THROMBOSIS RESEARCH 62; 6574,199l 0049-3848191 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.
CIRCADIAN VARIATIONS OF PLATELET AGGREGABILITY AND FIBRINOLYTIC ACTIVITY IN HEALTHY SUBJECTS 1
1 2 JoviEid A., 2Mandid S., Clinic of Neurology, Clinical chemical laboratory, Military Medical Academy, Beograd, Yuqdavia
(Received 17.12.1990; accepted in revised form 7.1.1991 by Editor H.A. Vinazzer)
ABSTRACT With a view to investigating whether in circadian variations of platelet aggregation (PA) and fibrinolytic activity there is an elevated risk period for incidence and development of ischemic stroke, 25 healthy subjects (5 females and 20 males), their age ranging from 29 to 51, were exposed to the analysis of PA, euglobulin lysis time (ELT), fibrinogen degradation products (FDP), antithrombin III (AT III) and heparin tolerance test (WIT) in blood samples drawn by venepuncture at 08.00, 11.00, 13.00, 15.00, 17.00 and 18.00h; beside these intervals in the case of 10 healthy males, whose age ranged from 32 to 45, blood samples were taken at midnight as vell. The group of 25 subjects comprised those who usually worked daily and nightly shifts, as well as those who were either at bed rest or doing their duties during daytime. The findings of this investigation have demonstrated that all the parameters studied exhibited circadian variations irrespective of sex, age or daily/nightly activities of the subjects. The most pronounced PA interval, which was not accompanied by corresponding increase of fibrinolytic activity, was that around ll.OOh and it is marked as the highest risk period for onset of ischemic stroke. INTRODUCTION The cycle as recurrence of phenomena and activity levels at regular intervals is a general regulation of non-living nature. During the new entities formation process and formation of biophysical systems, the regulations of cyclic activities are inbuilt in these systems (1). The notion of biorhythm refers to cyclic functioning of certain functional systems in the organism as a whole. That principle of certain biological systems functioning occurs in various astronomical cycles, the best known of which are the daily or circadian and the seasonal ones (2).
Key words: Circadian, Platelet aggregability, Fibrinolytic activity. 65
66
CIRCADIAN VARIATIONS...
Vol. 62, Nos. II2
The knowledge of these processes may be of importance when preventive and therapeutic programmes are being scheduled in clinical medicine. Starting from the data on circadian variations in frequency of ischemic stroke and myocardial infarction (1, 3, 4, 5) and causal connectedness of disturbance of the haemostasis process and the incidence of ce&raland myocardial infarction as well as the data that in healthy persons there is also circadian fluctuation of some haemostatic processes, we set up a hypothesis that in the course of the day there is an elevated risk period for onset and development of cerebral and myocardial infarction. With respect to it the purpose of this investigation was to simultaneously study the features of circadian variation of platelet aggregability and some parameters of fibrinolytic activity in healthy subjects.
METHODS The investigation was carried out in healthy volunteers,non smokers who, at least for two weeks prior to the investigation had not been taking any medicaments and had not been exposed to ony stress situations. All the subjects studied were recruited from the medical staff. The analysis was made of blood samples from 25 subjects (5 females and 20 males) whose age ranged from 29 to 51; the blood samples were drawn by venepuncture at 08.00, 11.00, 13.00, 15.00, 17.00 and 18.00h, and in the case of 10 males, whose age ranged between 32 and 45, blood samples were also taken at these intervals and at midnight as well. In the group of 25 subjects, 5 worked night shifts before the investigation and then stayed in the clinic throughout the investigation period. Of the remaining 20 subjects, five were at bed rest throughout the investigation period and would get out of bed for their elementary physiologic needs only. The remaining subjects performed their regular daily duties at the clinic after their night's sleep. Throughout the investigation period all the subjects were staying in identical microclimatic conditions and consuming identical daily meals. The group of subjects whose blood samples were also taken at midnight worked their regular daily shifts, after their night's sleep, and then stayed on the same premises till 24.00h with a brief rest between 18.00 and 20.00h. The blood samples were always drawn from all the subjects by the same medical technician and the blood analyses were all made by the same biochemist. In the group of 25 subjects whose blood samples were taken at afore mentioned intervals from 08.00 till 18.00h the following analyses were made: platelet aggregability, euglobulin lysis time, fibrinogen degradation products, heparin tolerance test and antithrombin III. In the group of 10 subjects, whose blood samples were also drawn at midnight, only PA and ELT were analysed. The blood analyses were made by following methods: PA was carried out by the Caen method (6) according to which the time of pla-
Vol. 62, Nos. l/2
67
CIRCADIAN VARIATIONS...
