STUDIES ON T H E THROMBOPLASTIN GENERATION TEST
II. BASIC MECHANISMS AND THEORETICAL ASPECTS
JOHN B. MIALE, M.D., AND MARJORIE P. WILSON, M.D.
WITH TECHNICAL ASSISTANCE FROM VIRGINIA R. GARRETT, M.T.
(ASCP)
Department ofPathology, University of Miami School of Medicine, and the Clinical Pathology
Laboratories, Jackson Memorial Hospital, Miami, Florida
Received, May 7, 1956; revision received, June 4; accepted for publication, June 19.
Dr. Miale is Professor of Pathology, University of Miami School of Medicine, and Director of Clinical Pathology, Jackson Memorial Hospital. Dr. Wilson is Resident in Clinical
Pathology, and Miss Garrett is Research Technician in Hematology.
Supported in part by a grant from Eli Lilly and Co.
984
Downloaded from http://ajcp.oxfordjournals.org/ by guest on March 5, 2016
In the preceding paper16 we described the method and discussed the applications of the thromboplastin generation test (TGT). In this paper we deal with
some theoretical aspects, and we present experimental data that bear on the
fundamental reactions involved in the evolution of measurable thromboplastic
activity.
The classic concept of blood coagulation is based on the assumption that
thromboplastin, in the presence of calcium ions, is active in the conversion of
prothrombin to thrombin. It has been suggested that in human hemostasis
thromboplastin is supplied to the coagulation system from the injured tissues.
However, if extreme precautions are taken to avoid contamination with tissue
juice while drawing blood, blood will still clot spontaneously when it is placed
in an untreated glass container. This suggests that an intrinsic mechanism exists
for the formation of thromboplastin derived only from components present in
normal blood. Therefore, in considering thromboplastic activity, a distinction
should be made between extrinsic thromboplastic activity, which is derived from
tissue extracts, and intrinsic thromboplastic activity, which is derived from
blood constituents.
Extrinsic thromboplastic activity is present in various tissue extracts such as
human and rabbit brain, human placental tissue, and beef lung. Russell viper
venom and trypsin also exhibit thromboplastic activity. Experience with these
substances indicates that, although each has thromboplastic activity, it is probable that they differ from each other. For example, although different thromboplastic substances may lead to identical "prothrombin times" when added to
normal recalcified plasma, in abnormal plasma (such as that from a patient receiving dicumarol) the results are variable, and measurements made with one
substance may have no relation to those made with another.
The conversion of prothrombin to thrombin can be effected by tissue extracts
plus calcium, but the reaction can be markedly accelerated if plasma or serum is
added. This has been interpreted to mean that plasma and serum contain accelerator substances (designated Factors V and VII in our outline).15 Factor V
has been adequately studied and has been isolated in moderately pure form from
Sept. 1956
THROMBOPLASTIN GENERATION TEST. II '
985
In addition to plasma and serum factors, platelets are necessary for the generation of intrinsic thromboplastic activity. Platelet derivatives have various effects,
and have been tentatively classified on the basis of these effects. Platelet factor
1 has accelerator properties similar to those of Factor V,27 and actually may be
nothing more than adsorbed Factor V.10 Platelet factor 2 is involved in the reaction between thrombin and fibrinogen.27 Platelet factor 3 26 is necessary for intrinsic thromboplastic activity, and is probably the active platelet substance involved in the in vitro test. Platelet factor 4 has anti-heparin activity.27 Platelet
factor 3 reacts with Factor VIII, and probably also with Factors IX and X, to
produce thromboplastic activity which is maximal in the presence of the plasma
and serum accelerators.
