Download A multiparameter test of clot formation and fibrinolysis for in

Original article 611
A multiparameter test of clot formation and fibrinolysis for
in-vitro drug screening
Barbara Kostkaa, Jadwiga Paraa and Joanna Sikorab
A large spectrum of methods has been used in both
routine and scientific studies of the hemostatic system.
The particular interest of the investigators has been
focused on methods simultaneously evaluating clotting
and fibrinolysis processes. The aim of the present study
was to develop an optical method for overall evaluation of
clot formation and lysis (CL-test) that could be used in
drug screening. The CL-test was performed in citrate
plasma diluted with Tris-buffered saline. Thrombin was
applied for plasma clotting (0.5 IU/ml), while tissue
plasminogen activator (60 ng/ml) was used for
fibrinolysis activation. Clot formation and lysis were
monitored in thermostatic conditions (37-C) as a
continuous record of transmittance change. By means of
own computer program, kinetic parameters of the
processes studied and plasma overall clot formation and
fibrinolysis potential, expressed as the area under the
clotgeneration and fibrinolysis curves, were calculated.
The CL-test was developed and checked by evaluation of
the effect, on clot formation and lysis, of various
Introduction
Fibrin formation and degradation are the final steps in
blood coagulation and they involve sequential activation
of multiple proenzymes circulating in the blood. The
hemostatic balance between these processes is regulated
by numerous mechanisms that control fibrin formation
(most important factors are antithrombin, the protein
C/protein S system, and tissue factor pathway inhibitor)
and its degradation (the activities of plasmin and
plasminogen activators are regulated by serpins and
a2-macroglobulin). Defects in the mechanisms responsible for fibrin turnover might lead to thrombosis or
bleeding. Many studies have shown that the hemostatic
balance depends on physical, biochemical, and genetic
factors. Numerous chemical compounds and drugs also
have a profound influence on fibrin structure and clot
formation and lysis [1]. A large spectrum of laboratory
tests exists to identify this imbalance in the hemostatic
system. In recent years, growing interest in a global assay
of coagulation and fibrinolysis that uses optical measurements of clot formation and lysis has been observed [2].
As early as in 1975, Glover and Warner [3] used an
aggregometer for turbidimetric measure of clot formation
and fibrinolysis dynamics. They determined that curve
segments, such as clotting time, maximum optical density
and lysis time, correlated respectively with the activated
concentrations of acetylsalicylic acid (a drug that
affects hemostasis), aprotinin (fibrinolysis activator)
and venoruton (fibrinolysis inhibitor). The obtained results
confirmed that the test we propose for monitoring clot
formation, stabilization and lysis is sensitive and
enables precise estimation of the processes studied.
In our opinion, it can be a useful tool in drug
screening investigations. Blood Coagul Fibrinolysis
18:611–618 ß 2007 Lippincott Williams & Wilkins.
Blood Coagulation and Fibrinolysis 2007, 18:611–618
Keywords: acetylsalicylic acid, clot formation, fibrinolysis, in-vitro method
a
Department of Pharmaceutical Biochemistry and bDepartment of Pharmaceutical
Chemistry and Drug Analysis, Medical University of Lodz, Lodz, Poland
Correspondence to Dr Joanna Sikora, Department of Pharmaceutical Chemistry
and Drug Analysis, Muszynskiego 1, 90-151 Lodz, Poland
Tel: +48 42 677 92 95; fax: +48 42 677 92 50; e-mail: [email protected]
Received 25 January 2007 Revised 10 May 2007
Accepted 10 May 2007
partial thromboplastin time, fibrinogen concentration and
plasminogen level [3].
He et al. [4,5] also applied optical measurements of clotting
and fibrinolysis in their studies. They introduced a new
parameter that allows for an overall evaluation of the
processes studied, and calculated the area under coagulation and fibrinolysis curves to determine overall hemostasis potential. This method with some modifications was
used in both routine and research studies [2,6–9].
Although 100 years have passed since the invention of
acetylsalicylic acid (ASA), research assessing its properties and therapeutic action is still conducted. Emphasis
has been recently placed on ASA application in cardiovascular prophylaxis [10]. On the other hand, the amount
of alarming reports on ASA resistance has increased and
this phenomenon may independently correlate with the
increase in cardiovascular events risk [11]. Moreover,
numerous studies confirm that ASA, apart from antiplatelet action, also affects clot formation and lysis [12,13].
ASA causes lysine residue acetylation of various proteins
of procoagulative (fibrinogen and prothrombin) as well as
anticoagulative action (antithrombin). Acetylation of
lysine residues in fibrinogen influences the clot structure
and stabilization through factor XIII-mediated formation
of cross-link bonds between fibrin fibers [14]. The results
0957-5235 ß 2007 Lippincott Williams & Wilkins
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
612 Blood Coagulation and Fibrinolysis 2007, Vol 18 No 7
of the studies evaluating the ASA effect on fibrinolysis,
however, are ambiguous. The impact of ASA activity is
thought to be dependent on the initial plasma fibrinolytic
activity, the dose, and the time of its administration [6].
