EFFECTS OF SEITZ FILTRATION ON PLASMA TRACE PROTEINS
CONCERNED WITH BLOOD COAGULATION
A QUANTITATIVE STUDY
COLIN FELL, M.S., J. FREDERIC JOHNSON, M.D., AND WALTER
H. SEEGERS, D.Sc.
Department of Physiology and Pharmacology, Wayne University College of Medicine,
Detroit, Michigan
In the study of the blood coagulation mechanisms the Seitz filter is a tool that
has been employed with increasing frequency. This device has been used especially in experiments involving the study of the accelerator of prothrombin
conversion, which among other designations has been called proconvertin, plasma
precursor of "serum prothrombin conversion accelerator" (SPCA precursor), and
Factor VII. Owren4, 5 has used the Seitz filter to produce what he believes to be
"proconvertin free plasma." He makes the claim that proconvertin is quantitatively removed from ox plasma by a filter pad containing 20 per cent asbestos,
while some of the plasma prothrombin is able to pass through. He also concludes
that prothrombin is completely removed by pads containing 50 per cent asbestos.
These results essentially have been confirmed by Koller,3 who states that filtration once through, a 20 per cent asbestos pad, followed by filtration through a
30 per cent asbestos pad, removes all Factor VII (proconvertin), from ox plasma,
while permitting 10 to 20 per cent of the prothrombin to remain in the filtrate.
Koller also agrees that prothrombin will be completely removed by pads containing 50 per cent asbestos.
In a study 6 of a new antithrombin reaction that can apparently be inhibited
by certain plasma factors and by platelet derivatives, we attempted to investigate
the effects of Seitz filtration on these and other substances. It soon became evident that the ability of Seitz filter pads to adsorb materials depended in part on
the amount of fluid filtered. It seemed that factors that were adsorbed when a
small amount of material was filtered reappeared in the filtrate if the volume of
fluid filtered was increased significantly. This is theoretically what might be expected, and makes the control of work with such filters difficult. We therefore
decided to investigate the relationship between the amount of plasma filtered
and the degree of adsorption of a number of factors associated with coagulation,
in order to determine more precisely the usefulness of Seitz filtration. It was
also hoped that this technic might furnish valuable starting material for the
preparations of certain plasma components already freed of other factors by
filtration.
Received for publication September 29, 1953.
This work was aided by a grant from the Michigan Heart Association.
Mr. Fell is Research Associate, Dr. Johnson is Instructor and Dr. Seegers is Professor of
Physiology and Chairman of the Department of Physiology and Pharmacology.
153
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FELL ET
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AL.
METHODS
Frozen bovine plasma, oxalated as previously described,7 was thawed and
centrifuged once in a Sharpless centrifuge at 5500 G, and again at 13,000 G. Two
liters of the clarified plasma were then passed through a Seitz filter with a pad
of 14 cm. diameter, containing 20 per cent asbestos. The plasma was filtered
under air pressure, and an attempt was made to keep the rate of flow constant;
however, as more material passed through the filter, the rate of flow was decreased somewhat by materials adhering to the pad (Table 1). These changes
in rate occur repeatedly. We believe that this variation cannot be avoided, and
that it is one of the important limitations affecting filtration-adsorption. The
filtration was carried out at room temperature (21 C ) . The first 100 ml. was
collected as a separate fraction, and then each subsequent 200 ml. was collected
separately, and at the end 100 ml. was collected. In this manner a number of
fractions were obtained that differed by the amount of plasma that had already
passed through the filter. Each fraction was placed in test tubes and snap-frozen
in a freezing mixture of dry ice and alcohol, and the samples were stored in the
deep freeze until they could be analyzed. The activity of a variety of factors
concerned with the clotting phenomenon was determined in several of the fractions described above.
Prothrombin. Prothrombin concentration was determined by the modified 2stage method of Ware and Seegers.13
Fibrinogen. Quantitative determinations of plasma fibrinogen were done by
the method of Ware, Guest and Seegers.11
Antithrombin-III. The activity of antithrombin-III (natural antithrombin)
was determined by a method described by Seegers and his associates.8
Ac-globulin. Plasma Ac-globulin activity was assayed by the method of Ware
and Seegers.12
TABLE 1
R A T E O F F L O W AND A I B P R E S S U R E A P P L I E D D U R I N G F I L T R A T I O N OP 2 L I T E R S
OF B O V I N E PLASMA
SAMPLE
Control
1
2
3
4
5
6
7
8
9
10
11
AMOUNT
RATE OF FLOW
AIR PRESSURE
ml.
ml./sec.
Ib./in*
—
—
—
1
1
1
0.5
0.5
1
1
1
0.25
0.15
0.15
3-10
10
10
10
10
25
25
25
25
25
25
First
Next
Next
Next
Next
Next
Next
Next
Next
Next
Last
100
200
200
200
200
200
200
200
200
200
100
FEB. 1954
PLASMA TRACE PROTEINS IN COAGULATION
155
SPCA precursor. The method of De Vries, Alexander and Goldstein1 was used,
except that BaSCU adsorbed bovine plasma and bovine lung-extract thromboplastin were used in place of human material.