The proposed basis for the studies on the generation of plasma thromboplastic
activity is the identification of such activity by adding the substance in question
to recalcified plasma. Under normal conditions the addition of calcium chloride
alone to citrated plasma will produce clotting in 3 to 5 minutes. It is thought
that, when calcium is restored, all the elements required for thrombin formation
are present, and the time required for the evolution of thrombin is an expression
of time-consuming reactions: first, the development of plasma thromboplastin;
secondly, the conversion of prothrombin to thrombin. Therefore, when citrated
plasma is recalcified, the development of thrombin can be followed by transferring
aliquots at 1-minute intervals to tubes containing a solution of fibrinogen and
noting the clotting times. This has been referred to as the "thrombin generation
test." A reduction in the amount of generated thrombin indicates that an abnormality is present but, since thrombin is the end result of a series of reactions, the
defect cannot be localized. On the other hand, if prothrombin is absent, so that
thrombin cannot be formed, it is possible to study reactions prior to conversion
of prothrombin. Biggs and Macfarlane7 report a case of idiopathic prothrombin
deficiency in which the level of prothrombin was 11 per cent of normal. In this
patient thrombin generation was much diminished, and they concluded that it
Downloaded from http://ajcp.oxfordjournals.org/ by guest on March 5, 2016
bovine plasma. 17 ' 29 It is converted by the action of thrombin, freezing and thawing, and during spontaneous blood coagulation to a more active prothrombin
conversion accelerator,28 Factor VI. Some investigators' • l8 believe that a second
system of plasma-serum accelerators exists, Factor VII being present in serum
and derived from pro-Factor VII in the plasma. Recently Seegers and associateshave claimed that Factor VII is a derivative of prothrombin (autoprothrombin)
rather than a special plasma precursor, and suggest that the deficiencies thought
to be due to a lack of Factor VII are deficiencies in conversion of prothrombin to
autoprothrombin. 20 The concept of "autoprothrombin" is derived from observations that, in vitro, purified prothrombin forms derivatives that are not acted
on by any of the known activators of prothrombin, but that are themselves
activators of prothrombin. 12 ' 21 The observations that the thromboplastin generation, test (TGT) is abnormal in cases of Factor V or VII deficiencies have been
interpreted as showing that Factors V and VII are concerned with the development of intrinsic thromboplastic activity. 6, 6 Although these observations are
valid they are subject to other interpretations (see discussion).
986
MIALE AND WILSON
VoL 26
was then possible to study the development of thromboplastic activity in recalcified plasma with relatively little interference by thrombin formation. These
conditions are reproduced in the TGT.
The value of the TGT in the laboratory diagnosis of hemorrhagic disease has
been amply documented. 4 ' 9 ' 15 The fundamental reactions which the test
measures are, however, still a matter of speculation and controversy. The following experiments were performed in an attempt to study some of the basic reactions. The methods used are given in detail in the preceding paper.15
E F F E C T OF D I L U T I O N OF S E R U M
E F F E C T O F PLASMA
DILUTION
As with serum, marked dilution of the alumina plasma can be achieved before
abnormal results are obtained (Fig. 2). When 1:10 plasma is substituted, its
activity is normal and the results are within the normal limits for 1:5 plasma.
Plasma diluted 1:60 represents a final plasma concentration of 0.417 per cent in
the incubation mixture and this again represents a rather remarkable activity of
the Factors V and VIII which are supposedly present. Furthermore, platelet
suspensions exhibit Factor V activity, even when washed 10 times.27 This activity amounts to about 6 per cent of plasma.10 With regard to Factor VIII, a 10
per cent concentration of normal plasma fails to correct completely the defective
thrombin generation in hemophilic blood, and a concentration of 0.2 per cent of
normal plasma is completely ineffective.7 In view of this, it is difficult to believe
Downloaded from http://ajcp.oxfordjournals.org/ by guest on March 5, 2016
In the method outlined by Biggs and Douglas,4 a serum dilution of 1:10 is
specified. This is equivalent to a final serum concentration of 2.5 per cent in the
incubation mixture in which thromboplastin is generated. A final serum dilution
of 2.5 per cent seems to represent fairly minute amounts of Factors VII, IX,
and X. If the serum is diluted so that it is added in dilutions of 1:5, 1:10, 1:20,
1:40, and 1:60 (Fig. 1), it will be noted that thromboplastin generation is as
efficient with a 1:20 dilution (final concentration 1.25 per cent), and slightly impaired with a 1:40 dilution (final concentration 0.625 per cent). The striking
degree of activity shown by these high dilutions is not in line with observations
of deficiency states, where relatively large amounts of the factors are required
in order to correct the defect completely in vitro and in vivo. It has been suggested
that many phenomena attributed to "factors" are actually the result of imbalances in activator-anticoagulant systems. Since, with normal blood, progressive
dilution of the fractions leads to diminished activity, it is difficult to relate thromboplastic activity to progressive dilution of anticoagulants under the conditions
of this test. One interesting observation is that, although progressive serum
dilution leads to little loss in thromboplastin generation, more time is required
for maximal activity to develop as higher dilutions are used. Figure 1 also illustrates the results when the test is carried out by substituting saline for serum.
The recorded activity is minimal if it is referred to the thromboplastin dilution
curve but, considering that one important fraction is absent, a clotting time of
about 2 minutes appears to be significant.
Sept.