Despite the fact that all the mechanisms by which ASA
affects hemostasis are not clear, it is well established that
this drug influences both coagulation and fibrinolysis
[6,10–14]. In the present study, our aim was to develop
a simple, routine and accurate test for overall evaluation
of clot formation and lysis (CL-test) that allows for
continuous registration and for precise estimation of
the processes studied, and can be used in in-vitro drug
screening. ASA was used as a model drug to check the
usefulness of our CL-test.
Materials and methods
Sample preparation
Venous blood was obtained from healthy human volunteers, who had abstained from aspirin and other drugs for at
least 2 weeks. Platelet-poor plasma was obtained by
centrifugation (1500 g, 20 min, 48C) of platelet-rich
plasma. The platelet-rich plasma was obtained by centrifugation (200 g, 10 min, room temperature) of anticoagulated blood (3.2% buffered sodium citrate used in a 1 : 9
volume ratio, Vacuette Coagulation Tubes; Greiner
Bio-One, Kremsmünster, Austria). The study protocol
complies with the Declarations of Helsinki and Tokyo.
Reagents
Reagents used for the study were freshly prepared from
the stock solutions. Thrombin (400 IU) (Biomed, Lublin,
Poland) was dissolved in 0.15 mol/l NaCl, giving a concentration of 400 IU/ml. Stock solution was stored in
small portions (20 ml) for up to 1 month at 208C. Then
20 mg actilyse recombinant tissue plasminogen activator
(t-PA) (Boehringer, Ingelheim, Germany) was dissolved
in 20 ml manufacturer’s solvent, giving a concentration of
1 mg/ml, and was stored in small aliquots at 708C until
further use. Fresh reactant solutions of thrombin and
t-PA were maintained during the assay in an ice-water
bath for no longer than 1 h.
Tris-buffered saline (TBS) was prepared by adding
90 ml of 0.15 mol/l NaCl into 10 ml of 0.15 mol/l buffer
Tris–HCl, pH 7.4, at 378C (adjusted with HCl). Sodium
chloride, Tris, and HCl were obtained from Polish
Chemical Reagents (Gliwice, Poland). TBS was stored
for up to 1 month at 48C.
Aprotinin (Traskolan) was obtained from Jelfa S.A.
(Jelenia Góra, Poland), venoruton from Zyma SA (Nyon,
Switzerland), ASA from Serva (Heidelberg, Germany),
and D,L-lysin-mono-(acetylsalicylate) (Aspisol) from
Bayer AG (Leverkusen, Germany). ASA solution was
prepared before the assay commenced: 9.01 mg ASA
was dissolved in 10 ml deionized water, giving a concentration of 5 mmol/l.
A Cecil CE 1011 spectrophotometer (Cecil, London, UK)
(378C, with stirring) was computerized with the
authors’ own program, which enables not only continuous registration but also quick and easy estimation of
the investigated processes.
Technical procedure
The CL-test with our own computer program, which
evaluates the kinetic parameters of the examined process,
was used in in-vitro screening studies. The test was based
on the assumption of the global assay of coagulation and
fibrinolysis with the use of optical measurements [2–5].
To obtain a clotting and lysis curve, 167 ml platelet-poor
plasma was diluted in a disposable plastic cuvette with
313 ml (control) or 288 –308 ml TBS, 5–30 ml examined
drug and 5 ml t-PA (final concentration, 60 ng/ml). It is
also possible to obtain only a fibrin clot formation curve.
In this case, 5 ml TBS was used instead of t-PA. Transmittance changes were measured continuously by a
spectrophotometer at 405 nm and were registered in
the computer program. The instrument was set for
100% transmission with platelet-poor plasma diluted
with TBS prior to each reading. The examined sample
was preincubated at 378C for 3 min. Then (when baseline stabilized) 10 ml thrombin (final concentration,
0.5 IU/ml) was added to start the process. The sample
was stirred at 800 rpm for 10–15 s during thrombin
addition.
As shown in Fig. 1, the curve is generated over the course
of the assay reactions and has a baseline transmittance
with a peak at the moment of thrombin addition
(indicator 0), and three consequent phases. Phase 1
(Fig. 1, light gray) corresponds to plasma fibrin generation
triggered by exogenous thrombin and is reflected by a
progressive decrease in transmittance to a point of minimum. Phase 2 (Fig. 1, gray) stands for the plateau of
maximal decrease in transmittance and is the moment of
stable clot formation. A final increase in transmittance
(phase 3; Fig. 1, white) indicates the fibrinolytic potential
in response to exogenous t-PA. Curves were recorded
continuously and evaluated by the computer program.
