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Automated fluorogenic methods for the evaluation of the extrinsic coagulation reactions in human plasma
THROMBOSIS RESEARCH 50; 77-90, 1988 0049-3848188 $3.00 + .OO Printed in the USA. Copyright (c> 1988 Pergamon Press plc. All rights reserved.
AUTOMATED FLUOROGENIC METHODS FOR THE EVALUATION OF THE EXTRINSIC COAGULATION REACTIONS IN HUMAN PLASMA
Hisao Kato and Kagehiro Uchida National Cardiovascular Center Research Institute and Clinical Laboratory, Fujisirodai-5, Suita, Osaka 565, Japan (Received 18.9.1987; Accepted in revised form 21.10.1987 by Editor H. Yamasaki) (Received in final form by Executive Editorial Office 11.1.1988)
ABSTRACT Highly sensitive automated methods for the evaluation of the extrinsic coagulation reactions in human plasma were developed by the combination of fluorogenic peptide substrate(MCA) for thrombin and a centrifugal autoanalyzer (Cobas Bio). Prothrombin time (PT) was measured by the reaction time to reach 0.1 relative fluorescence which was caused by the action of thrombin generated after the activation of 3 ~1 plasma with human placental tissue factor (Thromborel S). Factors X and VII contents in plasma were measured by the same method after mixing diluted plasma with each factor deficient plasma, tissue factor,calcium and MCAinwhich lo-800 % of each factor was quantitatively measured. Prothrombin content in plasma was quantitated by measuring thrombin activity after the activation with human activated Factor X in the presence of phospholipid and calcium in which 10 -160 % of prothrombin was measured.Bythe application of these fluorogenic methods to the patients with cardiovascular diseases, it was demonstrated that these methods are highy sensitive not only to hypocoagulable state, but also to hypercoagulable state, particularly to the increase of Factors X and VII concentrations in plasma. KEYWORDS; Fluorogenic Automation.
substrate. Partial thromboplastin time. Hypercoagulable state. Extrinsic reaction.
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INTRODUCTION Chromogenic assay methods for coagulation factors and for activated partial thromboplastin time and prothrombin time have been developed using peptidyl-p-nitro anilide substrates for thrombin (l-9). These methods were proved to be useful for automation comparing with the conventional methods to detect fibrin formation. However, these methods reported so far are sensitive only to the hypocoaulable state due to the decrease of coagulation factors. Therefore, they are not clinically applicable to the detection of hypercoagulable state in thrombotic diseases. We devised the automated methods using fluorogenic substrate for thrombin, particularly to develop the sensitive method for hypercoagulable state in human plasma. In the present study, we developed the automated fluorogenic methods for the evaluation of the extrinsic pathway, viz., the measurement of prothrombin time of plasma and measurements of functional activities of Factors X and VII and prothrombin in plasma. These fluorogenic automated methods were applied to the analysis of plasmas from the patients with cardiovascular diseases and it was demonstrated that they are highly sensitive to hypercoagulable state, particulary to the increase of Factors VII and X concentrations in plasma.
MATERIALS AND METHODS The following materials were purchased from the manufacturers as follows: bovine serum albumin from Sigma Chemical Co., 8t. and Louis, fluorogenic peptide substrates (Boc-Val-Pro-Arg-MCA Boc-Ile-Glu-Gly-Arg-MCA-) from Peptide Research Institute, Osaka, human placental tissue factor (Thromborel S) from Behringwerke AG, Marburg, West Germany, specific factor-deficient plasmas, Protein C-depleted plasma and Warfarin-treated plasma from George King Biomedical, Overland Park, KS, fibrinogen grade L from AB Kabi, Stockholm, Sweden. Purification of Factor X and Factor IX (10) and prothrombin (11) from human plasma was performed as reported previously. Factor VII was isolated by DE-52 ion-exchange column chromatography (11) and gel-filtration on Sephacryl S-200 column. The specific activities ( unit per absorbance unit) of the purified proteins and contamination of each factors in these preparations are shown in Table I. Activated Factor X, Xa, was prepared by the activation of Factor X with an activator from Russell's Viper venom which was kindly supplied by Prof. T. Morita, Meiji College of Pharmacy. The spesific activity of Factor Xa was calculated to be 1.36 umole AMC per min per absorbance unit at 280 nm toward Boc-Ile*MCA; 4-methylcoumaryl-7-amide,
AMC;
7-amino-4-methylcoumarin.
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Glu-Gly-Arg-MCA. The protein concentration was calculated from the extinction coefficient to be 9.6 (12). Phospholipid suspension was made by dissolving one vial of rabbit brain cephalin (Sigma Chemical Co.) in 10 ml of saline and stored at 20°C in aliquots. Tissue factor suspension was made by dissolving in 4 ml of distilled water and stored at one vial of ThromborelS -7O'C in aliquots. Buffers used are 0.02 M Tris-HCl, pH 8.0, containing 0.15 M NaCl (Tris-saline) and the buffer containing 0.1 mg of bovine serum albumin per ml (Tris-saline-BSA). All the experiments using fluorogenic peptide were performed at 37°C using a centrifugal autoanalyzer (Cobas Bio) with FIA module. Relative fluorescence intensity was expressed using 0.1 mM AMC as a standard with excitation at 380 nm and filter position 2. Fluorescence readings at 60 or 30 set intervals were sent to a computer, NEC PC-9801Vm, and were transformed into reaction times which were defined as the time elapsed to reach 0.1 relative fluorescence, using a program developed by us. Venous blood was drawn from normals and patients into a siliconized Venoject tubes (Terumo Co., Ltd., Tokyo) and anti-coagulated with sodium citrate (0.38 % final concentration). Plasma was prepared by centrifugation at 2300g x 15 min at room temperature and stored at -70°C until use. Pooled plasma was prepared from 10 normal individuals and used as control plasma. TABLE I Activities of Factors X, VII and IX and Prothrombin This Paper. Preparation Factor X Factor IX Factor X Factor VII Prothrombina
3 100 (78) N.D. N.D.
Activity Factor IX Prothrombin 100 (26.7)
Used in
Factor VII
N.D.
N.D.
1
N.D.
0.1
2.4
N.D.
100 (85)
N.D.
100 (20.1)
N.D.