THROMBOPLASTIN GENERATION7 TEST. II
1956
OF INCUBATION
MINUTES
OF INCUBATION
F I G . 2. T h e effect of various dilutions
of plasma on the thromboplastin generation test.
that Factor VIII is present in the incubation mixture in adequate amounts. I t is
possible that the test is a more sensitive index of the activity of Factor VIII than
the others, but even if this is so it would be necessary to assume that in hemophilia
the deficiency of Factor VIII lies somewhere between 0.4 per cent and 0—a conclusion not in line with other quantitative determinations of Factor VIII.
When saline is substituted for plasma so that no plasma is present (Fig. 2), the
clotting activity is much greater than for the comparable experiment where serum
is excluded (Fig. 1). If Factor VIII per se is the chief limiting factor in thromboplastin formation this observation is difficult to explain.
EFFECT OF CONCENTRATION OF PLATELETS
In performing the standard test, we adjust the concentration of the stock suspension of platelets at approximately 1 million per cu. mm., so that the concentration in the incubation mixture is normally about 250,000 per cu. mm. If the
concentration is serially reduced, the effect of platelet concentration on thromboplastin generation may be determined (Fig. 3). In several experiments we found
that the critical concentration in the incubation mixture is in the range of 62,000
platelets per cu. mm. Below this level thromboplastin generation is very poor.
Thromboplastin generation is poor if saline is substituted for platelet suspension,
but still this does not represent a platelet-free system, inasmuch as the alumina
plasma (as prepared) contains approximately 3000 platelets per cu. mm., and
the serum approximately 2500 per cu. mm. (direct counts under phase microscopy). The critical level of platelets in the TGT agrees closely with critical levels
in purpura, but not with the reported relationship between prothrombin con-
Downloaded from http://ajcp.oxfordjournals.org/ by guest on March 5, 2016
MINUTES
F I G . 1. T h e effect of various dilutions
of serum on the thromboplastin generation test.
987
988
MIALE AND WILSON
Vol. 26
EFFECT OF ANTICOAGULANTS
Blood normally contains active anticoagulants that have antithrombic and
antithromboplastic activity. Occasionally one encounters a hemorrhagic diathesis that is the result of an abnormally high concentration of anticoagulants. When
this occurs, the TGT is an excellent method of demonstrating the abnormality.
As expected, the greatest abnormality in thromboplastin generation is produced by the presence of abnormal amounts of antithromboplastin. Indeed, the
presence of such an anticoagulant in plasma or serum may at first glance be
interpreted as a demonstration that one of the plasma or serum factors is deficient. The presence of an anticoagulant may be demonstrated by repeating the
test using normal reagents with the addition to the system of serial dilutions of
plasma or serum. If an anticoagulant is present, thromboplastin generation will
be inhibited by a low dilution and will approach normal with high dilutions.4 • 9 • 15
After detection of the presence of an anticoagulant in the TGT, specific tests may
be done to identify the anticoagulant as an antithrombin or an antithromboplastin.
The presence of large amounts of heparin-like inhibitors will also reduce the
amount of thromboplastin generated. As illustrated in Figure 4, if heparin is
added to freshly drawn blood in various concentrations, no change occurs if the
concentration of heparin is not over 2 units per ml. of blood. This amount prolongs the clotting time to 45 minutes. However, at a concentration of heparin of
3 units per ml. (clotting time 2 hours and 15 minutes), only 20 per cent generation
of thromboplastin is produced, and its generation is delayed. At a level of 3 units
per ml. in the original blood 0.0075 units of heparin is transferred to the substrate
plasma when the test is performed, and the final concentration is 0.0075 heparin
units in 0.3 ml, or 0.025 units per ml. This amount of heparin, when added to the
substrate plasma, is not sufficient to affect the normal clotting times obtained
with a normal system.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on March 5, 2016
sumption and platelets, in which abnormal consumption occurs with only
moderate platelet reduction.
The TGT does not shed any further light on the role of platelets in blood coagulation, although possibly it provides a method for the study of platelets and
platelet factors. According to Seegers and associates,2 prothrombin reacts with
platelet factor 3, calcium ions, and Factor V to form an accelerator. Most workers
accept the concept that platelets react with Factor VIII in the generation of
thromboplastin. The observation that crude cephalin (extracted from an emulsion
of brain) may be substituted for platelets in the TGT 3 does not necessarily mean
that platelet phospholipid and crude cephalin from brain can be substituted for
one another in all environments. Nevertheless, the observation may prove to be
a significant contribution to the understanding of platelet function. From the
practical standpoint we do not feel at this time that there is anything to be
gained by substituting crude cephalin for platelets in the routine investigation of
a hemorrhagic diathesis by means of the TGT. A platelet suspension is simple to
prepare, and the use of crude cephalin introduces another substance whose
mechanism of action has not yet been completely studied.