The calculated parameters of clotting and fibrinolysis are
shown in Fig. 1. Introduction of 10 marks (To, R, 0–7),
automatically placed in the appropriate points of study
curves, enables calculation of 15 kinetic parameters
of clot formation and lysis. There is also the possibility
of manual change in the position of marks, in case of
nontypical curves.
The computer program can also be used to record platelet
shape change, aggregation [15,16], and clot formation and
fibrinolysis curves with the use of different activators
(such as calcium ions, thrombin, t-PA, streptokinase,
urokinase).
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Test of clot formation and fibrinolysis Kostka et al. 613
Fig. 1
that examines clot solubility in 5 mol/l urea solution
[17] – that the amount of calcium ions present in citrate
plasma was sufficient for activation of factor XIII and
stabilization of fibrin structure.
0
Light transmission (%)
20
Thrombin
Tt
Tf
Ti
Tc
The experimental conditions we proposed enable observation of a possible drug influence on all three phases of
this process at a time that does not exceed 30 min.
40
60
Sr
Sc
Sf
Lv
°
80
To provide an optimal condition similar to the physiological environment, thermostating (378C) and buffer
(pH 7.4 at 378C) were used.
Fmax
Lmax
Fv
°
100
0 To R12
5
3
4
10
5
6
7
15
20
Typical curve of clotting and fibrinolysis with parameters evaluated in
the CL-test demonstrated. Phase I – clot formation (light gray): Tt (s),
thrombin time [time elapsed from thrombin addition (indicator 0) to
coagulation beginning (indicator R)]; Fmax (%T), maximum clotting [i.e.
the transmittance difference between the baseline (indicator To) and
minimum transmittance (indicator 4)]; Tf (s), plasma clotting time
(from indicator 1 – means 10% of Fmax – to indicator 3 – means 90%
of Fmax); Fvo (%T/min), initial plasma clotting velocity corresponds
to the site of the quickest transmittance change (indicator 2); Sr (%T x
min), area under the clot formation curve. Phase II – forming a stable
clot (gray): Tc (s), clot stabilization time (from indicators 3 to 5); Sc (%T
x min), area under the curve of a stable clot formation. Phase III –
fibrinolysis (white): Lmax (%T), maximum lysis [i.e. the transmittance
difference between the minimum transmittance (indicator 4) and the
baseline (indicator 7)]; Tl (s), fibrinolysis time (from indicators 5 to 7);
Lvo (%T/min), initial clot fibrinolysis velocity corresponds to the site of
the quickest transmittance change (indicator 6); Sf (%T x min), area
under the fibrinolysis curve; CLAUC (%T x min), overall potential of
clot formation and lysis (i.e. the areas under clotting, fibrinolysis and
transition curves. CLAUC ¼ Sr þ Sc þ Sf). To, R, 0–7,– indicators used
in the CL-test computer program.
To check the method, the influence of various concentrations of model drugs has been examined. We have
used aprotinin as an in-vitro model of fibrinolysis inhibitor [18] and venoruton [0-(b-hydroxyethyl)-rutoside] as
the fibrinolysis activator [19]. In the CL-test, we have
confirmed that aprotinin, depending on the concentration
used, decreases the fibrinolytic capacities of plasma
(Table 1) whereas venoruton causes fibrinolysis acceleration (Table 1).
The intra-assay coefficients of variation for the CL-test
were established for 10 samples of normal plasma. Each
plasma sample was analyzed in triplicate or duplicate
under the same assay conditions. Intra-assay coefficients
of variation for clotting parameters were between 1.1 and
4.4%, for the clot stabilization phase were between 6.7
and 7.9%, and for fibrinolysis parameters were between
0.9 and 9.7%.
Effect of acetylsalicylic acid on clot formation
(phases I and II) and fibrinolysis (phase III)
Data analysis
The results are presented as the mean SD. Significance
of differences between groups was established by Student’s t-test. A value of P less than 0.05 was considered
statistically significant.
Results
Clotting and lysis test
We have performed several experiments to check how
the system behaves at different thrombin and t-PA
concentrations, with and without calcium ions. The
effect of various concentrations of calcium ions alone
(3.75–17.5 mmol/l) and with thrombin (0.04-1 IU/ml; data
not shown), and of thrombin alone (0.125–1 IU/ml)
and with t-PA (30–1400 ng/ml), was investigated by the
CL-test (Fig. 2a,b). We have experimentally found that
the concentrations of 0.5 IU/ml thrombin and 60 ng/ml
t-PA are optimal in in-vitro drug screening. Exogenous
calcium ions were not added to the sample, and high
thrombin concentration (0.5 IU/ml) was used to directly
initiate fibrin formation. This eliminates the effect of
thrombin generation on the processes studied [12].
