THROMBOSIS RESEARCH 62; 83-92,199l 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press plc. All rights reserved.
SIMPLIFICATION OF A VENOUSOCCLUSIONTEST
A. Mizuno, J. Isobe*
and K. Shima
Department of Laboratory Medicine, School of Medicine, The University of Tokushima, Tokushima, Japan. *School of Medical Sciences, The University of Tokushima, Tokushima, Japan (Received
19.4.1990;
accepted
in revised form 8.1 .1991 by Editor H. Yamazaki)
ABSTRACT
Chronological changes in plasma or serum concentrations of various fibrinolytic parameters, such as tissue-type plasminogen activator, plasminogen activator inhibitor, fibrinopeptide B ,9 I s--42 and FDP, before and after venous were examined in 31 healthy volunteers occlusion to determine a suitable time of venous stasis and a good parameter for use in a simplified venous occlusion test. After increase in the serum venous stasis for 5 min. a significant concentration of FDP (from 38.3 I 21.1 to 100.9 + 87.2 n&ml (n=24)) was observed, and the increase was parallel with changes in the plasma concentrations of other fibrinolytic parameters. The serum FDP level after 10 min VO was not significantly higher than that after 5 min VO. A significant decrease in ADP-induced platelet aggregation was observed after venous occlusion when measured by the impedance method, but not by the turbidimetric method. Therefore, as a simplified test, the change in the serum concentration of FDP after VO for 5 min was measured. The clinical significance of this simplified test was evaluated in fourteen patients with stenosis of cerebral artery. The mean percentage change in serum FDP concentration after venous stasis in the patients, 34.5 f 54.8%, was significantly less than that in normal subjects, 156.5 _+ 90.9%. Similar results were obtained on changes in plasma responses of other fibrinolytic parameters. Measurement of increase in serum FDP concentration after venous occlusion for 5 min should be useful as a screening tests for the hypofibrinolytic or thrombotic state due to vascular dysfunction.
Key words: plasminogen
Venous occlusion, FDP, platelet activator, plasminogen activator 83
aggregation, inhibitor
tissue-type
84
VENOUS OCCLUSION TEST
Vol. 62, Nos. l/2
INTRODUCTION Many factors are involved in blood coagulation and fibrinolysis, some of which are released from vascular endothelial cells into the circulation. Measurement of the responses of various factors derived from the vessel wall to various stimuli [ desmopressin acetate, exercise and venous occlusion (l-4) ] have been used in vascular function tests. Measurements of the plasma concentration of tissue-type plasminogen activator ( t-PA ), and/or plasminogen activator inhibitor-l ( PA1 ) after venous occlusion have been widely used as standard tests. However, venous occlusion for 10 or 20 min (5-7), which is conventionally used in these tests, is very painful for the patients, and so is unsuitable in a routine test. Moreover, the measurements of t-PA and PAI, which are parameters in these tests (5-9) take time, and prolonged venous occlusion is not suitable as a routine test on outpatients with thrombotic diseases. Therefore, in the present study, we examined whether the period of venous occlusion could be shortened and whether some parameter that was easier to measure than t-PA or PAI could be used routinely. We then used our modified venous occlusion test for detecting abnormalities in the fibrinolytic response of patients with stenosis of cerebral artery who were easily fallen into a thrombotic state due to systemic atherosclerosis.