Sept. 1956
THROMBOPLASTIN GENERATION TEST. II
989
iaoi
160
|:I0 serial dil'ns used
Vi alumina plasma dilhs used
140
A-|:IOsenim-hi
AI0H| plasm)
saline substituted
ftr platelets
B- 4,000 platelets
C- 9.000
•
D-16,000
"
E -68,500
F-125,000
G-250,000
'
H-500.000
1-1,000,000 '
120
100
<o
a
o
80
to
60
40
Downloaded from http://ajcp.oxfordjournals.org/ by guest on March 5, 2016
20
1
2
3
4
5
6
MINUTES OF INCUBATION
FIG. 3. The effect on the thromboplastin generation test of serial reduction in
the number of platelets in the incubation
mixture.
90
ao
70 -
in
60
o
111
<n
40 30 20 10 -
—r—I
1 2
MINUTES
1
r
T
3
4
5
OF INCUBATION
1
6
FIG. 4. Effect of increasing amounts of heparin in
vitro on the thromboplastin generation test.
ACTIVATING E F F E C T O F T H R O M B I N ON T H R O M B O P L A S T I N
GENERATION
If aliquots of the incubation mixture are transferred into a fibrinogen solution
and into the substrate plasma at the same time, it is possible to measure simultaneously the degrees of thromboplastic and of thrombic activity of the incuba-
990
MIALE AND WILSON
Vol. 26
When the basic incubation mixture containing added thrombin is preincubated
for 15 minutes and 30 minutes, thromboplastin generation is poor as compared
to the control values. It is presumed that loss or neutralization of thrombin takes
place on prolonged incubation, inasmuch as the 15-minute and 30-minute mixtures contain less thrombin than the fresh mixture.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on March 5, 2016
tion mixture. The results of one such experiment are shown in Figure 5. The
amount of thrombin formed is small, equivalent to 0.4-0.7 units per ml. according to a previously determined thrombin-fibrinogen curve. I t must be supposed
that this thrombin is derived from prothrombin remaining in the serum and in
the plasma after adsorption. It is claimed by Biggs and co-workers that this
amount of thrombin is insignificant. I t is thought that thrombin acts as a catalyst
in almost all of the blood coagulation reactions, and it seems possible that even
this small amount of thrombin could have a significant effect on the results. Accordingly, the following experiments were performed:
1. Effect of addition of thrombin to the incubation mixture. Crude thrombin
(Parke-Davis) was added to the incubation mixture in a final concentration of
0.25 units per ml. of the incubation mixture after recalcification. The total
volume of incubation mixture was the same as for all other studies (0.9 ml.).
Three identical incubation mixtures containing thrombin were used for TGTs.
The first was tested immediately after thrombin was added, the second after
incubation for 15 minutes at 37 C , and the third after incubation for 30 minutes
at 37 C. Incubation was carried out prior to recalcification.
The results are illustrated in Figure 6. When the mixture was tested immediately after thrombin was added, a marked acceleration of clotting time was
noted. Most notable was the elimination of the usual lag indicating that at the
stated concentration of thrombin a marked acceleration of thromboplastin
generation is produced. The short clotting times that are obtained are not the
results of a direct thrombin effect, inasmuch as the amount of thrombin transferred to the substrate plasma (0.025 units in 0.1 ml.) plus spontaneously generated thrombin (about 0.04 to 0.07 units in 0.01 ml.) can not account for a clotting time of 10 seconds. It is obvious, however, that relatively small amounts of
thrombin exert a striking influence on the results obtained in the TGT. When
this finding is considered in the light of the heparin experiments (Fig. 4), it is
reasonable to suggest that heparin retards thromboplastin generation by inhibiting the action of thrombin, as well as by an antithromboplastic action, as previously proposed,13 This same study demonstrated that the serum of patients who
are receiving heparin contains unutilized Factor VIII, a finding interpreted as an
indication of poor formation of thromboplastin. It is not possible at this time to
say which of the two mechanisms is more important. We feel justified in emphasizing the activating role of thrombin in all blood coagulation reactions. We have
found that when thromboplastin generation is poor, as in one of the various
plasma or serum defects, no thrombin is generated in the incubation mixture. I t
is known that the thrombin generation test in these deficiency states is abnormal.
Both of these defects may account for apparently poor thromboplastin generation, and it is difficult to distinguish one effect from the other.