We have experimentally confirmed – by the method
In the second part of this study, we performed several
experiments with ASA to check whether our proposed invitro model for drug screening can be a useful tool to
evaluate changes in plasma clot formation and fibrinolysis. ASA was added to diluted plasma at concentrations
of 50, 100, 200 and 300 mmol/l and the parameters of
the three phases together were measured (Fig. 3 and
Table 2). The range of ASA concentrations used in our
study was approximate to the maximal plasma concentrations reached after oral administration [6,10] and
approximate to the concentrations (1–1000 mg/ml) used
by other authors in in-vitro experiments [6,20].
When only clot formation was examined, there was no
significant influence of different ASA concentrations on
the evaluated parameters of phases I and II (data not
shown).
Figure 3 presents the influence of different concentrations of ASA on clotting and fibrinolysis processes
induced by thrombin and t-PA (data obtained with
the same plasma). Table 2 presents the means SD
of the examined CL-test parameters for 50, 100, 200
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
614 Blood Coagulation and Fibrinolysis 2007, Vol 18 No 7
Fig. 2
(a)
0
1 175 ng/ml t-PA
2 300 ng/ml t-PA
3 700 ng/ml t-PA
4 1400 ng/ml t-PA
Light transmission (%)
20
40
1
60
80
100
2
3
4
120
0
5
10
15
20
25
Time (min)
(b)
Light transmission (%)
0
1 0.125 IU/ml thrombin
2 0.250 IU/ml thrombin 3 0.5 IU/ml thrombin 4 1.0 IU/ml thrombin
20
40
60
80
4
100
2
1
3
120
0
5
10
15
20
25
Time (min)
Curve
Tt (s)
Fmax (%T)
Tf (s)
20.1
19.5
19.9
20.3
63.7
62.6
60.8
64.1
89.9
86.2
72.9
65.7
(b) Thrombin
1
325.0
2
134.0
3
52.6
4
32.6
17.3
41.3
58.0
60.6
88.5
86.8
79.7
65.0
(a) t-PA
1
2
3
4
Tc (s)
Lmax (%T)
Tl (s)
Lvo (%T/min)
CLAUC (Sr þ Sc þ Sf) (%TMmin)
242
258
263
277
772
548
166
62
30.9
56.0
59.3
63.9
774
431
160
79
5.1
13.1
26.4
57.3
1224.5 (73.3 þ 803.2 þ 348.0)
832.5 (69.6 þ 52.8 þ 234.1)
273.5 (56.4 þ 150.6 þ 66.5)
153.0 (55.4 þ 62.0 þ 35.6)
12.8
43.4
132.0
195.0
26
31
166
219
17.2
42.3
59.5
62.0
115
122
133
145
8.2
24.1
27.6
26.2
41.9 (13.7 þ 8.1 þ 20.1)
101.6 (36.4 þ 19.6 þ 45.6)
274.9 (53.8 þ 155.4 þ 65.5)
338.2 (47.4 þ 215.5 þ 75.3)
Fvo (%T/min)
Effects of various (a) tissue-type plasminogen activator (t-PA) concentrations (175–1400 ng/ml) and (b) thrombin concentrations (0.125–1 IU/ml)
on CL-test parameters (data obtained with the same plasma). For definitions, see Fig. 1.
Table 1
Effects of various concentrations of aprotinin (fibrinolysis inhibitor) and venoruton (fibrinolysis activator) on CL-test parameters
Aprotinin (n ¼ 2)
0 (control)
1 KIU/ml
10 KIU/ml
100 KIU/ml
Venoruton (n ¼ 2)
0 (control)
100 mmol/l
200 mmol/l
Tt (s)
Fmax (%T)
Tf (s)
Fvo (%T/min)
Tc (s)
Lmax (%T)
Tl (s)
Lvo (%T/min)
23.2
26.3
28.8
29.9
54.3
56.0
55.1
59.5
44.0
52.6
68.8
87.7
222
205
197
201
218
242
332
665
61.8
63.5
59.0
142
152
332
38.2
31.6
11.5
M
M
M
31.8
34.1
33.3
37.1
37.9
38.5
53.5
50.4
44.3
123
126
132
286
259
193
33.7
36.9
39.5
470
415
231
5.6
7.0
17.7
CLAUC (Sr þ Sc þ Sf) (%T min)
277.6 (28.7 þ 191.4 þ 57.5)
316.7 (35.5 þ 219.3 þ 61.8)
473.9 (46.5 þ 295.3 þ 132.0)
1122.1 (65.7 þ 1056.4 þ 0M)
360.4 (23.5 þ 171.7 þ 165.2)
324.5 (23.0 þ 161.0 þ 140.5)
209.2 (19.8 þ 120.0 þ 69.4)
See Fig. 1 for definitions.
