ACTIVATION OF P U R I F I E D PROTHROMBIN WITH
HEMOPHILIC PLASMA
SHIRLEY A. JOHNSON, PH.D.
Department of Physiology and Pharmacology, Wayne University College of Medicine,
Detroit, Michigan
The literature indicates that the blood coagulation mechanisms in hemophilia
probably show deviations concerned with thromboplastin activity. In extravascular clotting of shed blood, thromboplastin from the surrounding tissue is
added to the blood as it flows over the wound and undoubtedly contributes to
the acceleration of blood coagulation. The same type of thromboplastin can be
extracted from other tissues, especially the lungs, to give a powerful coagulant
which also enables hemophilic blood to clot fully as rapidly as normal blood. But
this tissue thromboplastin is evidently not indispensable for blood coagulation
to take place, since it is probably absent in the vascular system and still blood
clots within the vessels themselves. Moreover, we6 have observed that thromboplastin-like activity can arise after the interaction of platelet extract and a
plasma globulin. This activity, if not exactly equivalent to that of lung extract
thromboplastin, can substitute for such activity. For example, purified prothrombin is activated rapidly in combination with calcium, platelet-extract and lungextract thromboplastin. It is also activated rapidly in combination with calcium,
platelet extract and a partially purified globulin fraction of plasma. Evidently,
in the test tube, one combination of activators may substitute for the other. In
the first combination, lung-extract thromboplastin is the substance which can
be added to enable hemophilic blood to clot rapidly. In the second combination,
purified globulin fraction is the substance which may be especially concerned
with the bleeding tendency of the hemophiliac. It is probably this substance
that is commonly referred to by the terms "antihemophilic globulin," "antihemophilic factor" and "plasma euglobulin." In a recent study we used the term
platelet co-factor for this substance and described how it may be prepared in concentrated form. We also described some important details of its interaction with
platelets, calcium and purified prothrombin.
Brinkhous and his associates1"3 have been especially concerned with the study
of hemophilia from the viewpoint that it is related to a deficiency of this plasma
globulin. This concept has been accepted widely, because, in addition to the
experiments carried out by many investigators, the therapeutic effect of normal
plasma on the bleeding hemophiliac suggests that the normal plasma contributes
some important component to the hemophiliac.
Tocantins and his colleagues11' I2 have discovered a powerful inhibitor of
Received for publication April I, 1953.
This investigation was supported in part by a grant from the Medical Research and
Development Board, Office of the Surgeon General, Department of the Army, Contract
Number DA-49-007-MD-194, to Dr. Charles L. Schneider, Wayne University, Detroit.
Dr. Johnson is Research Associate.
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JOHNSON
blood coagulation. It is normally found in blood and has been called antithromboplastin. In applying this discovery to experiments with hemophilic blood the
view developed that antithromboplastin is present in greater quantity in the
plasma and other tissues of hemophiliacs than in normal individuals. Thus,
Tocantins does not believe that the hemophiliac lacks the platelet co-factor; and
he is, in fact, able to make concentrates of it from hemophilic plasma.
All these important observations have been concerned with hemophilia, but
thus far no one has been able to correlate the lack of antihemophilic globulin
with the idea of an excess of antithromboplastin. The experiments described
below are based on the use of purified prothrombin and enable us to propose
the theory that antihemophilic globulin is present in the hemophiliac, but its
activity is depressed by antithromboplastin. It can be shown that purified prothrombin is not activated as rapidly by a combination of calcium, platelet extract
and diluted hemophilic plasma as by a combination of calcium, platelet extract
and diluted normal plasma. However, when hemophilic plasma is thoroughly
extracted with ether it can be used in combination with calcium and platelet
extract to activate purified prothrombin fully as rapidly as normal plasma or
normal plasma extracted in the same way. Our interpretation is that ether removes the antithromboplastin from hemophilic plasma and thus permits the
platelet co-factor of plasma to manifest its full activity.
In another communication,6 it was shown that normal serum possessed the
same amount of platelet co-factor activity as hemophilic serum or plasma. On
the basis of that observation it was postulated that normal plasma contained 2
platelet co-factors, and that one of these disappeared or was masked when
blood clotted. Therefore, this one was not found in serum. We now find that
thorough ether extraction of normal serum restores the platelet co-factor activity
to the equivalent strength found in plasma.