4n
THROMBIN
• FORMATION
3-
2-
NORMAL
THROMBOPLASTIN
GENERATION
T
OF I N C U B A T I O N
70
60
50-
40
A-
U
5
0 MIN INCUBATION WITH THROMBIN
Q - 15 MIN INCUBATION WITH THROMBIN
30-
C - 3 0 MIN INCUBATION WITH THROMBIN
20
10
MINUTES OF INCUBATION
F I G . 6. Effect of thrombin added to the incubation mixture in the thromboplastin generation
test. Thrombin added to the incubation mixture
to make a concentration of 0.25 thrombin units per
ml. after recalcification. This mixture was tested
a t 0 time (A), and after incubation at 37 C. for 15
minutes (B), and for 30 minutes (C).
991
Downloaded from http://ajcp.oxfordjournals.org/ by guest on March 5, 2016
MINUTES
F I G . 5. T h e amount of thrombin formed at various
times during the thromboplastin generation test.
992
MIALE AND WILSON
Vol. 26
DISCUSSION
Most of our concepts of blood coagulation are based on inferences drawn from
experimental observations, and the validity of the inferences is limited by the
lack of purity of the reagents. I t is possible to describe the activity of thromboplastin by defining it as the substance that is responsible for the rapid conversion
of prothrombin to thrombin in the presence of calcium ions. With some possible
exceptions, 8,23 a single substance fulfilling these criteria has not been isolated;
therefore it is difficult to be certain that in the TGT such a substance is "generated," or what the precursors may be. It does seem justified to refer to the
generation of thromboplastic activity, and this more guarded wording then
permits some conclusions that are less open to question.
It is agreed that thromboplastic activity of considerable magnitude develops in
a system composed of normal platelets, normal citrated plasma from which most
Downloaded from http://ajcp.oxfordjournals.org/ by guest on March 5, 2016
2. Effect of prmicvbation of alumina •plasma with thrombin. In order to test
further the suggestion that thrombin is required for the reaction to occur at normal speed, the above experiment was repeated by preincubating alumina plasma
with thrombin, before adding it to the serum and platelet reagents. Again a striking accelerating effect was produced in thromboplastin generation (Fig. 7). The
lag phase was eliminated, and a flat curve was obtained. The effect was rapid,
and it disappeared on longer incubation; as in the experiment above, longer incubation resulted in abnormally long clotting times. In this instance the accelerating
effect of thrombin may be dependent on the activation of Factor V in the plasma,
although there is evidence to show that thrombin is also capable of activating
Factor VIII. 19
8. Effect of preincubation of serum with thrombin. The experiment with serum
was performed as outlined above for plasma. The results (Fig. 8) indicate again
a marked accelerating effect of thrombin, this time more sustained, so that, even
after 30 minutes of incubation, the activity is better than the control. I t is
perhaps not justified to draw too rigid conclusions from this experiment, but
previous observations24 justify the suggestion that thrombin is responsible for
some activation of serum factors, as well as of plasma factors.
4- Effect of preincubation of platelets with thrombin. When set up in the same
way as for the plasma and serum fractions, the presence of thrombin has a
striking accelerating effect when added to the platelet suspension (Fig. 9). The
"labilizing" effect of thrombin on platelets is a generally accepted concept,24 and
it is thought that the results may be interpreted in this light. One other consideration should be mentioned. Platelet suspensions exhibit Factor V activity regardless of repeated washings, and thrombin converts Factor V to the active
serum factor. In discussing the increased activity of platelet suspensions after
exposure to thrombin, it is necessary to consider that thrombin is a powerful
activator for Factor V as well, a consideration discussed in relation to the activating effect of thrombin on alumina plasma. We have not had an opportunity
to study a pure deficiency of Factor V, but such a study might clarify some of the
questions raised.
50
A-CONTROL
B - 0 MIN INCUBATION WITH THROMBIN
C * 15 MIN INCUBATION WITH THROMBIN
40
D * 3 0 MIN INCUBATION WITH THROMBIN
30-
3 20
10'