MATERIALSANDMlTMODS Subjects: Blood samples were taken from 31 normal volunteers ( 19 males and 12 females of 22 to 48 years old ). These subjects had not taken any drugs affecting blood coagulation, fibrinolysis or platelet aggregation for at least 2 weeks before the test. In addition tests were made on 14 patients ( 9 males and 5 females of 33 to 68 years old 1 with stenosis of cerebral artery who were attending the outpatient clinic of the Department of Neurosurgery of Tokushima University Hospital. The patients were not diabetic, and their mean body mass index was less than 23.0. All subjects gave informed consent to participate in the study. Blood samples and venous occlusion: Venous blood was obtained at rest and after various periods of up to 10 min of venous occlusion (VO) by inflating a sphygmomanometer cuff applied unilaterally to the upper arm and maintained midway between the systolic and diastolic pressure. Blood samples were taken before ( without stasis ) and after VO between lo:30 and 11:00 AM. We chose this time to reduce the influence of meals and the circadian variations in t-PA and PAI (10) on measurements of platelet aggregation. Part of each blood samle was containing l/10 volume of 3.8% sodium citrate for put into a tube measurementof the plasma levels of t-PA, PA1 and fibrinopeptide B B I CS--~Z ( B B ,6--42 ) and platelet aggregation, and the rest of the whole blood was put into a tube containing thrombin and aprotinin for measurement of the serum Platelet aggregation and FDP were measured within one hour level of FDP. The titrated blood samples for measurements after obtaining blood samples. of t-PA, PA1 and B /3 16--4z were promptly cooled on ice, and the plasma was separated by centrifugation at 1000 x g for 30 min at 4°C and stored at -70 “C until assayed. Assay methods: FDP was measured with a latex photometric immunoassay system (
Vol. 62, Nos. l/2
VENOUS OCCLUSION TEST
85
MITUBISHIYUKA co LTD. Japan 1 using a monoclonal antibody for the E fragment of fibrin or fibrinogen which reacted with all the fragments except fragment D. t-PA and PA1 were measured by an enzyme-linked immunosorbent assay using to the assay Sweden ) according a commercially available kit ( Biopool, procedure given in the manual. B B 16--4~ was assayed immunochemically with a kit ( AMKOCo, Switzerland 1. 12=?-B B 15--42 was prepared by the chloramine-T method. ADP-induced platelet aggregation was measured by turbidimetry ( final concentration of ADP, 5 /L M ) as well as by impedance aggregometry ( final concentration of ADP, 12.5 ,u M 1. The former method was performed according to a conventional procedure with an aggregometer ( PAP III RICOH, Japan 1, and the latter was done with a whole blood aggregometer ( Model II Chronolog Co, Sweden 1. FDP, B B 16--41 and platelet aggregation were Expression of measured values: expressed as absolute values or percentage increases following VO. t-PA and between ( t-PA - PA1 PA1 were expressed in d ( t-PA - PA1 1; i.e., the difference ) before and after venous stasis. Statistical analysis was performed by Student’s t-test Statistical analysis: or the Wilcoxon matched-pair and non-paired signed-rank test. Data in the text and figures are presented as means4 SD unless otherwise indicated.
RESULTS Changes in fibrinolytic parameters during VO for various times: the serum FDP concentration increased signifiIn seven healthy subjects, cantly from a basal level of 31.7-t 10.2 ng/ml to 62.7* 17.9 ng/ml after VO for 5 min, but did not increase significantly more after VO for 10 min (81.4f 28.7 ng/ml). On the basis of this result, a period of VO of 5 min, not 10 min, was used in subsequent studies. Changes in serum FDP and plasma B B 16--4Z levels were determined after VO for 5 min in 24 normal subjects. In five of these subjects, the values of these parameters in the blood were determined after 1 and 3 min as well as at rest and after 5 min. As shown in Table 1, the mean concentrations of serum FDP and plasma B ,8 I =--4~ both increased significantly during VO .
TABLE1 Changes in serum FDP and plasma BB ,---42 levels for 5 min in normal subjects (n=24)
FDP (ng/ml) B B 16-42 (mmol/ml)
Basal 38.3+ 21.1 0.64+ 0.36
5 min 100.9f 87.2 1.59 + 1.69
after
VO
p values co. 01 co. 05
Figure 1 shows the chronological changes of individual serum FDP and plasma B B 16-4~ levels during VO in 5 normal subjects. The FDP levels increased with the time of VO. The mean value of FDP after VO for 5 min but not after VO for
86
VENOUS OCCLUSION TEST
one or 3 min. was significantly higher than that at rest, B ,9 , e--45?levels were similar to those in FDP.
FDP
I 150 -
”
0
1
DB15-42
3
5
, 0
1
1
Vol. 62, Nos. l/2 and changes
in plasma
72.5
FIG 1 Chronologicalchanges of serum or plasma levels of FDP and B B 1e_4z during venous occlusion for 5 min in 5 patients.
n -0
3
5
Time aflervenous occlusion(min.)