Each value represents relative activity, taking the specific activity of each factor shown in parentheses as 100. N.D.; not detectable. a; specific activity of thrombin derived from prothrombin by the action of Factor Xa was calculated to be 89.8 umoles AMC per absorbance unit toward Boc-Val-Pro-Arg-MCA. Fluorogenic Prothrombin Time (FPT) Three 1.11 plasma was mixed with 300 ulof reagent for FPT which was made by mixing 5 ul of Thromborel S, 100 ul of phospholipid suspension, 1 ml of 0.02 M CaCl and 10 ml of Tris-saline. Then, 3 ul of 10 mM Boc-ValPro- z rg-MCA (dissolved in Tris-saline) was added. Fluorogenic Assay Methods for Factor X and Factor VII in Plasma Thirty ~1 of one-hundredth-diluted plasma was mixed with ~-
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300 ~1 of reagent which was made by mixing 120 ~1 of Factor Xdeficient or Factor VII-deficient plasma with 6 ml of Trissaline-BSA. Then, 75 ~1 of starting reagent which was made by mixing 400 1.11 of Thromborel S, 3 ml of 0.02 M CaC'12 and 300 )..! of 10 mM Boc-Val-Pro-Arg-MCA was added. Standard curves for each factors were obtained using diluted control plasma, assuming that ten-fold diluted plasma contains 100 % coagulation factors. Measurement of Prothrombin in Plasma Five ~1 of one-hundredth-xilutedplasma was mixed with 300 r;T-of reagent which was made by mixing 9 ml of Tris-saline-BSA, 10 1.11 of human Factor Xa (A2So=0.025), 0.1 ml of phospholipid suspension and 1 ml of 0.02 M CaC12: Then, 3 ~1 of 10 mM Boc-Val-Pro-Arg-MCA was added. Conventional PT Method In a disposable plastic sample cup for KC-10 coagulomet~~inrich Amelung GmbH, West Germany), 0.1 ml of plasma was prewarmed at 37°C for 1 min. After adding 0.2 ml of Thromborel S suspension, clotting time was measured using KC-1C coagulometer. Data were converted to per cent activity of standard human plasma from Behringwerke AG, Marburg, West Germany.
RESULTS FPT of Normals, Patients and Coagulation Factor-deficient Plasmas Fig. 1 shows the increase of fluorescence intensity by fluorogenic PT(FPT) method of normal pooled plasma and coagulation factors-deficient plasmas. Thrombin activity was not generated significantly in Factor V-, Factor VII- and Factor Xdeficient plasmas and Warfarin-treated plasma in this experimental conditions. On the other hand, thrombin generation in the intrinsic coagulation factors-deficient plasmas was delayed to some extent. As shown in Fig. 2, the addition of Factor X to control plasma or Factor X-deficient plasma accelerated significantly their FPT. The addition of Factor VII moderately accelerated the FPT of control and Factor VII deficient plasmas. The addition of prothrombin did not accelerated significantly FPT of normal plasma. Although the generation of thrombin in Factor IX-deficient plasma was significantly slower than control plasma (Fig. l), the addition of Factor IX to control plasma and Factor IX-deficient plasma did not accelerated the FPT of each plasma. These results indicate that the FPT method is sensitive not only to the decrease of the concentration of Factors X and VII and prothrombin, but also sensitive to the increase of the concentration of Factor X and Factor VII in plasma. FPT of fibrinogen-deficient plasma did not change by the addition of fibrinogen (data not shown). The precision study of FPT method is shown in Table II. When the FPT method was applied to patient plasmas and FPT was calculated from the incubation time to reach 0.1 relative fluorescence, many plasmas from the patients with cardiovascular diseases gave shorter FPT than control plasma. Generally, PT has
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been expressed as per cent of control plasma by calculating from the standard curve which was obtained by diluting the control plasma with buffer. Therefore, it is impossible to calculate PT which is shorter than control plasma. We divided FPT of control and expressed as per cent for plasma with FPT of patient plasma convenience. The per cent values of each plasma which were thus calculated with two different concentrations of tissue factor were approximately the same in spite of the different reaction times as shown in Table III. The FPT's of patient plasmas were then compared with PT of the same plasmas which were determined by conventional PT method. The per cent values of patient plasmas from the two methods which were calculated by dividing clotting time or reaction time of control plasma with that of patient plasma were correlated with the coefficient of 0.90, as shown in Fig. 3. However, 9 samples gave more than 150 % in FPT, while they were between100 and130 % in PT and therefore, they were excluded for the calculation of the correlation coefficient. This result indicates that the FPT method is sensitive not only to hypocoagulability, but also to hypercoagulability, while the conventional PT method is not relevant for the detection of hypercoagulability.
INCUBATION
TIME
(min)
FIG. 1 FPT of coagulation factor deficient plasmas. (1) protein C depleted plasma, (2) control plasma, (3) Factor XI deficient plasma, (4) prekallikrein deficient plasma, (5) fibrinogen, Factor VIII and HMW kininogen deficient plasmas, (6) Factor XII deficient plasma, (7) Factor IX deficient plasma, (8) Factor V deficient plasma and (9) Factor VII and Factor X deficient plasmas and Warfarin-treated plasma.
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TABLE II Precision Studies Methods within Run.
Mean
N=lO (set)
with
SD
CV
the Automated
(%)
FPT
Factor
249.8 308.1 440.0 619.8 1063.0 1382.1
6.0 9.8 7.7 14.9 10.1 13.6
2.4 3.2 1.7 2.4 1.0 1.0
X assay 244.5 298.7 410.3 487.8 561.8 859.1 1286.1
2.0 2.7 2.5 3.3 3.5 7.4 17.3
0.8 0.9 0.8 1.0 0.6 0.9 1.3
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Fluorogenic
N=8 Relative Fluorescencea) Prothrombin 0.122 0.075 0.043
SD
assay 0.0040 0.0017 0.0045
W(8)
3.2 2.2 10.5
a) prothrombin concentration in plasma was determined as described in METHCDS and relative fluorescence intensity after 5 min incubation was calculated. TABLE III FPT of Patient Plasmas as Per Cent of Control (1) No.
1
2
8 9
10 11
Reaction time 1303 911 779 675 550 490 522 437 383 343 206
as Expressed Plasma (2)
%
40 57 67 77 94 106 100 119 136 152 254
Reaction time 976
661 595 453 392 358 353 294 274 237 147
%
36 53 59 78 90 99 100 120 129 149 241
FPT reagent was made by mixing 5 1.11(1) or 10 1_t1(2)of Thromborel S solution with 100 1.11of phospholipid solution, 1 ml pf 0.02 M Percentage was calculated by CaC12, and 10 ml of Tris-saline. dividing reaction time of control plasma (No. 7) with reaction time of sample plasma.
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o-
83
i
012
34 FACTOR
5
VII
0
I2345 FACTOR
(winI)
X
lu/ml)
PROTHRCMBlN
(u/ml1
FACTOR
IX
(u/ml)
FIG. 2 Effect of the purified Factors VII, X and IX and prothrombin on FPT of control plasma and each factor-deficient plasmas. The purified Factor VII, Factor X, prothrombin or Factor IX was mixed with normal plasma ( G ) or each factor-deficient plasma(+) and their FPT's were calculated as described in METHODS.
