Chromogenic Substrate (S-2238) Prothrombin Assay in
Prothrombin Deficiencies and Abnormaiities
Lack of Identity with Clotting Assays in Congenital
Dysprothrombinemias
ANTONIO GIROLAMI, M.D., GIOVANNI PATRASSI, M.D., FIORENZO TOFFANIN, M.D., AND LEOPOLDO SAGGIN, M.D.
Girolami, Antonio, Patrassi, Giovanni, Toffanin, Fiorenzo,
and Saggin, Leopoldo: Chromogenic substrate (S-2238) prothrombin assay in prothombin deficiencies and abnormalities.
Lack of identity with clotting assays in congenital dysprothrombinemias. Am J Clin Pathol 74: 83-87, 1980. Prothrombin was assayed using chromogenic substrate S-2238
for patients who were being treated with coumarin, for patients
who had liver disease, and for patients who had congenital
hypoprothrombinemias and dysprothrombinemias. In coumarin therapy and in patients with liver disease the levels found
correlated well with the one-stage clotting methods. The same
was true for heterozygous and homozygous "true" prothrombin deficiency. In the case of congenital dysprothrombinemias the levels observed with the chromogenic substrate
were higher than the clotting counterparts, particularly so in
the case of prothrombin Padua. In the latter case the levels
observed were always about 100% of normal, as compared with
the levels of about 50% of normal found with clotting methods.
These data indicate that chromogenic substrates are not always
equivalent to "clotting" substrates, namely, that amidolytic
activity is not always equivalent to clotting activity. Therefore the two methods cannot be used interchangeably, lest
some defects escape detection. (Key words: Prothrombin
deficiencies; Prothrombin assay; Chromogenic substrate
(S-2238); Congenital dysprothrombinemias.)
University of Padua Medical School, Institute of
"Semeiotica Medica," Padua, Italy
is maintained to be more specific. A similar substrate,
chromozym TH, has been used by others.12 All these
substrates have been or are still used also for antithrombin-III assays.
The activating agents employed with chromogenic
substrates for prothrombin assays were: tissue thromboplastins,1 intrinsic system prothrombin-converting
principle, namely, prothrombinase,2,3 Echis carincitus
viper venom,14 Xa,11,12 and a Russell viper venomcephalin mixture.'3 In the last case, prothrombin is
estimated together with factors V and X. The present
report deals with the study of prothrombin levels of
patients having acquired and congenital hypoprothrombinemias and dysprothrombinemias with a synthetic chromogenic substrate (substrate 2238).
Materials and Methods
Materials and methods have been dealt with in detail
in previous papers.7,8,10 Only pertinent data will be
supplied here. Fresh or deep-frozen plasmas of the following subjects were used: three patients with homozygous prothrombin deficiency, four patients with
heterozygous prothrombin deficiency, five patients
with prothrombin Padua (II and IIA), and four patients
with prothrombin Molise. Two types of patients with
prothrombin Molise were included, namely, the double
heterozygotes for the abnormality and for' 'true" factor
II deficiency (II0, HA) and the heterozygotes for the
abnormality (IIN, IIA). All these patients with congenital
prothrombin disorders have been extensively studied
by us and previously reported.4-6 Seventeen patients
receiving long-term coumarin therapy and 16 patients
having liver disease were also studied. The coumarintreated patients were patients receiving long-term
anticoagulant therapy and having a patient/normal
(P/N) ratio varying between 1.8 and 2.3 (rabbit brain
and lung thromboplastin). The patients who had liver
THE ACTIVATING AGENTS used during the past
decades in prothrombin activity assays have been:
tissue thromboplastins, viper venoms, a Stypvencephalin mixture, staphylocoagulase, intrinsic prothrombin-converting principle, or prothrombinase. All
these methods were developed in a one-stage, twostage, or three-stage technic. In every instance the
end substrate was human or bovine fibrinogen.