These data show that the levels of these fibrinolytic parameters were elevated after VO for 5min. Figure 2 shows the changes in platelet aggregation evaluated by turbidimetry and impedance aggregometry after venous stasis in 19 normal subjects. After VO, a significant decrease in ADP-induced platelet aggregation was observed by the impedance method (p
SIMPLIFICATION OF A VENOUSOCCLUSIONTEST
A. Mizuno, J. Isobe*
and K. Shima
Department of Laboratory Medicine, School of Medicine, The University of Tokushima, Tokushima, Japan. *School of Medical Sciences, The University of Tokushima, Tokushima, Japan (Received
19.4.1990;
accepted
in revised form 8.1 .1991 by Editor H. Yamazaki)
ABSTRACT
Chronological changes in plasma or serum concentrations of various fibrinolytic parameters, such as tissue-type plasminogen activator, plasminogen activator inhibitor, fibrinopeptide B ,9 I s--42 and FDP, before and after venous were examined in 31 healthy volunteers occlusion to determine a suitable time of venous stasis and a good parameter for use in a simplified venous occlusion test. After increase in the serum venous stasis for 5 min. a significant concentration of FDP (from 38.3 I 21.1 to 100.9 + 87.2 n&ml (n=24)) was observed, and the increase was parallel with changes in the plasma concentrations of other fibrinolytic parameters. The serum FDP level after 10 min VO was not significantly higher than that after 5 min VO. A significant decrease in ADP-induced platelet aggregation was observed after venous occlusion when measured by the impedance method, but not by the turbidimetric method. Therefore, as a simplified test, the change in the serum concentration of FDP after VO for 5 min was measured. The clinical significance of this simplified test was evaluated in fourteen patients with stenosis of cerebral artery. The mean percentage change in serum FDP concentration after venous stasis in the patients, 34.5 f 54.8%, was significantly less than that in normal subjects, 156.5 _+ 90.9%. Similar results were obtained on changes in plasma responses of other fibrinolytic parameters. Measurement of increase in serum FDP concentration after venous occlusion for 5 min should be useful as a screening tests for the hypofibrinolytic or thrombotic state due to vascular dysfunction.
Key words: plasminogen
Venous occlusion, FDP, platelet activator, plasminogen activator 83
aggregation, inhibitor
tissue-type
84
VENOUS OCCLUSION TEST
Vol. 62, Nos. l/2
INTRODUCTION Many factors are involved in blood coagulation and fibrinolysis, some of which are released from vascular endothelial cells into the circulation. Measurement of the responses of various factors derived from the vessel wall to various stimuli [ desmopressin acetate, exercise and venous occlusion (l-4) ] have been used in vascular function tests. Measurements of the plasma concentration of tissue-type plasminogen activator ( t-PA ), and/or plasminogen activator inhibitor-l ( PA1 ) after venous occlusion have been widely used as standard tests. However, venous occlusion for 10 or 20 min (5-7), which is conventionally used in these tests, is very painful for the patients, and so is unsuitable in a routine test. Moreover, the measurements of t-PA and PAI, which are parameters in these tests (5-9) take time, and prolonged venous occlusion is not suitable as a routine test on outpatients with thrombotic diseases. Therefore, in the present study, we examined whether the period of venous occlusion could be shortened and whether some parameter that was easier to measure than t-PA or PAI could be used routinely. We then used our modified venous occlusion test for detecting abnormalities in the fibrinolytic response of patients with stenosis of cerebral artery who were easily fallen into a thrombotic state due to systemic atherosclerosis.