200
0, 0
40 FPT
60 (%I
120
160
200
240
FIG. 3 Correlation of FPT with conventional PT. Percentages of PT of patients which were calculated by dividing the clotting time of control plasma with that of patient plasma were compared with the percentages of FPT which were calculated by dividing reaction time of control plasma with that of patient plasma.The correlation of the samples( n=75) was calculated as follows; Y = 0.58 X + 34, r=0.895 0; samples excluded for the calculation.
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Measurements of Factor X, Factor VII and Prothrombin in Plasma Fig. 4 shows the generation of thrombin activity in the mixture of diluted control plasma and Factor X-deficient plasma (A) and the calibration curves for Factor X and Factor VII in plasma (B). Ten-fold diluted control plasma was assumed to contain 100 % of Factor X or Factor VII.Each diluted plasma was mixed with each factor-deficient plasma and the reaction time for the generation of thrombin activity was calculated as described in METHODS. Figure 4(B) shows that the concentration of Factor X and Factor VII in plasma can be measured from 10 5;to 800 8 by this method. The precision study with this method was performed on Factor X assay and shown in Table II.
A
0
20 TIME
IO INCUBATION
CONCENTRATION
OF
FACTORS
30 (mid
VII
AND
X
(X)
FIG. 4 Standard curves for the estimation of Factor X and Factor VII in plasma. Control plasma was diluted with Tris-saline-BSA and the concentration of each coagulation factors were measured using each coagulation factor deficient plasma as described in METHODS. (A) Generation of thrombin activity in the mixture of diluted control plasma and Factor X deficient plasma. (B) Standard curves for Factors X and VII. Factor X ;-O_ Factor VII;+
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Fig. 5 shows calibration curve for prothrombin in plasma. Prothrombin was activated by Factor Xa in the presence of phospholipid and calcium as described in METHODS. After the addition of Boc-Val-Pro-Arg-MCA, fluorescence intensity increased linearly as shown in Fig. 5(A). The concentration of lo-fold diluted control plasma was defined as 100 %. Linear standard curve was obtained from 10 % to 160 % as shown in Fig. 5(B). The addition of further phosphlipid and Factor Xa did not increase significantly generation of thrombin activity. Shorter preincubation time decreased generation of thrombin activity. Since the amounts of Factor V in samples were supposed to affect the generation of thrombin activity in this experimental condition, various amounts of prothrombin-deficient plasma were added to Factor V-deficient plasma and thrombin activity was measured. The result indicated that the presence of 20 % Factor V was enough for the maximum generation of thrombin in the experimental condition, assuming that Factor V concentration in prothrombin-deficient plasma was 100 % ( data not shown). These results indicate that prothrombin concentration in plasma can be quantitatively measured using this method, unless Factor V concentration in samples is quite low. Precision of this method is shown in Table II.
,(B)
, INCUBATION
TIME
(mln)
50
CONCENTRATION
100
150
OF PROTHROMBIN
WJ
FIG. 5. Calibration curve for prothrombin in plasma. Diluted normal plasma was activated with Factor Xa and thrombin activity generated was measured as described in METHODS. The fluorescence intensity due to AMC increased linearly with the incubation time (A) and the intensity after 5 min incubation was plotted versus concentration of prothrombin assuming that ten-fold diluted plasma contains 100 % of prothrombin (B).
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Factors X and VII and Prothrombin in Plasmas from Patients with Cardiovascular Diseases The automated fluorogenic methods for the evaluation of Factors X and VII and orothrombin in plasma as described in the previous section were applied to the analysis of plasmas from the inpatients with cerebral and myocardial infarctions. Eighty one patients plasmas were classified into two groups, one with higher FPT than 120 % and another with lower FPT than 80 %. As shown in Fig. 6, the concentrations of Factors X and VII and prothrombin in these two groups were compared with those in normal plasmas. The concentrations of Factors X and VII and prothrombin in a group with higher FPTthan 120 % were almosthigherthan100 %,while other group with lower FPT than 80 % showed lower concentrations than 100 %. In the latter group, plasmas from warfarin-treated patients showed lower concentrations than 50 %. These results demonstrate that concentrations of Factors X and VII and prothrombin are higher in plasmas with shorter FPT and lower in plasmas with longer FPT, as expected. These results also support that the automated fluorogenic PT method is sensitive to the hypercoagulable state due to the increase of Factor VII and Factor X or prothrombin, in addition to the hypocoagulable state due to the decrease of these coagulation factors.
(Cl
(6)
: :. . i
f 7 :
&
:
.
i
i
;
p
. .
. I
VII
x
II
VII
x
II
FIG. 6 Concentration of Factors X and VII and prothrombin in patients plasmas and normal plasmas. Concentrations of Factors VII and X were measured using deficient plasma as described in METHODS. Prothrombin was measured after the activation with Factor Xa as described in METHODS. (A): Patient plasmas with higher FPT than 120 %. (B): Patient plasmas with lower FPT than 80 8. (C): Normal plasmas.
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DISCUSSION Thrombosis is caused by complicated in vivo reactions which involve the damage or loss of endothelial cells, activation of platelets and leukocytes, activation of coagulation and fibrinolysis systems in plasma and alteration of their regulation systems. For the prognosis, diagnosis and therapy of the thrombotic diseases of cerebral and coronary arteries and lower limbs veins, the development of the highly sensitive method to detect the prethrombotic state is essential, together with the analysis of the prethrombotic state which precedes thrombus formation. In spite of the contribution of the hypercoagulability in plasma to the prethrombotic state, the conventional PT and APTT and also the chromogenic method recently developed, are not relevant to detect the hypercoagulability. The chromogenic substrates are now widely used in clinical laboratory for the estimation of coagulation factors and proved to be useful for Factor X and prothrombin. However, it is not still possible to estimate directly other coagulation factors, since the specific and sensitive substrates have not been developed except Factor Xa and thrombin. Even the estimation of Factor X and prothrombin is required to be improved to measure their functional activities, particularly in Warfarin-treated plasma. In the present work, we intended to develop a sensitive automated methods to estimate hypercoagulability, using fluorogenic peptide substrate for thrombin and a centrifugal autoanalyzer. This paper described a fluorogenic PT method to detect hypercoagulability of plasma and sensitive method for the estimation of functional activities of Factor X, Factor VII and prothrombin. In the fluorogenic PT method, the amounts of tissue factor (Thromborel S) were smaller than those used in the conventional and chromogenic PT methods, so that, the lag time before the generation of thrombin was longer. In the FPT, thrombin was not generated significantly in Factors V-, X- and VII-deficient plasmas. Althogh the generation of thrombin in the intrinsic factors-deficient plasmas, was delayed to some extent comparing withthatof control plasma, it may not be due to the deficiency of each factor. It may be rather due to the lower content of Factor V or Factor VIII than those of control plasma. In fact, FPT of Factor IX-deficient plasma was not accelerated by the addition of Factor IX. In the chromogenic methods, turbidity due to the fibrin formation disturbed the spectrophotometric mesurement. In the FPT, plasma was diluted 100-fold, therefore, no visible fibrin formation was observed. The effect of fibrinogen on thrombin activity toward fluorogenic subtrate is supposed to be negligible, since the final concentration of fluorogenic substrate is lO,OOO-fold higher than that of fibrinogen, while Km values of thrombin toward both of substrates are comparable (13). In the present paper, FPT was expressed as per cent of control plasma by dividing FPT of control plasma with that of sample