Recently, chromogenic substrates have been proposed as a useful substrate for "thrombin" activity.19
A "specific" method for prothrombin was developed
using substrate 2160 or substrate 2238.1-3-13-'4-17-20
The former seems now abandoned because the latter
Received June 21, 1979; received revised manuscript and accepted
for publication December 18, 1979.
Supported by grants from the M.P.I., Rome (Grant 1592+1978),
from the C.N.R., Rome (Grant 78.02123.04), and from the Venetian
Region, Venice.
Address reprint requests to Dr. Girolami: University of Padua
Medical School, Institute of "Semeiotica Medica," Padua, Italy.
0002-9173/80/0700/0083 $00.75 © American Society of Clinical Pathologists
83
carried in percentage of normal plasma according to the
following formula:
100
100 x
iv
Closing
tchvity
in %
FIG. 1. Prothrombin levels determined by the one-stage and
chromogenic methods for a group of coumarin-treated patients. The
correlation is statistically significant.
100-
5^
A.J.C.P. • July 1980
GIROLAMI ETAL.
84
80-
B
N - B
where P = patient plasma, N = normal plasma, and B
= blank (citrated saline solution). The activating agent
used in both instances was the ampoule P reagent
supplied by Imco Laboratories,t as proposed by
Noren.15 Such ampoule P contains factor Xa together
with suited amounts of factor V and phospholipid,
namely, a prothrombinase. Such activator shows no
direct effect on fibrinogen or on the chromogenic substrate in the absence of plasma.
Reconstituted ampoule P and test plasma were constantly kept in a melting ice bath. One hundred and
fifty microliters of ampoule P reagent was incubated in
a plastic tube with 10 fjd of test plasma for 5 min at 37 C.
One milliliter of 6 M carbamide in 1.2 M Tris buffer, pH
8.2, diluted 1 to 8 with distilled water was added,
immediately followed by 200 /A1 of the chromogenic
substrate solution. The sample was quickly mixed and
immediately placed in a Beckmann® model 25 spectrophotometer with cuvette at 37 C, and the recorder
was started.
t Imco Laboratories, Stockholm, Sweden.
Table 1. Prothrombin Levels Found in the Different
Groups of Patients
No. of
Cases
Average*
(%)
Range
(%)
SD
Coumarin
17
(a) 23.0
(b) 29.7
7-47
8-40
10.76
13.89
Liver disease
16
(a) 39.2
(b) 45.2
13-62
17-70
14.01
13.56
Congenital hypoprothrombinemia (homozygotes)
3
(a) 13.6
(b) 14.6
(c) 9.0
10-16
14-16
8-10
3.21
1.15
1.00
Congenital hypoprothrombinemia (heterozygotes)
4
(a) 49.3
(b) 55.2
(c)45.1
45-52
52-57
40-50
2.99
2.22
5.06
Prothrombin Padua
5
(a) 52.6
(b) 95.6
(c) 43.0
46-60
86-106
40-52
5.27
7.67
6.92
Prothrombin Molise
(double heterozygotes)
2
(a) 12.5
(b) 23.0
(c) 8.0
12-13
22-24
—
Prothrombin Molise
2
(a) 59.5
(b) 69.5
(c) 63.5
54-65
69-70
60-67
—
Groups of Patients
1
CIorHnq " acMvily
%
FIG. 2. Correlation between chromogenic and one-stage clotting
assays for a group of patients with liver disease. The correlation
is statistically significant.
disease were all histologically proven to have either
alcoholic or postnecrotic cirrhosis. All these patients
were treated with 10 mg of Vitamin K for a few days
before study in order to eliminate the possibility of a
concomitant Vitamin K deficiency.
The chromogenic prothrombin assay was carried out
using Kabi S-2238 (H-D-Phe-Pip-Arg-pNA) substrate.*
Two methods were used: (1) the end-point method, in
which the reaction was stopped by the addition of acetic
acid, and (2) the initial-rate method. The latter method
was carried out using a Hitachi Perkin-Elmer® recorder.
In both instances spectrophotometric readings were
* Kabi Diagnostics, Stockholm, Sweden.