M
100% inhibition.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Test of clot formation and fibrinolysis Kostka et al. 615
Fig. 3
0
1
Control
2
3
50 micromol/l ASA
100 micromol/l ASA
4
200 micromol/l ASA
5
300 micromol/l ASA
Light transmission (%)
20
40
60
80
4
100
1
5
3 2
120
0
5
10
20
15
25
Time (min)
Curve
Tt (s)
Fmax (%T)
Tf (s)
Fvo (%T/min)
Tc (s)
Lmax (%T)
Tl (s)
Lvo (%T/min)
1
2
3
4
5
140
140
149
128
132
37.1
36.4
37.8
37.3
38.9
159
145
145
129
144
33.5
32.2
32.0
35.8
36.7
335
270
248
207
280
34.6
35.1
37.8
37.6
38.2
258
209
211
197
226
8.8
10.8
11.6
12.1
10.9
CLAUC (Sr þ Sc þ Sf) (%TMmin)
391.1
305.0
283.5
245.1
325.6
(67.9 þ 205.1 þ 118.1)
(58.7 þ 159.4 þ 86.9)
(59.8 þ 150.8 þ 72.9)
(53.1 þ 125.4 þ 66.6)
(62.1 þ 175.7 þ 87.8)
Effects of 50, 100, 200 and 300 mmol/l acetylsalicylic acid (ASA) on clotting and fibrinolysis curves. The final concentrations of exogenous thrombin
and t-PA were 0.5 IU/ml and 60 ng/ml, respectively (data obtained with the same plasma). For definitions, see Fig. 1.
and 300 mmol/l ASA. For 200 mmol/l ASA there were
significant differences, in relation to the control, in the
areas under the clotting curve (#Sr) and stable clot
formation curve (#Sc). Moreover, the overall clot formation and fibrinolysis potential (areas under clotting,
fibrinolysis and transition curves, #CLAUC), the time of
stable clot formation (#Tc) and the time of clot lysis
(#Tl) decreased statistically significantly. When
100 mmol/l ASA was used, we observed a significant
decrease only in CLAUC and Sc. In the case of 50 and
300 mmol/l ASA, there was no significant influence on
Table 2
the coagulation and fibrinolysis parameters. Owing to
weak ASA solubility in water and the possibility of
precipitation, however, we performed additional tests
with D,L-lysin-mono-(acetylsalicylate) (Aspisol), which
is water-soluble and hydrolyzes to ASA. Preliminary
results obtained with Aspisol were parallel to those with
ASA. Aspisol concentrations of 100 and 200 mmol/l
(concentrations converted and expressed as ASA concentrations in the sample) accelerated fibrinolysis, whereas
higher concentrations (300–3000 mmol/l) did not cause
such (as above) effect (data not shown).
In-vitro effects of different acetylsalicylic acid (ASA) concentrations on CL-test parameters
Tt (s)
Fmax (%T)
Tf (s)
Fvo (%T/min)
Sr (%T x min)
Tc (s)
Sc (%T x min)
Lmax (%T)
Tl (s)
Lvo (%T/min)
Sf (%T x min)
CLAUC (%T x min)
Control (n ¼ 13)
50 mmol/l ASA (n ¼ 10)
100 mmol/l ASA (n ¼ 7)
200 mmol/l ASA (n ¼ 10)
300 mmol/l ASA (n ¼ 10)
111.2 13.3
47.9 8.2
153.2 12.1
44.1 15.1
80.7 13.5
352.8 65.4
277.6 96.5
46.9 7.9
314.7 23.1
9.9 1.4
148.3 35.32
506.6 142.2
114.0 33.5
44.8 8.6
152.9 23.6
41.8 17.3
75.5 42.0
334.1 7.0
213.8 57.0
42.0 5.7
272.4 61.0
9.9 2.0
128.8 55.5
430.3 97.0
119.1 30.7
44.0 8.5
149.7 24.1
39.1 16.5
71.7 13.5
292.9 78.4
178.5 43.1MM
44.0 8.2
269.9 65.6
10.8 2.4
111.0 44.2
344.3 68.1MM
104.4 29.7
44.3 5.1
141.4 17.8
40.5 9.0
68.4 13.5MM
255.3 27.5MM
182.7 31.8MM
44.3 5.2
251.1 51.5MM
11.5 2.8
103.0 30.8
351.0 72.9MM
100.9 22.8
48.5 10.0
150.0 23,8
44.7 22.9
77.0 20.1
304.7 99.4
249.4 125.3
47.9 9.5
319.2 62.5
9.7 2.1
147.5 54.4
473.9 192.8
See Fig. 1 for definitions.
MM
P ¼ (0.05–0.001) versus control.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
616 Blood Coagulation and Fibrinolysis 2007, Vol 18 No 7
Discussion
In recent years, growing interest in a global assay
of coagulation and fibrinolysis that uses optical measurement of clot formation and lysis can be observed [2].