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plasma. We used the calculation method for convenience, since the method for hypercoagulable plasma has not been established. However, the per cent values for each plasma were approximately the same in each experiment with different concentration of tissue factor, in which the reaction time of control plasma was divided with the reaction time of each plasma. The percentage of each patient plasma correlated with that from the conventional PT with high correlation coefficient if the per cent values higher than 150 % in FPT were excluded. The sensitivity of the FPT to hypercoagulable state was demonstrated by the findings that the reaction times of FPT of control plasma were accelerated by the addition of Factors VII and X and that 25 out of 81 patient plasmas gave more than 120 % of FPT, whereas they were less than 120 % in the conventional PT method. On the other hand, the FPT method was also sensitive to hypocoagulability as shown by no generation of thrombin in Warfarin-treated plasma, although less than 30 % in the conventional PT could not be measured. The concentarations of Factors VII and X in plasma were measured by the application of FPT method. One of the characteristics of the method is the automated measurement of the functional activities of Factors VII and X using small amounts of sample (30 1.11 of 100-fold diluted plasma) and small amounts of factors-deficient plamsas ( 6 ul for each sample). By the methods, linear standard curves were obtained from 10 % to 800 % by the dilution of control plasma. The concentration of prothrombin in plasma was measured after the activation of prothrombin by Factor Xa and linear standard curve was obtained from 10 % to 160 %. In the method, Factor V was not added, because the presence of 20 % of Factor V in plasma was sufficient for the maximum activation of prothrombin in plasma. Factor X and prothrombin in plasma have been measured using chromogenic substrates after the activation with enzymes from snake venoms. However, these methods do not necessarily measure the functional activities of prothrombin and Factor X. The functional activities of prothrombin and Factor X have been measured recently using chromogenic substrates after the addition of tissue factor (14). This method depends on the concentration of Factors VII and or X andV, which has been overcomed by the addition of Factor VII- or Factor X-deficient plasma. The present fluorogenic methods are similar to the above method in principle, but, the functional activities of Factors X and VII and prothrombin were measured using the fluorogenic substrate for thrombin after the addition of deficient plasmas and tissue factor or after the activation by Factor Xa. The fluorogenic methods for PT and for the estimation of Factors X and VII and prothrombin concentrations in plasma were applied to the analysis of the hypercoagulability of plasmas from patients with cardiovascular diseases. Twenty five samples out of 81 samples gave higher FPT than 120 %. The concentration of Factors X and VII and prothrombin in those plasmas were higher than those of normal plasmas. On the other hand, their concentrations in the samples with lower FPT than 80 % were lower than those of normal plasmas. As described above, FPT was
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sensitive to the increase of the concentration of Factors X and VII, but not prothrombin. Therefore, the hypercoagulability of many samples as shown by the shorter FPT is supposed to be in part due to the increase of the concentration of Factors X and VII. These results indicate that the fluorogenic methods are useful for the analysis of hypercoagulability in plasma. However, it is clear that other factors in plasma, particulary, Factor V, protein C and protein S and protease inhibitors such as antithrombin III, may affect the hypercoagulability in plasma, hence, FPT. The effect of these factors on FPT remains to be established. ACKNOWLEDGMENTS This study was supported in part by a Grant-in-Aid for Scientific Research from Japan Foundation for Applied Enzymology. The authors wish to thank Dr. Junzo Kodama, Hoken Iryo center, Sanyo Denki Co., Ltd. for helpful discussion and suggestion during this work and Mr. Yoshiyuki Shigeyasu of Baxter-Travenol, Japan for developing the computer program to calculate reaction times of FPT.
REFERENCES 1. ZOLTON, R. P. Synthetic peptide substrate assays for hemostasis testing. Advances -in Clin. Chem., 25, 117-168, 1986. 2. YAMADA, K. and MEGURO, T. A new APTT assay employing a chromogenic substrate and a centrifugal autoanalyzer. Thrombos. Res., 15, 351-358, 1979. 3. BAUGHMAN, D. J. and LYTWYN, A. Thrombin activation rate constant: one-stage chromogenic assay for the extrinsic system. Thrombos. Res., 26, l-12, 1982. 4. BECKER, U., JERING, H., BAETL, K. and JILEK, F. Automated prothrombin time test with use of a chromogenic peptide substrate and a centrifugal analyzer. Clin. Chem., 30, 524-528, 1984. 5. DUNCAN, A., BOWIE, E. J. W., OWEN, Jr., C. A. and FASS, D. N. A clinical evaluation of automated chromogenic tests as substitutes for conventional prothrombin time tests. Clin. Chem., 2, 853-855, 1985. 6. BECKER, U., BARTL, K., LILL, H. and WAHEFELD, A. W. Development of a photometric assay for activated partial thromboplastin time and its application to the Cobas Bio centrifugal analyzer. Thrombos. Res., 40, 721-730, 1985. 7. KOLDE, H. J. Chromo time system-A new generation of coagulation analyses. Behring Inst. Mitt., No. 78, 176-187,
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1985.
8. DATI, F., KOLDE, H. J. and Heimburger, N. Methodische Aspekte zur photometrischen Bestimmung der Prothromibinzeit mittels chromogener Substrate. J. Clin. Chem. Clin. Biochem., 24, 877-888, 1986. - ___ -9. YAMAMOTO, J., ASADA, N., MIYATAKE, Y., MASUYA, M., YAMASHITA, T. and OKAMOTO, U. New modified activated partial thromboplastin time and prothrombin time method using a synthetic chromogenic substrate in comparison with diazotization. Thrombo. Res., 46, 225-231, 1987. 10.
THEODORSSON, B., HEDNER, U., NILSON, I. M., KISIEL , W. A technique for specific removal of factor IX alloantibodies from human plasma; partial characterization of the alloantibodies. Blood, 61, 973-981, 1983.
11.
MILETICH, J. P., BROZE, G. J., MAJERUS, P. W. The synthesis of sulfated dextran beads for isolation of human coagulation factors II, X and IX. Anal. Biochem., 105, 304-310, 1980.
12. JESTY, J. Measurement of the kinetics of inhibition of activated coagulation factor X in human plasma; The effect of plasma and inhibitor concentration. Anal. Chem., E, 402-411, 1986. 13. CHANG, J-Y. The structure and proteolytic specificities autolysed human thrombin. Biochem. J., 240, 797-802, 1986.