* (a) One-stage method; (b) Chromogenic method; (c) Prothrombinase-clotting method.
BRIEF SCIENTIFIC REPORTS
Vol. 74 • No. 1
85
A
100-
/.*
1?
FIG. 3. Correlation between chromogenic and onestage activities for congenital hypoprothrombinemias
and dysprothrombinemias. A discrepancy between
clotting and chromogenic activities is evident for
both dysprothrombinemias, being more pronounced
for prothrombin Padua. For congenital "true"
prothrombin deficiency there is a good correlation
between the two methods.
80O
60-
Q/
o oo/
• Homozygous prothrombin deficiency
o Heterozygous prothrombin deficiency
40-
A Prothrombin P»du* ( l l 4 1I N )
20-
/
/
A Prothrombin Molise ( I I 0 I I A )
•yf
O Prothrombin Molise (1I 4 II N )
20
40
60
80
"Clotting" ictivity
For the end-point method the procedure was the
same but for the blocking of the reaction with 150 fjd of
50% acetic acid solution 2 min after the addition of the
chromogenic substrate. The reading in the case was
carried out within 45 min.
For patients with congenital disorders prothrombin
was also assayed in a two stage "clotting" system
using the same prothrombinase reagent and fibrinogen
as substrate, as originally proposed by Noren. 15
Results
Results are summarized in Table 1.
In coumarin-treated patients and in patients with liver
damage, prothrombin levels were low. The levels were
observed correlated well with those obtained using the
classic one-stage method (r = 0.695; P < 0.01 and
r = 0.553; P < 0.05, respectively) (Figs. 1 and 2).
In patients with congenital homozygous or heterozygous prothrombin deficiency, prothrombin levels
were low, and the levels also correlated very well with
those found by means of clotting assays (r = 0.985;
P < 0.001) (Fig. 3).
In patients with prothrombin Padua the chromogenic
level was normal and therefore approximately twice
as much as the clotting activity. There is in fact no
significant correlation between the two series of values
(r = 0.269; P > 0.80) (Fig. 3).
In patients with prothrombin Molise the levels observed with chromogenic substrate were also higher, as
a whole, then those observed by means of clotting
methods, but the correlation between the two methods
was still statistically significant (r = 0.983; P < 0.01)
(Fig. 3).
A good correlation was found between the end-point
100
in %
and the initial-rate methods. A few curves obtained
by means of the latter method are shown in Figures
4 and 5. The values obtained by the prothrombinase
two-stage clotting method were similar to those observed by the one-stage method in every instance.
Discussion
Prothrombin can be assayed using chromogenic substrate S-2238 with no major difficulty. There is a good
correlation with the one-step clotting method when used
with patients having liver disease or with patients being
treated with coumarin. The same seems to be true for
patients having isolated, congenital deficiency of prothrombin. This seems to suggest that the chromogenic
substrate is suited for the evaluation of prothrombin
for both isolated and combined defects. These observations appear to support the claims that chromogenic
substrate assays have several advantages, namely,
easy adaptation to automated technics easy standardization, and avoidance of conditions interfering with
fibrin formation. n The results also demonstrate that the
chromogenic substrate is unable to detect coumarininduced preprothrombin. However, there are some
limitations to the use of these chromogenic substrates, as demonstrated by the data pertaining to the
two congenital dysprothrombinemias, namely, prothrombin Padua and prothrombin Molise. In both
instances, the chromogenic assays yielded levels higher
than those obtained using tissue thromboplastins. In
the case of prothrombin Padua, normal prothrombin
levels were observed, as compared with levels of about
50% found using clotting tests. In the case of prothrombin Molise, the difference was more pronounced
for the double heterozygotes (II A , Ho) than for the
86
GIROLAMI ETAL.
A.J.C.P. • July 1980
tissue thromboplastin one-stage methods.1516 Furthermore, the levels observed by the prothrombinase
clotting method in prothrombins Padua and Molise
were similar to those obtained by the one-stage method.
Therefore, the reagent responsible for the discrepancy
appears to be the chromogenic substrate.