In the present study we have developed an optical
method for overall evaluation of clot formation and
fibrinolysis (the CL-test) in human plasma, which can
be used in in-vitro drug screening. Implementation of
continuous registration and our own computer program
that calculates 15 kinetic parameters (Fig. 1) allowed for
overall, as well as detailed, evaluation of the examined
processes.
As it is of crucial importance that the test used for drug
screening should be sensitive enough to catch even
subtle changes induced by studied compounds, we have
performed several experiments to choose the most suitable concentrations of thrombin and t-PA, and other
experimental conditions, and have checked the results
using aprotinin, venoruton and ASA, drugs with a known
effect on hemostasis.
Various environmental factors, such as salt and protein
concentrations, ion contents, and so on, exert an influence
on fibrin clot formation and its structure [21–23]. While
performing measurements, the maintenance of constant
pH and temperature levels, due to their impact on protein
involved in molecular interactions such as thrombin–
fibrinogen binding, are of crucial significance [22]. In
our experiments, in order to provide an optimal, similar
to physiological environment, thermostating (378C) and
buffer (pH 7.4 at 378C) were used. We did not add
calcium ions to the sample and applied a high concentration of thrombin (0.5 IU/ml). Addition of small
amounts of thrombin (0.05 IU/ml) does not result in
coagulation initiation but induces a feedback reaction
leading to endogenous thrombin generation, and by that
to clot formation [12]. The concentration of thrombin
used in our method induces clotting directly, and thus we
eliminate the effect of thrombin generation on the processes studied. For the same reason, application of
exogenous calcium was ceased.
We confirmed that, by means of the method that
examines clot solubility in 5 mol/l urea solution [17],
the amount of calcium ions in diluted citrate plasma
was sufficient to activate factor XIII and to stabilize
the fibrin structure.
Under such experimental conditions, fibrinogen function
should be the main contributor to variations in fibrin
structure.
in the amounts of thrombin and t-PA used (Fig. 2a,b). In
our proposed experimental conditions, the thrombin and
t-PA concentrations are constant and ensure that even
subtle changes in all three phases of examined processes
can be detected, as confirmed with aprotinin and venoruton, which is important in drug screening tests.
We used area under the curve for the overall evaluation of
clotting, clot stabilization and fibrinolysis phases. Owing
to the continuous recording and the computer evaluation
of the processes, we introduced our own parameters
assessing the overall potential of clotting and fibrinolysis
(CLAUC), the overall clotting potential (Sr), the clot
stabilization phase (Sc) and the overall fibrinolysis potential (Sf). The computer program also enables one to
determine kinetic parameters such as the times and
initial velocities of those processes (Fig. 1). In our
opinion, such a form of clot formation and fibrinolysis
evaluation may find application in drug screening.
There are numerous studies substantiating clinical efficacy of ASA as an antithrombotic drug. The majority of
these studies’ concerns the impact on platelet reactivity
[24], chiefly through irreversible inhibition of platelet
cyclooxygenase-1 and also through reduction of expression of adhesive molecule binding monocytes, neutrophils and platelets on the surface of endothelial cells [25].
The mechanism of ASA action on plasma coagulation
and fibrinolysis factors, however, has not so far been
completely elucidated.
We have used ASA, a model drug that influences both
coagulation and fibrinolysis, to check whether the CLtest is sensitive enough to catch its induced changes in
clot formation, stabilization and lysis. In the system
studied, ASA concentrations of 100 and 200 mmol/l
appeared to have a statistically significant influence on
CLAUC. Shortening of the clot stabilization time (#Tc)
and reduction of the area under the stable clot formation
curve (#Sc), when the final fibrin organization and stabilization occur, as well as fibrinolysis acceleration (#Tl, #Sf)
and reduction of the area under the clotting curve (#Sr)
affected the statistically significant decrease in CLAUC at
the concentration of 200 mmol/l.
In our experimental conditions we have found that the
ASA concentration of 200 mmol/l has the strongest action,
while higher (300 mmol/l) and lower (50 mmol/l) concentrations have not caused substantial changes in the
effect observed.
Preliminary
results
obtained
with water-soluble
(Aspisol) confirmed
dose–response to ASA (data not shown).
D,L-lysin-mono-(acetylsalicylate)
When assessing sensitivity of the CL-test to the different
concentrations of thrombin and t-PA, we observed
obvious interdependence that changes in the clot formation and fibrinolysis curves are correlated with changes
The results of our experiments are also supported by the
study of Bjornsson et al. [26]. They observed fibrinolysis
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Test of clot formation and fibrinolysis Kostka et al. 617
acceleration, manifested by shortening of the clot lysis
time, after treatment with aspirin. What is more, those
authors reported an inverse dependence between the
fibrinogen acetylation level and clot lysis time in in-vitro
studies [26].