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14. ASAI, K. Vitamin K dependent factors-F.11, F.X. Rinsyo Byouri (The Japanese -J. of Clin. Pathol.), 4, No.70, 88-99, 1987.
AUTOMATED FLUOROGENIC METHODS FOR THE EVALUATION OF THE EXTRINSIC COAGULATION REACTIONS IN HUMAN PLASMA
Hisao Kato and Kagehiro Uchida National Cardiovascular Center Research Institute and Clinical Laboratory, Fujisirodai-5, Suita, Osaka 565, Japan (Received 18.9.1987; Accepted in revised form 21.10.1987 by Editor H. Yamasaki) (Received in final form by Executive Editorial Office 11.1.1988)
ABSTRACT Highly sensitive automated methods for the evaluation of the extrinsic coagulation reactions in human plasma were developed by the combination of fluorogenic peptide substrate(MCA) for thrombin and a centrifugal autoanalyzer (Cobas Bio). Prothrombin time (PT) was measured by the reaction time to reach 0.1 relative fluorescence which was caused by the action of thrombin generated after the activation of 3 ~1 plasma with human placental tissue factor (Thromborel S). Factors X and VII contents in plasma were measured by the same method after mixing diluted plasma with each factor deficient plasma, tissue factor,calcium and MCAinwhich lo-800 % of each factor was quantitatively measured. Prothrombin content in plasma was quantitated by measuring thrombin activity after the activation with human activated Factor X in the presence of phospholipid and calcium in which 10 -160 % of prothrombin was measured.Bythe application of these fluorogenic methods to the patients with cardiovascular diseases, it was demonstrated that these methods are highy sensitive not only to hypocoagulable state, but also to hypercoagulable state, particularly to the increase of Factors X and VII concentrations in plasma. KEYWORDS; Fluorogenic Automation.
substrate. Partial thromboplastin time. Hypercoagulable state. Extrinsic reaction.
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INTRODUCTION Chromogenic assay methods for coagulation factors and for activated partial thromboplastin time and prothrombin time have been developed using peptidyl-p-nitro anilide substrates for thrombin (l-9). These methods were proved to be useful for automation comparing with the conventional methods to detect fibrin formation. However, these methods reported so far are sensitive only to the hypocoaulable state due to the decrease of coagulation factors. Therefore, they are not clinically applicable to the detection of hypercoagulable state in thrombotic diseases. We devised the automated methods using fluorogenic substrate for thrombin, particularly to develop the sensitive method for hypercoagulable state in human plasma. In the present study, we developed the automated fluorogenic methods for the evaluation of the extrinsic pathway, viz., the measurement of prothrombin time of plasma and measurements of functional activities of Factors X and VII and prothrombin in plasma. These fluorogenic automated methods were applied to the analysis of plasmas from the patients with cardiovascular diseases and it was demonstrated that they are highly sensitive to hypercoagulable state, particulary to the increase of Factors VII and X concentrations in plasma.
MATERIALS AND METHODS The following materials were purchased from the manufacturers as follows: bovine serum albumin from Sigma Chemical Co., 8t. and Louis, fluorogenic peptide substrates (Boc-Val-Pro-Arg-MCA Boc-Ile-Glu-Gly-Arg-MCA-) from Peptide Research Institute, Osaka, human placental tissue factor (Thromborel S) from Behringwerke AG, Marburg, West Germany, specific factor-deficient plasmas, Protein C-depleted plasma and Warfarin-treated plasma from George King Biomedical, Overland Park, KS, fibrinogen grade L from AB Kabi, Stockholm, Sweden. Purification of Factor X and Factor IX (10) and prothrombin (11) from human plasma was performed as reported previously. Factor VII was isolated by DE-52 ion-exchange column chromatography (11) and gel-filtration on Sephacryl S-200 column. The specific activities ( unit per absorbance unit) of the purified proteins and contamination of each factors in these preparations are shown in Table I. Activated Factor X, Xa, was prepared by the activation of Factor X with an activator from Russell's Viper venom which was kindly supplied by Prof. T. Morita, Meiji College of Pharmacy. The spesific activity of Factor Xa was calculated to be 1.36 umole AMC per min per absorbance unit at 280 nm toward Boc-Ile*MCA; 4-methylcoumaryl-7-amide,
AMC;
7-amino-4-methylcoumarin.
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Glu-Gly-Arg-MCA. The protein concentration was calculated from the extinction coefficient to be 9.6 (12). Phospholipid suspension was made by dissolving one vial of rabbit brain cephalin (Sigma Chemical Co.) in 10 ml of saline and stored at 20°C in aliquots. Tissue factor suspension was made by dissolving in 4 ml of distilled water and stored at one vial of ThromborelS -7O'C in aliquots. Buffers used are 0.02 M Tris-HCl, pH 8.0, containing 0.15 M NaCl (Tris-saline) and the buffer containing 0.1 mg of bovine serum albumin per ml (Tris-saline-BSA). All the experiments using fluorogenic peptide were performed at 37°C using a centrifugal autoanalyzer (Cobas Bio) with FIA module. Relative fluorescence intensity was expressed using 0.1 mM AMC as a standard with excitation at 380 nm and filter position 2. Fluorescence readings at 60 or 30 set intervals were sent to a computer, NEC PC-9801Vm, and were transformed into reaction times which were defined as the time elapsed to reach 0.1 relative fluorescence, using a program developed by us. Venous blood was drawn from normals and patients into a siliconized Venoject tubes (Terumo Co., Ltd., Tokyo) and anti-coagulated with sodium citrate (0.38 % final concentration). Plasma was prepared by centrifugation at 2300g x 15 min at room temperature and stored at -70°C until use. Pooled plasma was prepared from 10 normal individuals and used as control plasma. TABLE I Activities of Factors X, VII and IX and Prothrombin This Paper. Preparation Factor X Factor IX Factor X Factor VII Prothrombina
3 100 (78) N.D. N.D.
Activity Factor IX Prothrombin 100 (26.7)
Used in
Factor VII
N.D.
N.D.
1
N.D.
0.1
2.4
N.D.
100 (85)
N.D.
100 (20.1)
N.D.