The main objections against the routine use of these
chromogenic substrates may be summarized along the
following lines. (1) The cost of the assay is about ten to
20 times that of a standard clotting test. (2) The substrate is often not specific, being sensitive, even though
to a lesser extent, to other enzymes.3'718 However,
in this regard one has to remember that specificity
of the substrate may be of relative importance when the
activator used is specific and only thrombin is gen-
FIG. 4. Chromogenic prothrombin assay by the initial-rate method
for normal, hypoprothrombinemic, and prothrombin Padua plasmas.
The slope of the curve for prothrombin Padua plasma is similar
to that for normal plasma. The curves obtained for prothrombin
Molise are not reported here, but they were also consistent with
the results obtained by the end-point method. The speed of the
recorder was 2 cm/min.
single heterozygotes, but the phenomenon was still
evident.
The present study clearly indicates that there is no
constant identity between "clotting" activity and
amidolytic activity. Since the physiologic substrate for
thrombin is fibrinogen, it is clear that this is the most
important function that has to be explored. Prothrombin Padua patients have a tendency toward a mild
bleeding, and this finding correlates well with the slight
decrease of prothrombin activity as found by onestage methods. Had we used the chromogenic substrate as a routine coagulation method we would have
missed the defect.
The possibility that the activator (prothrombinase)
used in the chromogenic assay may be responsible for
the discrepancy seems remote. The prothrombinase
method has been demonstrated to correlate well with
FIG. 5. Chromogenic prothrombin assay by the initial-rate method
for normal plasma and for acquired hypoprothrombinemias (coumarin treatment and liver disease). The levels observed in the latter
cases are similar to those observed by clotting methods. The speed
of the recorder was 2 cm/min.
Vol. 74 . No. l
BRIEF SCIENTIFIC REPORTS
87
Table 2. Behavior of Prothrombin Padua and Prothrombin Molise in Different Prothrombin Assays*
Condition
Prothrombin Padua
(heterozygotes)
Prothrombin Molise
(double heterozygotes)
Prothrombin Molise
(heterozygotes)
One-stage
Method
Two-stage
Method
(Iowa)
Staphylocoagulase
Tiger
Viper
Venom
Taipan
Viper
Venom
Echis
carinalus
Venom
Prothrombinase
(Clotting)
Chromogenic
Substrate
Immunologic
(Mancini)
53
49
100
47
43
99
45
96
100
12
11
50
11
10
14
9
23
52
59
56
105
58
46
62
63
69
98
* The values represent the average percentages approximated to eliminate decimal fractions.
erated. (3) The chromogenic assay is dependent on the
turbidity of plasma or serum, such as that caused by
lipemia, increased bilirubin, or nonspecific flocculations. This pitfall may be overcome by carrying on the
assay on an appropriate blank without the addition of
the activator. However, this procedure further increases
the cost of the assay. (4) There is a lack of absolute
identity between amidolytic and "clotting" activity.
For the above reasons and in agreement with others,18
for the time being we abstain from advocating the
routine use of these substrates. However, the reagents
may be very useful for research purposes in order
to clarify abnormal clotting factors.
The objections to the routine use of chromogenic
substrates apply to the case of abnormal factor X
Friuli as well as to prothrombin Padua.5 In this abnormality the factor X level is found to be low when
assayed using a chromogenic substrate and a Russell
viper venom-cephalin mixture as activating agent. This
was a surprising result, since factor X Friuli is known
to be normal when assayed using the same activating
agent and fibrinogen as a substrate.
In view of these results, the extensions of the use of
chromogenic substrate assay to all the remaining dysprothrombinemias so far reported seems indicated. The
behavior of the assay may differ in each case, and this
may help clarify the peculiarity of the defect. The
complete activation patterns of the two dysprothrombinemias we studied are summarized in Table 2.
Acknowledgments. Mr. G. Boeri and Mr. R. Gazzetta provided
valuable technical assistance.