2
3
4
5
6
MINUTES OF INCUBATION
FIG. 7. Effect of preincubation of alumina
plasma with thrombin in the thromboplastin
generation test. A 1:5 dilution of plasma was made
in a solution of thrombin; final concentration of
0.25 units of thrombin per ml. after recalcification
of the final incubation mixture. Plasma was tested
in the thromboplastin generation test immediately
after the thrombin was added after incubation at
37 C. for 15 minutes, and for 30 minutes.
50-
A,
A'CONTROL
B - 0 MIN INCUBATION WITH THROMBIN
Q
C-19 MIN INCUBATION WITH THROMBIN
g40u
D - 3 0 MIN INCUBATION WITH THROMBIN
UJ
z
UJ
2 30
P
20-
\
10-
B
^-—^
B-OSv^
D—~-S
I
2
3
4
MINUTES OF INCUBATION
5
6
Fro. 8. Effect of preincubation of serum with
thrombin in the thromboplastin generation
test. A 1:10 dilution of serum was made in a
solution of thrombin; final concentration of 0.25
thrombin units per ml. after recalcification of
the final incubation mixture. Serum was tested
in the thromboplastin generation test immediately after the thrombin was added, and after
15 minutes and 30 minutes of incubation at
37 C.
993
Downloaded from http://ajcp.oxfordjournals.org/ by guest on March 5, 2016
1
994
MIALE AND WILSON
Vol. 26
The question of the measurement of activity of a specific factor by the TGT
is also raised. For example, it is thought that platelet factor 3 is the platelet
component involved in the development of thromboplastic activity. 26 Purified
platelet factor 3 may be substituted for platelets in the test, but so may phospholipid from brain.3 We have demonstrated that preincubation of platelets with
thrombin eliminates the lag phase (Fig. 9), and feel that this phenomenon may
result from accelerated platelet lysis or from activation of the Factor V-like
activity of platelets. We have recently demonstrated that preincubation of brain
phospholipid with thrombin also eliminates the lag phase in the TGT, 14 and
Downloaded from http://ajcp.oxfordjournals.org/ by guest on March 5, 2016
of the prothrombin has been adsorbed by aluminum hydroxide, and normal
serum. The activity found in apparently normal individuals does show some
variation in rate of development,15 but, when maximally developed, it is uniformly high. I t must also be accepted that decreased activity results when the
TGT is done with blood that is abnormal. The diagnostic value of the test is
based on this observation, i.e., whenever the platelets, plasma, or serum yield
less than normal activity, the coagulation defect may be localized in one of these
fractions of blood.
We realize that a point of logic must be raised when presenting the results of
the test in various deficiency states, for the validity of these demonstrations depends on the correctness of several assumptions: (1) that the various blood coagulation factors are definite chemical and physiologic entities; (2) that the test is
able to measure in a specific manner the function of these factors; (3) that the
results of the test are due to the specific elaboration of thromboplastin, and that
this comes about from a predictable interaction of specific factors; and (4) that
the results are not modified by nonspecific reactions occurring in the course of
the test.
The answer to the first point is of course basic, but it is not available at present. Correlation between clinical and laboratory data suggests that abnormal
coagulation results from deficient function of certain blood fractions; where these
have been prepared in fairly pure form (such as prothrombin and Factor V)
their inclusion into the basic mechanism of blood coagulation is justified. When
hypothetical precursors of combinations are included, it must be considered a
matter of hypothesis, and this should not be used to explain the results obtained
in a test such as the TGT. Biggs and associates 5,6 believe that the blood components necessary for the generation of intrinsic thromboplastin are platelets,
Factors V and VIII in the plasma, and Factors VII and I X in the serum. They
state 4 that "these five factors react together in the presence of CaCl2 to form a
labile thromboplastin." Since then at least one other factor has been described—
•Factor X—which is thought to be similar in activity to Factors VIII and IX.
If this is so, this factor must also enter into the generation of thromboplastin.
The literature abounds with speculation on the interactions among these factors
and a review is beyond the scope of this paper. We feel that no analysis of the
TGT can contribute much to this basic problem, nor can the various hypotheses be
used to prove the reactions involved in the development of thromboplastic
activity.
Sept.
1956
THROMBOPLASTIN GENERATION' TEST. II
995
50
0 40UJ
to
z
UJ
130
20
10
2
3
4
MINUTES OF INCUBATION
5
6
F I G . 9. Effect of preincubation of platelets with
thrombin in the thromboplastin generation test.
A suspension of platelets, 1 million per cu. mm.,
was packed by centrifugation and resuspended in
a solution of thrombin; final concentration 0.25
thrombin units per ml. after recalcification of the
final incubation mixture. This platelet suspension
was tested in the thromboplastin generation test
immediately after the thrombin was added, and
after incubation a t 37 C. for 15, and for 30 minutes.