MATERIALS AND METHODS
Purified prothrombin. Purified prothrombin was prepared by the method of
Seegers and his associates.7' 8> u
Platelet extract. As in previous work, a platelet extract was made from bovine
platelets. Platelets were obtained by differential centrifugation, and washed 3
times with physiologic saline solutions. One part of packed platelets was then
mixed with 9 parts of saline and frozen in a "deep freeze" cabinet. Then 10 ml.
of the suspension was thawed, and centrifuged at approximately 1500 G for 30
minutes in an angle head, refrigerator centrifuge to remove suspended material.
The sediment was recovered, washed once in physiologic saline and resuspended
in 2 ml. of saline. This resuspended sediment contains the activity described by
Ware, Fahey and Seegers13 as platelet-AcG activity.
Preparation of plasma samples. Both normal and hemophilic blood samples
were collected and prepared in exactly the same way.6 A clean venipuncture,
using a siliconed syringe, was made and the first syringe was replaced by another
after 2 ml. of blood was withdrawn and discarded. Then 10 ml. of blood was
placed immediately in a siliconed centrifuge tube containing 1 ml. of 0.112 M
ACTIVATION OF PURIFIED PROTHROMBIN
877
potassium oxalate. The two were mixed and centrifuged at 1500 G at 8 C. for 30
minutes. The plasma was defibrinated by adding an equal volume of purified
thrombin containing 20 units per ml. To destroy the antithrombin the preparation4 was then mixed with an equal volume of ethyl ether and shaken for several
minutes. This was separated and the ether extraction repeated 3 times. The
material was stored in small aliquots in a "deep freeze" cabinet.
Thrombin activity. These determinations were carried out by the method of
Seegers and Smith.10
Serum. Blood was collected and permitted to clot at room temperature. After
2 hours it was centrifuged at 1500 G for 30 minutes.
Assay oj platelet co-factor {plasma euglobulin). As shown'in previous work,6
when purified prothrombin was activated by a combination of calcium ions,
platelet extract and a crude platelet co-factor preparation, thrombin was formed
rapidly. There was always a latent period of thrombin production of a few minutes' duration, followed by a more rapid activation of prothrombin, and a full
yield was obtained in about 30 minutes. Limited quantities of activator produced limited quantities of thrombin and the amount and rate of formation of
the latter served as a quantitative measure of the amount- of co-factor present.
If, in the place of partially purified platelet co-factor, defibrinated ethertreated plasma was used, a similar activation of purified prothrombin resulted.
Thus the fundamental reaction could be applied to plasma samples for purposes
of assay.
The incubation tube in the assay contained:
a. Purified prothrombin (about 3000 units per ml.)—1.00 ml.
b. Platelet extract—0.50 ml.
c. CaCl2(0.153M) (in imidazole buffer)—0.50 ml.
d. The unknown (such as plasma, diluted plasma or serum)—1.00 ml.
This arrangement enabled us to measure how much and how rapidly thrombin
formed when a mixture of purified prothrombin, platelet extracts and calcium
ions was tested with unknown materials.
RESULTS
Normal plasma or normal plasma shaken with ether, when assayed, produced
thrombin at an equal rate. This can be seen in Figure 1, Curve A.
Hemophilic plasma extracted 3 times with ether, for 3^ minute each, produced
thrombin at a slower rate than normal plasma. This is shown by Curve B of
Figure 1. However, when hemophilic plasma was extracted with ether for 15
minutes, 3 times, thrombin was produced at a more rapid rate. Also, a full yield
was obtained sooner than if the ether treatment of the hemophilic plasma was
less extensive. Extraction of the hemophilic plasma with ether for 30 minutes, 3
times, 90 minutes in all, yielded a plasma that produced thrombin as rapidly as
normal plasma or, in fact, as rapidly as normal plasma similarly treated with
ether.
Normal serum was equivalent to hemophilic plasma, i.e., the result was. equal
to curve B of the text figure. Extraction of the serum with ether for 30 minutes,
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JOHNSON
3 times or 90 minutes in all, yielded serum that produced thrombin as rapidly as
normal plasma or ether-extracted hemophilic plasma.
DISCUSSION
In commenting on these experiments some details have to be considered. For
example, the prothrombin of hemophilic plasma and of normal plasma or serum
is diluted so much that it could be neglected in these experiments. By comparison
1400
A - Normal plasma shaken \vz, 45, 90min. _
B - Hemophilic plasma shaken l'/2 min.