References
1. Axelsson G, Korsan-Bengsten K, Waldenstrom J: Prothrombin
determination by means of a chromogenic peptide substrate.
Thromb Haemost 36:517-524, 1976
2. Bergstrom K, Blomback, M: Determination of plasma prothrombin with a reaction rate-analyzer using a synthetic
substrate. Thromb Res 4:719-729, 1974
3. Bergstrom K, Egberg N: Determination of Vitamin K sensitive
coagulation factors in plasma. Studies on three methods using
synthetic chromogenic substrates. Thromb Res 12:531-547,
1978
4. Girolami A: The hereditary transmission of congenital "true"
hypoprothrombinemia. Br J Haematol 21:695-703, 1971
5. Girolami A, Bareggi G, Brunetti A, et al: Prothrombin Padua:
a "new" congenital dysprothrombinemia. J Lab Clin Med
84:654-666, 1974
6. Girolami A, Coccheri S, Palareti G, et al: Prothrombin Molise:
a "new" congenital dysprothrombinemia. Double heterozygosis with an abnormal prothrombin and true prothrombin
deficiency. Blood 52: 115-125, 1979
7. Girolami A, Molaro G, Lazzarin M, et al: A "new" congenital
hemorrhagic condition due to the presence of an abnormal
factor X (factor X Friuli): study of a large kindred. Br J
Haematol 19:179-192, 1970
8. Girolami A, Patrassi GM, Virgolini L, et al: The effect of several
viper venoms on prothrombin Padua. Blut 31:155-160, 1975
9. Girolami A, Saggin L, Boeri G: Factor X assays and chromogenic
substrate S-2222., Am J Clin Pathol 73:400-402, 1980
10. Girolami A, Sticchi A, Brunetti A: Prothrombin level and
activity in the abnormal factor X (factor X Friuli) hemorrhagic disorder. Thromb Diath Haemorrh 25:147-156, 1971
11. Kirckhof BRJ, Muller AD, Vermeer C, et al: Control of anticoagulant therapy with a chromogenic substrate. Haemostasis
8:1-7, 1979
12. Kirckhof BRJ, Vermeer C, Hemker HC: The determination of
prothrombin with synthetic chromogenic substrates, choice
of a suitable activator. Thromb Res 13:219-232, 1979
13. Korsan-Bengsten K, Axelsson G, Waldenstrom J: Bestimmung
von Plasma-Prothrombin mit dem chromogenen Peptid-Substrat: H-D-Phe-Pip-Arg-pNA (S-2238), New Methods for the
analysis of coagulation using chromogenic substrates. Edited
by I Witt. Berlin, W. De Gruyter, 1977, pp 145-154
14. Latallo ZS, Teisseyre E: Amidolytic assay of prothrombin
activated with Ecarin, a procoagulant from Echis Carinatus
venom, New methods for the analysis of coagulation using
chromogenic substrates. Edited by I Witt. Berlin, W. de
Gruyter, 1977, pp 182-199
15. Noren I: Specific assay of prothrombin. A method usinga freezedried reagent of intrinsic coagulation factors. Scand J Clin
Lab Invest 25:47-58, 1970
16. Noren I, Blomback M: Specific determination of prothrombin
during anticoagulant therapy. Acta Med Scand 192:77-83,
1972
17. Roka L, Koch R, Bleyl M: Studies on the determination of
prothrombin with S-2160, New methods for the analysis of
coagulation using chromogenic substrates. Edited by I Witt.
Berlin, W. de Gruyter, 1977, pp 171-180
18. Shapiro SS, McCord S: Prothrombin, Progress in hemostasis and
thrombosis. Volume 4. Edited by T Spaet. New York, Grune
and Stratton, 1978, pp 177-192
19. Stormorken M: A new area in the laboratory evaluation of coagulation and fibrinolysis. Thromb Haemost 36:229-230,
1976
20. Witt I: Determination of plasma protein with Cromozym TH,
New methods for the analysis of coagulation using chromogenic
substrates. Edited by I Witt. Berlin, W. de Gruyter, 1977,
pp 155-169.