these findings pose the question of how specific the test is for a measurement of
the activity of platelet factor 3. The question of specific activity can also be
raised with regard to other factors. Factor VIII is thought to react with platelet
factor 3,16 and these two, together with calcium ions and Factor V, rapidly
convert prothrombin to thrombin. 2 The reaction is accelerated by small amounts
of thrombin,16 which may act by activating either Factor VIII 19 or Factor V.28
Since small amounts of thrombin are formed in the TGT (Fig. 7), the final result
may not be a specific measure of the activity of any one of these factors, but may
reflect the action of thrombin on platelets, platelet factor 3, Factor V, and Factor VIII, singly or in various combinations. Furthermore, although the TGT is
abnormal when the serum from cases of Factor IX and X deficiencies is used, the
manner in which these factors enter into the formation of thromboplastic activity
is not known. As far as Factor VII is concerned, it has been suggested that the
activity attributed to this factor may result from the action of prothrombin
derivatives,20 so that an abnormal TGT in Factor VII deficiencies may measure
a basic defect in the conversion of prothrombin to "autoprothrombin" and thrombin. Finally, it has been suggested that, although there is no activity of Factor
VIII in serum, this may not be the result of an absence of the factor (in the
sense that it has been utilized to form something else), but rather the result of
its combination with an inhibitor.26 The accelerating'effects of dilution on the
Downloaded from http://ajcp.oxfordjournals.org/ by guest on March 5, 2016
1
996
MIALE AND WILSON
Vol. 26
SUMMARY
1. Data are presented which indicate that the reactions involved in the TGT
are complex and poorly understood.
2. Our studies confirm the concept that thrombin plays an important role in
all phases of blood coagulation. Thrombin generated during the test may affect
the apparent generation of a thromboplastic substance by influencing related
reactions.
SUMMARIO IN INTERLINGUA
1. Datos es presentate que indica que le reactiones del test de generation de
thromboplastina es complexe e mal comprendite.
2. Nostre studios confirma le conception que thrombina ha un rolo significative in omne phases del coagulation de sanguine. Thrombina generate in le curso
del test affice possibilemente le apparente generation de substantia thromboplastic per influentiar reactiones affin.
REFERENCES
1. A L E X A N D E R , B . , G O L D S T E I N . R . , AND L A N D W E H R , G . : T h e p r o t h r o m b i n conversion a c -
celerator of serum (SPCA): its partial purification and its properties compared with
serum Ac-globulin. J . Clin. Invest., 29: 881-895, 1950.
2. ALKJAERSIG, N . , AND SEEGERS, W. H . : Activation of purified prothrombin in association
with preparations of platelet factor 3, platelet cofactor I and Ac-globulin. Am. J .
Physiol., 182: 347-350, 1955.
3. B E L L , W. N . , AND ALTON, H . G.: A brain extract as a substitute for platelet suspensions
in t h e thromboplastin generation test. N a t u r e , London, 174: 880-881, 1954.
4. B I G G S , R., AND DOUGLAS, A. S.: T h e thromboplastin generation test. J . Clin. P a t h . ,
6: 23-29, 1953.
5. B I G G S , R., D O U G L A S , A. S., AND M A C P A R L A N E , R . G . : T h e formation of t h r o m b o p l a s t i n
in human blood. J . Physiol., 119: 89-101, 1953.
6. B I G G S , R., DOUGLAS, A. S., AND MACPARLANE, R . G.: T h e initial stages of blood coagulation. J . Physiol., 122: 538-553, 1953.
7. B I G G S , R., AND MACFARLANE, R . G . : H u m a n Blood Coagulation a n d I t s Disorders.
Springfield, 111.: Charles C T h o m a s , 1953, p . 210.
8. CHARGAPP, E . , B E N D I C H , A., AND C O H E N , S. S.: T h e thromboplastic p r o t e i n ; s t r u c t u r e ,
properties, disintegration. J . Biol. Chem., 166: 161-178, 1944.
9. E V A N S , D . M . D . : Haemorrhagic diathesis due t o a circulating anticoagulant investigated b y t h e thromboplastin generation test. J . Clin. P a t h . , 8 : 168-172, 1955.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on March 5, 2016
coagulation of normal and hemophilic plasma25 must be taken into account in
interpreting the basic mechanisms involved in the TGT, where the final concentration of plasma is 1:60, and the final concentration of serum is 1:120.
From the theoretical viewpoint, it is not possible at this time to evaluate the
TGT on the basis of specific precursors reacting to generate a specific thromboplastic substance. The reagents represent a mixture of components, and the
reaction is complicated by the presence of small amounts of thrombin. This does
not necessarily reduce the value of the test as a diagnostic tool. I t localizes the
defects in the platelet, plasma, or serum fractions, and, in this sense, it is superior
to any other single diagnostic procedure. In our laboratory it has yielded a great
deal more information than the prothrombin consumption test, which indicates
only that a coagulation defect of some type is present. We have found it useful
for the identification and rough titrations of circulating anticoagulants, as well
as for studies on the role of platelets, platelet products, and platelet substitutes.