Each value represents relative activity, taking the specific activity of each factor shown in parentheses as 100. N.D.; not detectable. a; specific activity of thrombin derived from prothrombin by the action of Factor Xa was calculated to be 89.8 umoles AMC per absorbance unit toward Boc-Val-Pro-Arg-MCA. Fluorogenic Prothrombin Time (FPT) Three 1.11 plasma was mixed with 300 ulof reagent for FPT which was made by mixing 5 ul of Thromborel S, 100 ul of phospholipid suspension, 1 ml of 0.02 M CaCl and 10 ml of Tris-saline. Then, 3 ul of 10 mM Boc-ValPro- z rg-MCA (dissolved in Tris-saline) was added. Fluorogenic Assay Methods for Factor X and Factor VII in Plasma Thirty ~1 of one-hundredth-diluted plasma was mixed with ~-
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300 ~1 of reagent which was made by mixing 120 ~1 of Factor Xdeficient or Factor VII-deficient plasma with 6 ml of Trissaline-BSA. Then, 75 ~1 of starting reagent which was made by mixing 400 1.11 of Thromborel S, 3 ml of 0.02 M CaC'12 and 300 )..! of 10 mM Boc-Val-Pro-Arg-MCA was added. Standard curves for each factors were obtained using diluted control plasma, assuming that ten-fold diluted plasma contains 100 % coagulation factors. Measurement of Prothrombin in Plasma Five ~1 of one-hundredth-xilutedplasma was mixed with 300 r;T-of reagent which was made by mixing 9 ml of Tris-saline-BSA, 10 1.11 of human Factor Xa (A2So=0.025), 0.1 ml of phospholipid suspension and 1 ml of 0.02 M CaC12: Then, 3 ~1 of 10 mM Boc-Val-Pro-Arg-MCA was added. Conventional PT Method In a disposable plastic sample cup for KC-10 coagulomet~~inrich Amelung GmbH, West Germany), 0.1 ml of plasma was prewarmed at 37°C for 1 min. After adding 0.2 ml of Thromborel S suspension, clotting time was measured using KC-1C coagulometer. Data were converted to per cent activity of standard human plasma from Behringwerke AG, Marburg, West Germany.
RESULTS FPT of Normals, Patients and Coagulation Factor-deficient Plasmas Fig. 1 shows the increase of fluorescence intensity by fluorogenic PT(FPT) method of normal pooled plasma and coagulation factors-deficient plasmas. Thrombin activity was not generated significantly in Factor V-, Factor VII- and Factor Xdeficient plasmas and Warfarin-treated plasma in this experimental conditions. On the other hand, thrombin generation in the intrinsic coagulation factors-deficient plasmas was delayed to some extent. As shown in Fig. 2, the addition of Factor X to control plasma or Factor X-deficient plasma accelerated significantly their FPT. The addition of Factor VII moderately accelerated the FPT of control and Factor VII deficient plasmas. The addition of prothrombin did not accelerated significantly FPT of normal plasma. Although the generation of thrombin in Factor IX-deficient plasma was significantly slower than control plasma (Fig. l), the addition of Factor IX to control plasma and Factor IX-deficient plasma did not accelerated the FPT of each plasma. These results indicate that the FPT method is sensitive not only to the decrease of the concentration of Factors X and VII and prothrombin, but also sensitive to the increase of the concentration of Factor X and Factor VII in plasma. FPT of fibrinogen-deficient plasma did not change by the addition of fibrinogen (data not shown). The precision study of FPT method is shown in Table II. When the FPT method was applied to patient plasmas and FPT was calculated from the incubation time to reach 0.1 relative fluorescence, many plasmas from the patients with cardiovascular diseases gave shorter FPT than control plasma. Generally, PT has
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been expressed as per cent of control plasma by calculating from the standard curve which was obtained by diluting the control plasma with buffer. Therefore, it is impossible to calculate PT which is shorter than control plasma. We divided FPT of control and expressed as per cent for plasma with FPT of patient plasma convenience. The per cent values of each plasma which were thus calculated with two different concentrations of tissue factor were approximately the same in spite of the different reaction times as shown in Table III. The FPT's of patient plasmas were then compared with PT of the same plasmas which were determined by conventional PT method. The per cent values of patient plasmas from the two methods which were calculated by dividing clotting time or reaction time of control plasma with that of patient plasma were correlated with the coefficient of 0.90, as shown in Fig. 3. However, 9 samples gave more than 150 % in FPT, while they were between100 and130 % in PT and therefore, they were excluded for the calculation of the correlation coefficient. This result indicates that the FPT method is sensitive not only to hypocoagulability, but also to hypercoagulability, while the conventional PT method is not relevant for the detection of hypercoagulability.
INCUBATION
TIME
(min)
FIG. 1 FPT of coagulation factor deficient plasmas. (1) protein C depleted plasma, (2) control plasma, (3) Factor XI deficient plasma, (4) prekallikrein deficient plasma, (5) fibrinogen, Factor VIII and HMW kininogen deficient plasmas, (6) Factor XII deficient plasma, (7) Factor IX deficient plasma, (8) Factor V deficient plasma and (9) Factor VII and Factor X deficient plasmas and Warfarin-treated plasma.
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TABLE II Precision Studies Methods within Run.
Mean
N=lO (set)
with
SD
CV
the Automated
(%)
FPT
Factor
249.8 308.1 440.0 619.8 1063.0 1382.1
6.0 9.8 7.7 14.9 10.1 13.6
2.4 3.2 1.7 2.4 1.0 1.0
X assay 244.5 298.7 410.3 487.8 561.8 859.1 1286.1
2.0 2.7 2.5 3.3 3.5 7.4 17.3
0.8 0.9 0.8 1.0 0.6 0.9 1.3
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Fluorogenic
N=8 Relative Fluorescencea) Prothrombin 0.122 0.075 0.043
SD
assay 0.0040 0.0017 0.0045
W(8)
3.2 2.2 10.5
a) prothrombin concentration in plasma was determined as described in METHCDS and relative fluorescence intensity after 5 min incubation was calculated. TABLE III FPT of Patient Plasmas as Per Cent of Control (1) No.
1
2
8 9
10 11
Reaction time 1303 911 779 675 550 490 522 437 383 343 206
as Expressed Plasma (2)
%
40 57 67 77 94 106 100 119 136 152 254
Reaction time 976
661 595 453 392 358 353 294 274 237 147
%
36 53 59 78 90 99 100 120 129 149 241
FPT reagent was made by mixing 5 1.11(1) or 10 1_t1(2)of Thromborel S solution with 100 1.11of phospholipid solution, 1 ml pf 0.02 M Percentage was calculated by CaC12, and 10 ml of Tris-saline. dividing reaction time of control plasma (No. 7) with reaction time of sample plasma.
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o-
83
i
012
34 FACTOR
5
VII
0
I2345 FACTOR
(winI)
X
lu/ml)
PROTHRCMBlN
(u/ml1
FACTOR
IX
(u/ml)
FIG. 2 Effect of the purified Factors VII, X and IX and prothrombin on FPT of control plasma and each factor-deficient plasmas. The purified Factor VII, Factor X, prothrombin or Factor IX was mixed with normal plasma ( G ) or each factor-deficient plasma(+) and their FPT's were calculated as described in METHODS.
200
0, 0
40 FPT
60 (%I
120
160
200
240
FIG. 3 Correlation of FPT with conventional PT. Percentages of PT of patients which were calculated by dividing the clotting time of control plasma with that of patient plasma were compared with the percentages of FPT which were calculated by dividing reaction time of control plasma with that of patient plasma.The correlation of the samples( n=75) was calculated as follows; Y = 0.58 X + 34, r=0.895 0; samples excluded for the calculation.