Numerous controversies on ASA’s effect on fibrinolysis
[6] exist in the literature. Some of the in-vitro studies
provide evidence that low ASA concentrations do not
have any influence on fibrinolytic action, whereas
high concentrations inhibit this process [6]. In other
studies, the application of low aspirin doses has been
found to impair thrombin generation and thrombincatalyzed reactions at the moment of blood vessel
damage [27,28].
Antovic et al. [8] examined the influence of various ASA
doses (37.5, 320 and 960 mg daily) on fibrin gel porosity,
and they observed that low doses of ASA induce more
favorable changes in the fibrin network than the higher
ones. Williams et al. [13] also proved that low-dose ASA
ingestion (75 mg daily) results in increased fibrin gel
porosity in healthy individuals. Moreover, they found a
tendency towards less efficient blockade of thromboxane formation with higher ASA dose (320 mg daily).
ASA application may change the clot structure, the
velocity of clot formation and fibrinolysis due to lysine
acetylation present in the structure of prothrombin,
fibrinogen and antithrombin III. Lysine occurring in
fibrinogen is essential to form cross-link bonds,
mediated by factor XIII, between the fibrin fibers. Its
acetylation results in blocking bond formation and
decreasing clot stabilization [14]. Acetylation of too
many lysine residues, however, may result in a suboptimal change of the fibrinogen molecule [13]. ASA
slows down conversion of prothrombin into thrombin,
which contributes to formation of thicker fibers and clot
structure loosening [12,29]. These changes enable t-PA
to penetrate the clot and finally to enhance fibrinolysis,
as lysis of clots with loose fibrin conformation made of
thick fibers is faster than those with tight fibrin conformation made of thin fibers [23].
He et al. [30] studied the effect of ASA on fibrinous clot
formation in vitro using the optical method and
pure fibrinogen, thrombin, calcium ions and factor XIII.
ASA was shown to reduce maximum absorbance in a
dose-dependent manner, and to prolong the coagulation time. Together with the increase in ASA concentration, a decline in the amount of the clot-insoluble
proteins affected by the alkaline urea solution was
observed. These observations corroborate the inhibitory properties of ASA in relation to factor XIII activity
[30].
In conclusion, the CL-test may be applied in drug screening tests evaluating their influence on the process of
fibrinous clot formation and lysis (compounds that affect
thrombin, fibrinogen and fibrinolysis). This has been
confirmed by our studies with ASA, which revealed that
the drug decreases overall clot formation and fibrinolysis
potential (CLAUC), probably by clot structure changes
that lead to fibrinolysis acceleration. The use of the
computer program to determine coagulation and fibrinolysis kinetic parameters, as well as the overall evaluation
of subsequent phases of the processes through calculation
of the area under the recorded curve, provide the possibility of a rapid and simple assessment of the activity of the
compound studied.
Acknowledgement
The present study was supported by grant No. 502-13229 of the Medical University of Łódź, Poland.
References
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Sidelmann JJ, Gram J, Jespersen J, Kluft C. Fibrin clot formation and lysis:
basic mechanisms. Semin Thromb Hemost 2000; 26:605–618.
Goldenberg NA, Hathaway WE, Jacobson L, Manco-Johnson MJ. A new
global assay of coagulation and fibrinolysis. Thromb Res 2005; 116:345–
356.
Glover D, Warner ED. The CLUE test – a multiparameter coagulation and
fibrinolysis screening test using the platelet aggregometer. Am J Clin
Pathol 1975; 63:74–80.
He S, Bremme K, Blombäck M. A laboratory method for determination of
overall haemostatic potential in plasma. I Method design and preliminary
results. Thromb Res 1999; 96:145–156.
He S, Antovic A, Blombäck M. A simple and rapid laboratory method for
determination of hemostasis potential in plasma. II Modifications for use in
routine laboratories and research work. Thromb Res 2001; 103:355–361.
Buczko W, Mogielnicki A, Kramkowski K, Chabielska E. Aspirin and the
fibrinolytic response. Thromb Res 2003; 110:331–334.
Antovic A, Blomback M, Sten-Linder M, Petrini P, Holmstrom M, He S.
Identifying hypocoagulable states with a modified global assay of overall
haemostasis potential in plasma. Blood Coagul Fibrinolysis 2005;
16:585–596.
Antovic A, Blomback M, Bremme K, He S. The assay of overall haemostasis
potential used to monitor the low molecular mass (weight) heparin,
dalteparin, treatment in pregnant women with previous thromboembolism.
Blood Coagul Fibrinolysis 2001; 13:181–186.
Curnow JL, Morel-Kopp M-C, Roddie C, Aboud M, Ward CM. Reduced
fibrinolysis and increased fibrin generation can be detected in
hypercoagulable patients using the overall hemostatic potential assay.