telet aggregability onset is measured when ADP solution at 2mg/ml is applied. ELT is determined by the Buckell method (7) according to which euglobulin is separated from the plasma at +4'C by changing pH levels when a solution of acetic acid is added. Euglobulin clots in the presence of borate solution when calcium chloride is added. The clot lysis is observed at 30 min. intervals. FDP were determined by the BlombGck method (7) on commercial Thrombo-Wellcotest. The suspension of latex particles in the buffer was covered by specific FDP antibodies. The suspension reacts when the concentration of these products is 2mg/ml or higher. Latex particles agglutinate producing particles perceivable by microscope. By carrying out the test with varying dilutions of two given samples it is possible to express FDP concentrations. HTT was made by adding to the plasma the solution of Ca-heparinchloride (0,2u/ml) and by measuring the time until clot formation the outcome being the information on the level of free endogeneous heparin (7). AT III was determined by commercial Berichrom AT III test (kinetic method Code No OUBP). AT III is transformed by heparin into an inhibitor which inactivates the alfa-thrombin added in abundance. The remaining quantity of thrombin is assessed by kinetic test when absorption is measured at 405 nm in the presence of D-Cvclohexvlalanil -2- aminobutyrylarginyl -p- nitroanilid (HD-CHA~But-Arg-PHA).-
RESULTS -The findings of some parameters at various intervals of the day, from 08,00h, till 18.00h, are presented in table 1 and the results of PA and ELT studies at the same intervals and at mindight as well are presented in table 2.
Table 1 Time (h)
AT (set)
ELT (min)
~ a
laW-13.9
z&.8);
11
lla+la
< lO(14); ,lO(ll)
1 13
7.8+/-0.4
lO4+/-X3.0
15
l2.1+/-1.4*
17 18
7lO(l2);
AT III (:
~.
8,6+/-0.9 --___ 7.1+/-0.4*
.o
HTT (set)
FDP (ng/ml) >lO(7)
7loQ3)
l52.5+15.5
0.74t/-O.l8
lU+/-24.1*
0.8l+/-Q.l5*
2ll.4t/-35.5 0.9WO.7
62.8+/-l2.5* '1002);
>lo(v)
3l2.8+/-32.5* 0.82+/-0.12+
9.4+/-1.6
W-19.4
~1002);
'lo(V)
26C.7+/-37.8 0.8f%/-o.l4
9.8+/-1.0
92+/-14.3
QO(l2); =O(l3)
222.0+?2.6
0.@3+O.u3
Circadian variation of PA, ELT, FDP, KIT and AT III All values are shown as mean +/- standard deviation *=
Statistically significant difference to previous term ( p < 0.005
CIRCADIAN VARIATIONS...
68
Vol. 62, Nos. 112
Tn none of all these groups any variabilities whatsoever were observable in levels of other parameters studied. Not one of the parameters studied revealed any variabilities in those subjects who worked night shifts and after that stayed awake during the day compared to the subjects who worked by day after their night's sleep. No significant variations were observed in the degree of any parameter's prominence between those subjects who, during the investigation, were at bed rest and those who were doing their regular jobs on one hand, nor between those who were working night shifts and those who worked by day on the other. Figure 1 presents the PA and ELT Z&hour
rhythm.
FIGURE 1. Circadian variation of PA and ELT - statistically significant difrerence to previous term (p
CIRCADIAN VARIATIONS OF PLATELET AGGREGABILITY AND FIBRINOLYTIC ACTIVITY IN HEALTHY SUBJECTS 1
1 2 JoviEid A., 2Mandid S., Clinic of Neurology, Clinical chemical laboratory, Military Medical Academy, Beograd, Yuqdavia
(Received 17.12.1990; accepted in revised form 7.1.1991 by Editor H.A. Vinazzer)
ABSTRACT With a view to investigating whether in circadian variations of platelet aggregation (PA) and fibrinolytic activity there is an elevated risk period for incidence and development of ischemic stroke, 25 healthy subjects (5 females and 20 males), their age ranging from 29 to 51, were exposed to the analysis of PA, euglobulin lysis time (ELT), fibrinogen degradation products (FDP), antithrombin III (AT III) and heparin tolerance test (WIT) in blood samples drawn by venepuncture at 08.00, 11.00, 13.00, 15.00, 17.00 and 18.00h; beside these intervals in the case of 10 healthy males, whose age ranged from 32 to 45, blood samples were taken at midnight as vell. The group of 25 subjects comprised those who usually worked daily and nightly shifts, as well as those who were either at bed rest or doing their duties during daytime. The findings of this investigation have demonstrated that all the parameters studied exhibited circadian variations irrespective of sex, age or daily/nightly activities of the subjects. The most pronounced PA interval, which was not accompanied by corresponding increase of fibrinolytic activity, was that around ll.OOh and it is marked as the highest risk period for onset of ischemic stroke. INTRODUCTION The cycle as recurrence of phenomena and activity levels at regular intervals is a general regulation of non-living nature. During the new entities formation process and formation of biophysical systems, the regulations of cyclic activities are inbuilt in these systems (1). The notion of biorhythm refers to cyclic functioning of certain functional systems in the organism as a whole. That principle of certain biological systems functioning occurs in various astronomical cycles, the best known of which are the daily or circadian and the seasonal ones (2).