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Measurements of Factor X, Factor VII and Prothrombin in Plasma Fig. 4 shows the generation of thrombin activity in the mixture of diluted control plasma and Factor X-deficient plasma (A) and the calibration curves for Factor X and Factor VII in plasma (B). Ten-fold diluted control plasma was assumed to contain 100 % of Factor X or Factor VII.Each diluted plasma was mixed with each factor-deficient plasma and the reaction time for the generation of thrombin activity was calculated as described in METHODS. Figure 4(B) shows that the concentration of Factor X and Factor VII in plasma can be measured from 10 5;to 800 8 by this method. The precision study with this method was performed on Factor X assay and shown in Table II.
A
0
20 TIME
IO INCUBATION
CONCENTRATION
OF
FACTORS
30 (mid
VII
AND
X
(X)
FIG. 4 Standard curves for the estimation of Factor X and Factor VII in plasma. Control plasma was diluted with Tris-saline-BSA and the concentration of each coagulation factors were measured using each coagulation factor deficient plasma as described in METHODS. (A) Generation of thrombin activity in the mixture of diluted control plasma and Factor X deficient plasma. (B) Standard curves for Factors X and VII. Factor X ;-O_ Factor VII;+
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Fig. 5 shows calibration curve for prothrombin in plasma. Prothrombin was activated by Factor Xa in the presence of phospholipid and calcium as described in METHODS. After the addition of Boc-Val-Pro-Arg-MCA, fluorescence intensity increased linearly as shown in Fig. 5(A). The concentration of lo-fold diluted control plasma was defined as 100 %. Linear standard curve was obtained from 10 % to 160 % as shown in Fig. 5(B). The addition of further phosphlipid and Factor Xa did not increase significantly generation of thrombin activity. Shorter preincubation time decreased generation of thrombin activity. Since the amounts of Factor V in samples were supposed to affect the generation of thrombin activity in this experimental condition, various amounts of prothrombin-deficient plasma were added to Factor V-deficient plasma and thrombin activity was measured. The result indicated that the presence of 20 % Factor V was enough for the maximum generation of thrombin in the experimental condition, assuming that Factor V concentration in prothrombin-deficient plasma was 100 % ( data not shown). These results indicate that prothrombin concentration in plasma can be quantitatively measured using this method, unless Factor V concentration in samples is quite low. Precision of this method is shown in Table II.
,(B)
, INCUBATION
TIME
(mln)
50
CONCENTRATION
100
150
OF PROTHROMBIN
WJ
FIG. 5. Calibration curve for prothrombin in plasma. Diluted normal plasma was activated with Factor Xa and thrombin activity generated was measured as described in METHODS. The fluorescence intensity due to AMC increased linearly with the incubation time (A) and the intensity after 5 min incubation was plotted versus concentration of prothrombin assuming that ten-fold diluted plasma contains 100 % of prothrombin (B).
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Factors X and VII and Prothrombin in Plasmas from Patients with Cardiovascular Diseases The automated fluorogenic methods for the evaluation of Factors X and VII and orothrombin in plasma as described in the previous section were applied to the analysis of plasmas from the inpatients with cerebral and myocardial infarctions. Eighty one patients plasmas were classified into two groups, one with higher FPT than 120 % and another with lower FPT than 80 %. As shown in Fig. 6, the concentrations of Factors X and VII and prothrombin in these two groups were compared with those in normal plasmas. The concentrations of Factors X and VII and prothrombin in a group with higher FPTthan 120 % were almosthigherthan100 %,while other group with lower FPT than 80 % showed lower concentrations than 100 %. In the latter group, plasmas from warfarin-treated patients showed lower concentrations than 50 %. These results demonstrate that concentrations of Factors X and VII and prothrombin are higher in plasmas with shorter FPT and lower in plasmas with longer FPT, as expected. These results also support that the automated fluorogenic PT method is sensitive to the hypercoagulable state due to the increase of Factor VII and Factor X or prothrombin, in addition to the hypocoagulable state due to the decrease of these coagulation factors.
(Cl
(6)
: :. . i
f 7 :
&
:
.
i
i
;
p
. .
. I
VII
x
II
VII
x
II
FIG. 6 Concentration of Factors X and VII and prothrombin in patients plasmas and normal plasmas. Concentrations of Factors VII and X were measured using deficient plasma as described in METHODS. Prothrombin was measured after the activation with Factor Xa as described in METHODS. (A): Patient plasmas with higher FPT than 120 %. (B): Patient plasmas with lower FPT than 80 8. (C): Normal plasmas.
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DISCUSSION Thrombosis is caused by complicated in vivo reactions which involve the damage or loss of endothelial cells, activation of platelets and leukocytes, activation of coagulation and fibrinolysis systems in plasma and alteration of their regulation systems. For the prognosis, diagnosis and therapy of the thrombotic diseases of cerebral and coronary arteries and lower limbs veins, the development of the highly sensitive method to detect the prethrombotic state is essential, together with the analysis of the prethrombotic state which precedes thrombus formation. In spite of the contribution of the hypercoagulability in plasma to the prethrombotic state, the conventional PT and APTT and also the chromogenic method recently developed, are not relevant to detect the hypercoagulability. The chromogenic substrates are now widely used in clinical laboratory for the estimation of coagulation factors and proved to be useful for Factor X and prothrombin. However, it is not still possible to estimate directly other coagulation factors, since the specific and sensitive substrates have not been developed except Factor Xa and thrombin. Even the estimation of Factor X and prothrombin is required to be improved to measure their functional activities, particularly in Warfarin-treated plasma. In the present work, we intended to develop a sensitive automated methods to estimate hypercoagulability, using fluorogenic peptide substrate for thrombin and a centrifugal autoanalyzer. This paper described a fluorogenic PT method to detect hypercoagulability of plasma and sensitive method for the estimation of functional activities of Factor X, Factor VII and prothrombin. In the fluorogenic PT method, the amounts of tissue factor (Thromborel S) were smaller than those used in the conventional and chromogenic PT methods, so that, the lag time before the generation of thrombin was longer. In the FPT, thrombin was not generated significantly in Factors V-, X- and VII-deficient plasmas. Althogh the generation of thrombin in the intrinsic factors-deficient plasmas, was delayed to some extent comparing withthatof control plasma, it may not be due to the deficiency of each factor. It may be rather due to the lower content of Factor V or Factor VIII than those of control plasma. In fact, FPT of Factor IX-deficient plasma was not accelerated by the addition of Factor IX. In the chromogenic methods, turbidity due to the fibrin formation disturbed the spectrophotometric mesurement. In the FPT, plasma was diluted 100-fold, therefore, no visible fibrin formation was observed. The effect of fibrinogen on thrombin activity toward fluorogenic subtrate is supposed to be negligible, since the final concentration of fluorogenic substrate is lO,OOO-fold higher than that of fibrinogen, while Km values of thrombin toward both of substrates are comparable (13). In the present paper, FPT was expressed as per cent of control plasma by dividing FPT of control plasma with that of sample