Heparin co-factor. Controls consisted of determining clotting times of a mixture of 0.4 ml. of plasma, 0.1 ml. of saline and 0.1 ml. of purified thrombin (100
units per ml.). These clotting times were compared with those obtained with a
mixture of 0.4 ml. of plasma, 0.1 ml. of heparin (Liquaemin-Roche-Organon,
diluted J^oo with, saline), and 0.1 ml. of the same thrombin as that used in the
control experiment. In this manner an estimate of heparin co-factor activity was
made on the basis of the increase in clotting times between the control and the
experimental sample.
Thrombin titer method. This method was developed initially to study the activity of a new antithrombin reaction.6 The results are also influenced by prothrombin concentration, the activity of accelerators of prothrombin conversion
and perhaps other variables. In this method defibrinated plasma is ether-treated
to destroy antithrombin-III activity. 8 This plasma is then incubated with lungextract thromboplastin and calcium. At various time intervals aliquots are removed, and put in tubes containing standardized fibrinogen solution. 9 - u The
results are expressed by plotting clotting times of the various samples on the
ordinate against activation time on the abscissa, as shown in Figure 1.
Anlihemophilia factor. The assay method for this factor is described in a recent
paper.2 The method consists of incubating purified prothrombin with defibrinated, ether-treated plasma, calcium and a platelet preparation. At various time
intervals the concentration of thrombin in the activation mixture is determined
quantitatively by the method of Seegers and Smith. 9 Results are expressed
graphically.
EXPERIMENTAL PROCEDURE
The first graph in Figure 2 shows the relationship between prothrombin concentration and the amount of plasma filtered. The concentration of prothrombin
was considerably reduced in the first 100 ml. of filtrate. However, in subsequent
fractions the prothrombin concentration was progressively increased, and by the
time approximately 1 liter of plasma had been filtered, the concentration of prothrombin was again equal to its concentration in the plasma before filtration.
The results for fibrinogen (Fig. 2) seem to indicate a more rapid recovery pattern than that obtained for prothrombin. About two-thirds of the protein was
absent from the first 100 ml. of filtrate, but, in subsequent fractions, its concentration was found to be near the control level.
Antithrombin-III is not adsorbed to any significant extent (Fig. 2). The concentration of the factor in the first 100 ml. of filtrate was slightly reduced, but
it may be that the difference from the control value lies within the range of experimental error for the antithrombin determination. In subsequent fractions
the concentration of the material was almost identical with that in the control.
Seegers and his co-workers,8 incidentally, have reported that antithrombin-III
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FELL ET AL.
VOL. 24
100
10
20
ACTIVATION TIME IN MINUTES
FIG. 1. Results of thrombin titer test on fractions
of Seitz-filtered plasma. The lengthening of clotting
times indicates loss of thrombin activity referable to
antithrombin.
is not adsorbed on washed, shredded asbestos, nor on a variety of other adsorbing
agents.
The action of the Seitz filter on Ac-globulin (Fig. 2) seemed to us to be especially novel. The concentration of this factor was reduced by about a third in
the first 500 ml. of filtrate. However, in later fractions its concentration not
only returned to the control level, but increased far beyond this level. In the last
500 ml. of filtrate the Ac-globulin concentration returned to the original control
level. We were unable to understand these fluctuations in Ac-globulin concentration, and repeated the analyses several times in order to reassure ourselves
of their validity.
Results for SPCA-precursor concentration in the various fractions are also
shown in Figure 1. These are expressed in terms of "per cent enhancement" of
prothrombin activity; that is, the degree to which "prothrombin activity" is
increased beyond an average of the values for a test sample and a constant plasma
prothrombin source. We have a certain lack of confidence in these results. It
FEB. 1954
PLASMA TRACE PROTEINS IN COAGULATION
400 i
1
i 300?-
1
157
1
C0NTR L
°
2 4 6 8 10 12 14 16 18 20
ml_. PLASMA FILTERED, multiplied by 100
'Fic.2. Activity of prothrombin, fibrinogen, antithrombin-III, Ac-globulin and SPCA precursor in
fractions of Seitz-filtered plasma.
may be that variations in the curve are due to our lack of familiarity with the
technic. Nonetheless, it would seem that SPCA precursor is removed from the
first 100 ml. of filtrate, and is present in subsequent fractions. However, wo are
inclined to wonder just how specific the SPCA analysis is, since it is based in part
on a 1-stage prothrombin determination that presumably would be influenced
by variations in SPCA-precursor concentration in the test sample.
Results as unexpected as those found for Ac-globulin are obtained for heparin
co-factor activity (Table 2). Apparently the plasma co-factor of heparin is not
adsorbed. On the contrary, Seitz filtration seems to render plasma far more sensitive to the action of heparin. With a concentration of heparin sufficient to
change the clotting time of a normal plasma-thrombin mixture from 12 to 30
seconds, the filtered plasma could be regarded as incoagulable with the same
thrombin.