J Thromb Haemost 2007; 5:528–534.
Cerletti C, Dell’Elba G, Pecce R, Di Castelnuovo A, Scorpiglione N,
Feliziani V, de Gaetano G. Pharmakokinetic and pharmacodynamic
differences between two low dosages of aspirin may affect therapeutic
outcomes. Clin Pharmakokinet 2003; 42:1059–1070.
Hankey G, Eikelboom JW. Aspirin resistance. Lancet 2006; 367:606–617.
Antovic A, Pernrby C, Jakobsson EG, Wallen HN, Hejemadahl P, Blomback
M, He S. Marked increase of fibrin gel permeability with very low dose ASA
treatment. Thromb Res 2005; 116:509–517.
Williams S, Fatah K, Hjemdahl P, Blomback M. Better increase in fibrin gel
porosity by low dose than intermediate dose acetylsalicylic acid. Eur Heart J
1998; 19:1666–1672.
Undas A, Sydor WJ, Brummel K, Musiał J, Mann KG, Szczeklik A. Aspirin
alters the cardioprotective effects of the factor XIII Val34Leu polymorphism.
Circulation 2003; 107:17–20.
Kostka B, Sikora J, Para J, Krajewska U, Korzycka L. A New nitrate derivative
of piperazine: its influence on platelet activity. Blood Coagul Fibrinolysis
2007; 18:151–156.
Kostka B, Sikora J, Aranowska K, Para J, Ochocki J. Effect of cis-platinum(II)
amine phosphonate complexes on in vitro platelets reactivity. Acta Toxicol
2005; 13:113–121.
Łopaciuk S. Blood coagulation tests. In: Pawelski S, editor. Laboratory
diagnostics in haematology [in Polish]. Warsaw: PZWL; 1990. pp. 226–
227.
Sodha NR, Boodhwani M, Bianchi C, Ramlavi B, Sellke FW. Aprotinin in
cardiac surgery. Expert Rev Cardiovasc Ther 2006; 4:151–160.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
618 Blood Coagulation and Fibrinolysis 2007, Vol 18 No 7
19
20
21
22
23
24
25
26
27
28
29
30
Kostka B, Michalska M, Krajewska U, Wierzbicki R. Blood coagulation
changes in rats poisoned with methylmercuric chloride (MeHg). Pol J
Pharmacol Pharm 1989; 41:182–189.
Kupryś I, Kurowski M, Kuźminska B, Zielińska-Wyderkiewicz E, Górski P,
Kuna P. Basophil degranulation test in the diagnosis of nonsteroidal antiinflammatory drug hypersensitivity [in Polish]. Pol Merkuriusz Lek 2005;
18:540–543.
Carr ME Jr, Alving BM. Effect of fibrin structure on plasmin-mediated
dissolution of plasma clots. Blood Coagul Fibrinolysis 1995; 6:567–573.
Weisel JW, Nagasawami C. Computer modeling of fibrin polimeryzation
kinetics correlated with electron microscope and turbidity observations:
clot structure and assembly are kinetically controlled. Biophys J 1992;
63:111–128.
Collet JP, Park D, Lesty C, Soria C, Soria G, Montalescot G, Weisel JW.
Influence of fibrin network conformation and fibrin fiber diameter on
fibrinolysis speed: dynamic and structural approaches by confocal
microscopy. Arterioscler Thromb Vasc Biol 2000; 20:1354–
1361.
Patrono C. Aspirin as an antiplatelet drug. N Engl J Med 1994; 330:1287–
1294.
Lopez-Ferre A, Caramelo C, Esteban A, Alberola ML, Millas I, Monton N,
Casado S. Effects of aspirin on platelet–neutrofil interactions. Circulation
1995; 91:2080–2088.
Bjornsson TD, Schneider DE, Berger H Jr. Aspirin acetylates fibrinogen
and enhances fibrinolysis. Fibrinolytic effect is independent of changes in
plasminogen activator levels [abstract]. J Pharmacol Exp Ther 1989;
250:154–161.
Undas A, Brummel K, Musial J, Mann KG, Szczeklik A. Blood coagulation at
the site of microvascular injury: effects of low-dose aspirin. Blood 2001;
98:2423–2431.
Szczeklik A, Musiał J, Undas A, Swadzba J, Gora PF, Piwowarska W,
Duplaga M. Inhibition of thrombin generation by aspirin is blunted in
hypercholesterolemia. Arterioscler Thromb Vasc Biol 1996; 16:948–954.
Veklich Y, Francis CW, White J, Weisel JW. Structural studies of
fibrinolysis by electron microscopy. Blood 1998; 92:4721–4729.
He S, Blombäck M, Yoo G. Modified clotting properties of fibrinogen in
presence of acetylsalicylic acid in purified system. Ann N Y Acad Sci 2001;
936:531–535.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.