypsin inhibitor.21
These blocking agents permit the citrate-autoprothrombin concentration to accumulate and
prevent its transformation to thrombin.
Citrate-autoprothrombin was prepared with
the use of 3,4,4'-triaminodiphenyl sulfone, and
was added to SPCA plasma. Then an analysis
for Ac-globulin was performed. The result found
was 9.9 units Ac-globulin per ml. plasma. This
value is comparable to that found with autoprothrombin and with BaCO3 eluate.
In the previous work of Johnson and
Seegers10 it was found that suspensions of disintegrated platelets could be added to SPCA
deficient plasma and that the apparent Acglobulin deficiency of the plasma was corrected.
We again performed that kind of experiment
and found 9.0 units of Ac-globulin with our
sample of plasma. Since it is specifically platelet
factor 3 which is needed to convert prothrombin
to autoprothrombin it was important to see
whether this factor could be used to develop
the Ac-globulin titre of the plasma. It was
found that this occurs with our purified preparation of platelet factor 3. The plasma Acglobulin concentration was found to be 9.7
units per ml. though the preparation of purified
platelet factor 3 itself does not contain any Acglobulin or autoprothrombin.
DISCUSSION
As in the work of Seegers and Johnson10 we
find that an analysis of SPCA plasma for Acglobulin gives low values and these are restored
to normal if platelets or BaCC>3 eluate are supplied. Furthermore, platelet factor 3, autoprothrombin or citrate-autoprothrombin serve
the same purpose. We believe that autoprothrombin, a substance derived from prothrombin, supplies the deficiency represented by
SPCA plasma. When platelets or platelet factor
3 are used to correct the deficiency in our Acglobulin analysis the autoprothrombin is
formed from the patient's own prothrombin by
means of platelet factor 3 as follows:
Ca++
Platelet factor 3
Ac-globulin
Prothrombin
Autoprothrombin
The data are consistent with the view that the
SPCA patient cannot convert prothrombin to
autoprothrombin in the same way as a normal
individual. The reasons for this inability are not
known.
Alexander,22 Owren,6 Roller, Loeliger and
Duckert6 and others found the accelerator
needed by the SPCA patient to be in normal
serum. They postulate a precursor in plasma
called proconvertin, pro-SPCA, etc. They have
made attempts to devise quantitative methods
for measuring the activity of this precursor and
have suggested mechanisms for its activation.
We do not believe that their viewpoints regarding the precursor can lie upheld despite the
extensive literature. We regard the postulated
precursor of the serum factor as prothrombin
itself. Their conclusions are actually extrapolations that reach a little far for critical
discernment. On the basis of the procedures
they employed their data can be given another
interpretation. It is interesting, for example,
that the methods of O\vren were so nonspecific and precarious that a complete transposition of the words "prothrombin" and "proconvertin" were made in a manuscript. A more
accurate correction would have been to say that
prothrombin and proconvertin are the same. It
is quite significant that no one has been able
to prepare a concentrate of the hypothetical
precursor and show that it is different from
prothrombin.
Owren24 has applied his methods for the
study of obstructive jaundice. The hypothetical
precursor of the accelerator is said to decrease
in concentration parallel with the decrease in
prothrombin concentration. This observation
corresponds with our view that prothrombin
and the precursor are one and the same substance. In this connection it is helpful to consider the experiments of Lasch and Roka26 who
were able to obtain prothrombin from a serum
precursor by means of liver mitochondria. It is
WALTER H. SEEGERS AND NORMA ALKJAERSIG
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thus possible to convert prothrombin to a derivative and to convert a derivative found in
serum to prothrombin.
Some authors6• 6• M• 26 believe that an activator of prothrombin decreases in concentration in plasma before prothrombin concentration drops appreciably when dicumarol is given.
Apparently this activator is supposed to be
identical with proconvertin, pro-SPCA, etc.
This does not necessarily follow from data thus
far presented. The facts are compatible with
the suggestion which we offer for the first time;
namely, that the accelerator deficient in dicumarol plasma is more than a deficiency in
prothrombin and its derivative autoprothrombin. The latter could hardly be found in dicumarol serum in normal concentration if prothrombin itself decreased in concentration.
Very likely, when more data become available,
it will be recognized that quite another accelerator that is not related to autoprothrombin
decreases in concentration in dicumarol plasma.
It is interesting, for example, that Johnson and
Seegers10 wrote: "Thus material adsorbed on
BaCOa and eluted with sodium citrate contains
very little if any Ac-globulin itself, but can
enable proconvertin deficient plasma to develop its full Ac-globulin titre. Platelet extracts develop a similar titre in proconvertin
deficient plasma. By contrast with these experiments our method for Ac-globulin analysis gives
normal values for dicumarol plasma without
addition of platelet extract and BaCO3 eluate
preparation. Nevertheless dicumarol plasma is
supposed to be deficient in proconvertin."
Further study of the properties of dicumarol
plasma are indicated from the newer knowledge
of the properties of prothrombin and its derivatives.
SUMMARY
When SPCA deficient plasma is analyzed for
its Ac-globulin content it apparently contains
only a small amount. The full titre can, however, be found if the analytic procedure is
modified by adding autoprothrombin, citrateautoprothrombin, platelets, platelet factor 3, or
a preparation containing SPCA. Since autoprothrombin is derived from prothrombin by
517
means of platelet factor 3, and since it can
supply the same activity as an SPCA concentrate from serum the two are regarded as the
same. The precursor of SPCA is considered to
be prothrombin and it is believed that the
SPCA patient cannot convert prothrombin to
autoprothrombin and thrombin in the same
way as a normal individual.
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Nature of the Blood Coagulation Mechanisms in SPCA Plasma
WALTER H. SEEGERS and NORMA ALKJAERSIG
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Circ Res. 1955;3:514-518
doi: 10.1161/01.RES.3.5.514
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