In the thrombin titer method (Fig. 1) the first fractions of filtered plasma
showed fully as much antithrombin-IV activity as later fractions. At the same
time, one finds that the amount of thrombin formed in the early fractions ap-
158
FELL ET
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AJ..
TABLE 2
H E P A R I N CO-FACTOR ACTIVITY IN FRACTIONS OF S E I T Z - F I L T E R E D P L A S M A , AS INDICATED
BY D I F F E R E N C E S B E T W E E N C L O T T I N G T I M E S OF SAMPLES W I T H AND
WITHOUT HEPARIN
SAMPLE
Control
1
2
3
4
5
6
11
CLOTTING TIME WITHOUT HEPARIN
CLOTTING TIME WITH HEPARIN
sec.
sec.
12
15
11
11
12
13
12
14
30
90
90
90
54
90
35
57
peared to be less than in the control plasma, and the rate of activation was markedly less rapid. Our interpretation is that antithrombin-IV is not adsorbed in the
Seitz filter, and that the reduced rates of activation associated with the lower
thrombin yields may be ascribed to a reduction in prothrombin concentration,
and perhaps a reduction in the concentration of SPCA precursor or other factors.
Previous work6 has shown that such changes in prothrombin and activator concentration will give results of this type in the thrombin titer test.
Our results with the antihemophilic factor assay did not permit us to draw any
definite conclusions. Indications were that the plasma fractions that had been
Seitz-filtered gave more activity than the control plasma. Such an observation
would not be too surprising, since it is known that an inhibitor of the antihemophilic factor exists (antithromboplastin), and that this inhibitor can be adsorbed
on asbestos.10 However, our results are not sufficiently unequivocal to permit us
to state that we have achieved this result.
DISCUSSION
The nature of the Seitz filtration set-up is such that it is difficult to understand
the relationship between the adsorbing agent and the material adsorbed. The
rate of flow will certainly affect this equilibrium, as well as the nature and amount
of material already adsorbed in the pad. It would seem, contrary to the suggestions made in some publications, that there is no such thing as an absolute or
complete adsorption of a particular factor, regardless of these variables.
For these reasons we find it difficult to evaluate work from other laboratories
that has been based on Seitz filtration. As indicated above, Owren4, 6 and Ivoller3
claim to have separated prothrombin and proconvertin by means of the Seitz
filter. While this may perhaps be true, we do not believe the data of these workers
adequate to establish their claim. In their publications they have not discussed
some of the variables with which we have concerned ourselves in this study. It
may be that the Seitz filter does offer a means of separation of prothrombin and
proconvertin. Despite the careful work of those authors, our view is simply that
FEB. 1954
PLASMA TRACE PROTEINS IN COAGULATION
159
we are as yet unable to evaluate such experiments on the basis of the existing
reports, because of the inherent complexity of the variables.
If it is desired to use asbestos as an adsorbing agent for proteins, it might be
best to stir a known amount of pure, shredded asbestos for a specified time under
specified conditions with the solvent from which the material is being removed.
The conditions are then at least to some extent controlled, and do not involve
the variable of filtration rate.
Filtration may well be an extremely useful tool in those instances where one
substance is adsorbed and another is not. For example, our results for Ac-globulin
suggest the possibility of separating it from prothrombin. Furthermore, the enhancement of Ac-globulin activity may be a distinct advantage, and suggests
the possibility that this increase may be associated with the removal of an inhibitor. Our results for heparin co-factor also suggest some interesting possibilities. The increased sensitivity of the plasma to heparin could also be based on
the removal of an inhibitor. While ideas such as these are as yet entirely speculative, they suggest promising areas for future work.
SUMMARY
Two liters of bovine plasma were filtered through a 20 per cent Seitz asbestos
pad of 14-cm. diameter. The first 100 ml. of filtrate and then each subsequent
200 ml. were collected as separate fractions and assayed for prothrombin, fibrinogen, antithrombin-III, Ac-globulin, SPCA precursor, heparin co-factor, antithrombin-IV and the antihemophilic factor.
The concentration of prothrombin and fibrinogen was markedly decreased in
the early fractions but rapidly returned to normal in subsequent fractions. SPCA
precursor seemed to adsorb in a manner similar to prothrombin and fibrinogen,
although the assay results were somewhat erratic. It is doubted that the Seitz
filter offers much opportunity for the satisfactory separation of prothrombin and
SPCA precursor. Antithrombins III and IV did not appear to be adsorbed to any
appreciable extent.
Ac-globulin and the heparin co-factor responded to Seitz filtration in an unexpected way. The concentrations of these factors in some of the fractions of filtered
plasma appeared to be considerably higher than their concentrations in unfiltered
plasma. The hypothesis is offered that these seeming increases in concentration
are associated with the removal of inhibitor material. The antihemophilic factor
apparently also increased in Seitz filtered plasma, but the results do not permit
the drawing of any definite conclusions.
It is also hoped that the seeming increases in concentration of Ac-globulin and
heparin co-factor in Seitz-filtered plasma will offer new approaches to work with
these factors.
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