MATERIALSANDMlTMODS Subjects: Blood samples were taken from 31 normal volunteers ( 19 males and 12 females of 22 to 48 years old ). These subjects had not taken any drugs affecting blood coagulation, fibrinolysis or platelet aggregation for at least 2 weeks before the test. In addition tests were made on 14 patients ( 9 males and 5 females of 33 to 68 years old 1 with stenosis of cerebral artery who were attending the outpatient clinic of the Department of Neurosurgery of Tokushima University Hospital. The patients were not diabetic, and their mean body mass index was less than 23.0. All subjects gave informed consent to participate in the study. Blood samples and venous occlusion: Venous blood was obtained at rest and after various periods of up to 10 min of venous occlusion (VO) by inflating a sphygmomanometer cuff applied unilaterally to the upper arm and maintained midway between the systolic and diastolic pressure. Blood samples were taken before ( without stasis ) and after VO between lo:30 and 11:00 AM. We chose this time to reduce the influence of meals and the circadian variations in t-PA and PAI (10) on measurements of platelet aggregation. Part of each blood samle was containing l/10 volume of 3.8% sodium citrate for put into a tube measurementof the plasma levels of t-PA, PA1 and fibrinopeptide B B I CS--~Z ( B B ,6--42 ) and platelet aggregation, and the rest of the whole blood was put into a tube containing thrombin and aprotinin for measurement of the serum Platelet aggregation and FDP were measured within one hour level of FDP. The titrated blood samples for measurements after obtaining blood samples. of t-PA, PA1 and B /3 16--4z were promptly cooled on ice, and the plasma was separated by centrifugation at 1000 x g for 30 min at 4°C and stored at -70 “C until assayed. Assay methods: FDP was measured with a latex photometric immunoassay system (
Vol. 62, Nos. l/2
VENOUS OCCLUSION TEST
85
MITUBISHIYUKA co LTD. Japan 1 using a monoclonal antibody for the E fragment of fibrin or fibrinogen which reacted with all the fragments except fragment D. t-PA and PA1 were measured by an enzyme-linked immunosorbent assay using to the assay Sweden ) according a commercially available kit ( Biopool, procedure given in the manual. B B 16--4~ was assayed immunochemically with a kit ( AMKOCo, Switzerland 1. 12=?-B B 15--42 was prepared by the chloramine-T method. ADP-induced platelet aggregation was measured by turbidimetry ( final concentration of ADP, 5 /L M ) as well as by impedance aggregometry ( final concentration of ADP, 12.5 ,u M 1. The former method was performed according to a conventional procedure with an aggregometer ( PAP III RICOH, Japan 1, and the latter was done with a whole blood aggregometer ( Model II Chronolog Co, Sweden 1. FDP, B B 16--41 and platelet aggregation were Expression of measured values: expressed as absolute values or percentage increases following VO. t-PA and between ( t-PA - PA1 PA1 were expressed in d ( t-PA - PA1 1; i.e., the difference ) before and after venous stasis. Statistical analysis was performed by Student’s t-test Statistical analysis: or the Wilcoxon matched-pair and non-paired signed-rank test. Data in the text and figures are presented as means4 SD unless otherwise indicated.
RESULTS Changes in fibrinolytic parameters during VO for various times: the serum FDP concentration increased signifiIn seven healthy subjects, cantly from a basal level of 31.7-t 10.2 ng/ml to 62.7* 17.9 ng/ml after VO for 5 min, but did not increase significantly more after VO for 10 min (81.4f 28.7 ng/ml). On the basis of this result, a period of VO of 5 min, not 10 min, was used in subsequent studies. Changes in serum FDP and plasma B B 16--4Z levels were determined after VO for 5 min in 24 normal subjects. In five of these subjects, the values of these parameters in the blood were determined after 1 and 3 min as well as at rest and after 5 min. As shown in Table 1, the mean concentrations of serum FDP and plasma B ,8 I =--4~ both increased significantly during VO .
TABLE1 Changes in serum FDP and plasma BB ,---42 levels for 5 min in normal subjects (n=24)
FDP (ng/ml) B B 16-42 (mmol/ml)
Basal 38.3+ 21.1 0.64+ 0.36
5 min 100.9f 87.2 1.59 + 1.69
after
VO
p values co. 01 co. 05
Figure 1 shows the chronological changes of individual serum FDP and plasma B B 16-4~ levels during VO in 5 normal subjects. The FDP levels increased with the time of VO. The mean value of FDP after VO for 5 min but not after VO for
86
VENOUS OCCLUSION TEST
one or 3 min. was significantly higher than that at rest, B ,9 , e--45?levels were similar to those in FDP.
FDP
I 150 -
”
0
1
DB15-42
3
5
, 0
1
1
Vol. 62, Nos. l/2 and changes
in plasma
72.5
FIG 1 Chronologicalchanges of serum or plasma levels of FDP and B B 1e_4z during venous occlusion for 5 min in 5 patients.
n -0
3
5
Time aflervenous occlusion(min.)
These data show that the levels of these fibrinolytic parameters were elevated after VO for 5min. Figure 2 shows the changes in platelet aggregation evaluated by turbidimetry and impedance aggregometry after venous stasis in 19 normal subjects. After VO, a significant decrease in ADP-induced platelet aggregation was observed by the impedance method (p