»
CD-
It
"
II
"
40
60
80
100
Prothrombin activation (min.)
II
"
.
c
•
45 mm,
90 "
120
FIG. 1. Activation of purified prothrombin with combinations of platelet
extract and diluted plasma. The plasma samples were diluted 1:12 in the reaction mixture. Temperature 28 C. The substrate was purified bovine prothrombin, but purified human prothrombin also gave similar results.
this contribution represented less than 2 per cent of the total prothrombin furnished as purified prothrombin. The shaking of plasma with ether for at least 1J^
minutes destroyed the antithrombin of plasma. This was first observed qualitatively by Grunning,4 and studied in more detail by Seegers, Miller, Andrews and
Murphy. 9 In experiments described above, antithrombin was destroyed, and
we see that hemophilic plasma in combination with purified prothrombin, calcium ions and platelet extract did not yield thrombin as rapidly as when normal
plasma was used. However, the treatment of hemophilic plasma with ether gave
it the same capacity as normal plasma or normal plasma also treated with ether.
As a theory we propose that ether removes antithromboplastin from plasma
and thus enables the globulin substance to exhibit its full activity. This view
then regards the blood coagulation mechanisms in hemophilia as being different
ACTIVATION OF PURIFIED PROTHROMBIN
879
from the normal, because of a close and important connection between antithromboplastin and the plasma globulin. Perhaps the concentration of the
globulin material in hemophilia is the same as in normal plasma. I t may, however, be associated with more antithromboplastin than in normal plasma. Evidently other quantitative combinations of antithromboplastin and the platelet
co-factor are also possible. In any case, we believe that the hemophilic problem
is primarily concerned with the- implications of these combinations. We must
consider the possibility that the titer of serum is lower in platelet co-factor activity than plasma because some mechanisms tend to neutralize it during clotting.
This neutralization of activity involves that substance critically involved in
hemophilia. But serum and hemophilic plasma still possess a large quota of substance that can interact with platelets to give the equivalent of thromboplastin
activity. One wonders whether some bleeding tendency may not be discovered
involving that characteristic of serum.
SUMMARY
A combination of platelet extract, calcium and plasma activates purified prothrombin to thrombin. When normal plasma is replaced with hemophilic plasma
or normal serum, thrombin is produced at a reduced rate. Shaking of the hemophilic plasma or normal serum with ether, however, changes the plasma and
serum so that purified prothrombin is activated at the same rate as with normal
plasma.
Hemophilic plasma and normal serum, without prior ether-extraction, possess
the capacity to act with platelets to activate purified prothrombin. This may
mean that another plasma substance occurs, which is not the same as the one
counteracted by an inhibitor in hemophilia or in normal serum.
These experiments suggest that a plasma component called platelet co-factor
(antihemophilic globulin or euglobulin) may not be missing in hemophilia or in
serum but instead may be masked by an inhibitor such as antithromboplastin.
It is possible that the inhibitor, antithromboplastin, is extracted or destroyed by
ether, leaving the platelet co-factor free to act with platelet extract to convert
prothrombin to thrombin.
Acknowledgment. The author is indebted especially to Dr. Walter H. Seegers for his
helpful guidance. Appreciation is extended to Dr. Leandro M. Tocantins for his suggestions
and the facilities of his laboratory, to Miss Ruth Holburn for technical assistance and to
Dr. Julius Rutzky.
REFERENCES
1. BRINKHOUS, K. M.: Clotting defect in hemophilia; deficiency in a plasma factor required for platelet utilization. Proc. Soc. Exper. Biol. & Med., 66:117-120, 1947.
2. BRINKHOUS, K. M., GRAHAM, J. B., PENICK, G. D., AND LANGDELL, R. D.: Studies on
canine hemophilia. New York: Josiah Macy, Jr., Conference, 51-118, 1951.
3. GRAHAM, J. B., PENICK, G. D., AND BRINKHOUS, K. M.: Utilization of the antihemo-
philic factor during clotting of canine blood and plasma. Am. J. Phvsiol., 164: 710715, 1951.
4. GRUNNING, W.: Zur Frage der Lipoidnatur des Antithrombins. Naturwissenschaften,
31: 299, 1943.
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JOHNSON
6. JOHNSON, S. A., SMATHERS, W. M., AND SCHNEIDER, C. L.: Platelets and their plasma
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