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plasma. We used the calculation method for convenience, since the method for hypercoagulable plasma has not been established. However, the per cent values for each plasma were approximately the same in each experiment with different concentration of tissue factor, in which the reaction time of control plasma was divided with the reaction time of each plasma. The percentage of each patient plasma correlated with that from the conventional PT with high correlation coefficient if the per cent values higher than 150 % in FPT were excluded. The sensitivity of the FPT to hypercoagulable state was demonstrated by the findings that the reaction times of FPT of control plasma were accelerated by the addition of Factors VII and X and that 25 out of 81 patient plasmas gave more than 120 % of FPT, whereas they were less than 120 % in the conventional PT method. On the other hand, the FPT method was also sensitive to hypocoagulability as shown by no generation of thrombin in Warfarin-treated plasma, although less than 30 % in the conventional PT could not be measured. The concentarations of Factors VII and X in plasma were measured by the application of FPT method. One of the characteristics of the method is the automated measurement of the functional activities of Factors VII and X using small amounts of sample (30 1.11 of 100-fold diluted plasma) and small amounts of factors-deficient plamsas ( 6 ul for each sample). By the methods, linear standard curves were obtained from 10 % to 800 % by the dilution of control plasma. The concentration of prothrombin in plasma was measured after the activation of prothrombin by Factor Xa and linear standard curve was obtained from 10 % to 160 %. In the method, Factor V was not added, because the presence of 20 % of Factor V in plasma was sufficient for the maximum activation of prothrombin in plasma. Factor X and prothrombin in plasma have been measured using chromogenic substrates after the activation with enzymes from snake venoms. However, these methods do not necessarily measure the functional activities of prothrombin and Factor X. The functional activities of prothrombin and Factor X have been measured recently using chromogenic substrates after the addition of tissue factor (14). This method depends on the concentration of Factors VII and or X andV, which has been overcomed by the addition of Factor VII- or Factor X-deficient plasma. The present fluorogenic methods are similar to the above method in principle, but, the functional activities of Factors X and VII and prothrombin were measured using the fluorogenic substrate for thrombin after the addition of deficient plasmas and tissue factor or after the activation by Factor Xa. The fluorogenic methods for PT and for the estimation of Factors X and VII and prothrombin concentrations in plasma were applied to the analysis of the hypercoagulability of plasmas from patients with cardiovascular diseases. Twenty five samples out of 81 samples gave higher FPT than 120 %. The concentration of Factors X and VII and prothrombin in those plasmas were higher than those of normal plasmas. On the other hand, their concentrations in the samples with lower FPT than 80 % were lower than those of normal plasmas. As described above, FPT was
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sensitive to the increase of the concentration of Factors X and VII, but not prothrombin. Therefore, the hypercoagulability of many samples as shown by the shorter FPT is supposed to be in part due to the increase of the concentration of Factors X and VII. These results indicate that the fluorogenic methods are useful for the analysis of hypercoagulability in plasma. However, it is clear that other factors in plasma, particulary, Factor V, protein C and protein S and protease inhibitors such as antithrombin III, may affect the hypercoagulability in plasma, hence, FPT. The effect of these factors on FPT remains to be established. ACKNOWLEDGMENTS This study was supported in part by a Grant-in-Aid for Scientific Research from Japan Foundation for Applied Enzymology. The authors wish to thank Dr. Junzo Kodama, Hoken Iryo center, Sanyo Denki Co., Ltd. for helpful discussion and suggestion during this work and Mr. Yoshiyuki Shigeyasu of Baxter-Travenol, Japan for developing the computer program to calculate reaction times of FPT.
REFERENCES 1. ZOLTON, R. P. Synthetic peptide substrate assays for hemostasis testing. Advances -in Clin. Chem., 25, 117-168, 1986. 2. YAMADA, K. and MEGURO, T. A new APTT assay employing a chromogenic substrate and a centrifugal autoanalyzer. Thrombos. Res., 15, 351-358, 1979. 3. BAUGHMAN, D. J. and LYTWYN, A. Thrombin activation rate constant: one-stage chromogenic assay for the extrinsic system. Thrombos. Res., 26, l-12, 1982. 4. BECKER, U., JERING, H., BAETL, K. and JILEK, F. Automated prothrombin time test with use of a chromogenic peptide substrate and a centrifugal analyzer. Clin. Chem., 30, 524-528, 1984. 5. DUNCAN, A., BOWIE, E. J. W., OWEN, Jr., C. A. and FASS, D. N. A clinical evaluation of automated chromogenic tests as substitutes for conventional prothrombin time tests. Clin. Chem., 2, 853-855, 1985. 6. BECKER, U., BARTL, K., LILL, H. and WAHEFELD, A. W. Development of a photometric assay for activated partial thromboplastin time and its application to the Cobas Bio centrifugal analyzer. Thrombos. Res., 40, 721-730, 1985. 7. KOLDE, H. J. Chromo time system-A new generation of coagulation analyses. Behring Inst. Mitt., No. 78, 176-187,
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8. DATI, F., KOLDE, H. J. and Heimburger, N. Methodische Aspekte zur photometrischen Bestimmung der Prothromibinzeit mittels chromogener Substrate. J. Clin. Chem. Clin. Biochem., 24, 877-888, 1986. - ___ -9. YAMAMOTO, J., ASADA, N., MIYATAKE, Y., MASUYA, M., YAMASHITA, T. and OKAMOTO, U. New modified activated partial thromboplastin time and prothrombin time method using a synthetic chromogenic substrate in comparison with diazotization. Thrombo. Res., 46, 225-231, 1987. 10.
THEODORSSON, B., HEDNER, U., NILSON, I. M., KISIEL , W. A technique for specific removal of factor IX alloantibodies from human plasma; partial characterization of the alloantibodies. Blood, 61, 973-981, 1983.
11.
MILETICH, J. P., BROZE, G. J., MAJERUS, P. W. The synthesis of sulfated dextran beads for isolation of human coagulation factors II, X and IX. Anal. Biochem., 105, 304-310, 1980.
12. JESTY, J. Measurement of the kinetics of inhibition of activated coagulation factor X in human plasma; The effect of plasma and inhibitor concentration. Anal. Chem., E, 402-411, 1986. 13. CHANG, J-Y. The structure and proteolytic specificities autolysed human thrombin. Biochem. J., 240, 797-802, 1986.
of
14. ASAI, K. Vitamin K dependent factors-F.11, F.X. Rinsyo Byouri (The Japanese -J. of Clin. Pathol.), 4, No.70, 88-99, 1987.