Sept.
1956
THROMBOPLASTIN GENERATION TEST. I I
997
10. H J O R T , P . , RAPAPORT, S. I., AND O W B E N , P . A . : Evidence t h a t - p l a t e l e t accelerator
(platelet factor 1) is adsorbed plasma proaccelerin. Blood, 10: 1139-1150, 1955.
11. MACFARLANE, R . G., AND B I G G S , R . : Thrombin generation t e s t ; application in haemophilia and thrombocytopenia. J . Clin. P a t h . , 6: 3-8, 1953.
12. MCCLAUGHRY, R . I . , AND SEEGEBS, W. H . : N a t u r e of an accelerator of prothrombin
activation arising during storage of purified prothrombin. Proc. Soc. Exper. Biol.
& Med., 80: 372-375, 1952.
13. M A C M I L L A N , R . L., AND B R O W N , K . W. G . : Heparin and t h r o m b o p l a s t i n .
J. Lab. &
Clin. Med., 44: 378-386, 1954.
14. M I A L E , J . B . , AND J O N E S , R . A . : Unpublished observations.
15. M I A L E , J . B . , AND WILSON, M . P . : Studies on t h e thromboplastin generation test. I .
Method and clinical applications. Am. J . Clin. P a t h . , 26: 969-983, 1956.
16. M U R B A Y , M . , JOHNSON, S. A., AND S E E G E B S , W. H . : Relationship of certain a n t i -
histamine drugs to t h e activation of purified prothrombin. Am. J . Physiol., 178:
10-16, 1954.
17. O W B E N , P . A . : Fifth coagulation factor ("factor V " ) ; preparation and properties.
Biochem. J., 43: 136-139, 1948.
18. O W R E N , P . A . : T h e prothrombin activating complex a n d i t s clinical significance. In
Proceedings of t h e Third International Congress of t h e International Society of
Hematology. New York: Grune & S t r a t t o n , 1951, p p . 379-390.
thrombin. Am. J . Physiol., 174: 123-126, 1953.
20. SEEGEBS, W. H . , AND ALKJAERSIG, N . : N a t u r e of t h e blood coagulation mechanisms in
SPCA plasma. Circulation Res., 3 : 514-518, 1955.
21. S E E G E B S , W. H . , A L K J A E R S I G , N . , AND J O H N S O N , S. A . : F o r m a t i o n of a u t o p r o t h r o m b i n
in solutions containing purified prothrombin and purified platelet factor 3. Am. J .
Physiol., 181: 589-594, 1955.
22. S E E G E R S , W. H . , ALKJAERSIG, N . , AND JOHNSON, S. A . : On t h e n a t u r e of t h e blood coagu-
lation mechanisms in certain clinical s t a t e s . Am. J . Clin. P a t h . , 25: 983-987, 1955.
23. SHINOWABA, G. Y . : Enzyme studies on human blood. X I . T h e isolation and characterization of thromboplastic cell and plasma components. J . L a b . & Clin. Med., 38:
11-27, 1951.
24. STEFANINI, M . : Autocatalytic formation of thrombin in blood coagulation. Acta med.
scandinav., 140: 290-306, 1951.
25. TOCANTINS, L . M., CARROLL, R . T . , AND H O L B U B N , R . H . : T h e clot accelerating effect
26.
27.
28.
29.
of dilution of blood and plasma. Relation t o the mechanism of coagulation of normal
and hemophilic blood. Blood, 6: 720-739, 1951.
VAN CBEVALD, S., AND P A U L S E N , M. M. P . : Isolation and properties of third clotting
factor in blood-platelets. Lancet, 1: 23-25, 1952.
W A R E , A. G., F A H E Y , J . C , AND S E E G E R S , W. H . : Platelet extracts, fibrin formation and
interaction of purified prothrombin and thromboplastin. Am. J . Physiol., 154: 140147, 1948.
W A R E , A. G., AND SEEGERS, W. H . : Serum Ac-globulin; formation from plasma Acglobulin; role in blood coagulation; partial purification; properties, and q u a n t i t a t i v e
determination. Am. J . Physiol., 152: 567-576, 1948.
W A R E , A. G., AND SEEGERS, W. H . : Plasma accelerator globulin: partial purification,
q u a n t i t a t i v e determination and properties. J . Biol. Chem., 172: 699-705, 1948.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on March 5, 2016
19. Q U I C K , A. J . , H U S S E Y , C. V., AND E P S T E I N , E . : A c t i v a t i o n of t h r o m b o p l a s t i n o g e n b y