Key words: Circadian, Platelet aggregability, Fibrinolytic activity. 65
66
CIRCADIAN VARIATIONS...
Vol. 62, Nos. II2
The knowledge of these processes may be of importance when preventive and therapeutic programmes are being scheduled in clinical medicine. Starting from the data on circadian variations in frequency of ischemic stroke and myocardial infarction (1, 3, 4, 5) and causal connectedness of disturbance of the haemostasis process and the incidence of ce&raland myocardial infarction as well as the data that in healthy persons there is also circadian fluctuation of some haemostatic processes, we set up a hypothesis that in the course of the day there is an elevated risk period for onset and development of cerebral and myocardial infarction. With respect to it the purpose of this investigation was to simultaneously study the features of circadian variation of platelet aggregability and some parameters of fibrinolytic activity in healthy subjects.
METHODS The investigation was carried out in healthy volunteers,non smokers who, at least for two weeks prior to the investigation had not been taking any medicaments and had not been exposed to ony stress situations. All the subjects studied were recruited from the medical staff. The analysis was made of blood samples from 25 subjects (5 females and 20 males) whose age ranged from 29 to 51; the blood samples were drawn by venepuncture at 08.00, 11.00, 13.00, 15.00, 17.00 and 18.00h, and in the case of 10 males, whose age ranged between 32 and 45, blood samples were also taken at these intervals and at midnight as well. In the group of 25 subjects, 5 worked night shifts before the investigation and then stayed in the clinic throughout the investigation period. Of the remaining 20 subjects, five were at bed rest throughout the investigation period and would get out of bed for their elementary physiologic needs only. The remaining subjects performed their regular daily duties at the clinic after their night's sleep. Throughout the investigation period all the subjects were staying in identical microclimatic conditions and consuming identical daily meals. The group of subjects whose blood samples were also taken at midnight worked their regular daily shifts, after their night's sleep, and then stayed on the same premises till 24.00h with a brief rest between 18.00 and 20.00h. The blood samples were always drawn from all the subjects by the same medical technician and the blood analyses were all made by the same biochemist. In the group of 25 subjects whose blood samples were taken at afore mentioned intervals from 08.00 till 18.00h the following analyses were made: platelet aggregability, euglobulin lysis time, fibrinogen degradation products, heparin tolerance test and antithrombin III. In the group of 10 subjects, whose blood samples were also drawn at midnight, only PA and ELT were analysed. The blood analyses were made by following methods: PA was carried out by the Caen method (6) according to which the time of pla-
Vol. 62, Nos. l/2
67
CIRCADIAN VARIATIONS...
telet aggregability onset is measured when ADP solution at 2mg/ml is applied. ELT is determined by the Buckell method (7) according to which euglobulin is separated from the plasma at +4'C by changing pH levels when a solution of acetic acid is added. Euglobulin clots in the presence of borate solution when calcium chloride is added. The clot lysis is observed at 30 min. intervals. FDP were determined by the BlombGck method (7) on commercial Thrombo-Wellcotest. The suspension of latex particles in the buffer was covered by specific FDP antibodies. The suspension reacts when the concentration of these products is 2mg/ml or higher. Latex particles agglutinate producing particles perceivable by microscope. By carrying out the test with varying dilutions of two given samples it is possible to express FDP concentrations. HTT was made by adding to the plasma the solution of Ca-heparinchloride (0,2u/ml) and by measuring the time until clot formation the outcome being the information on the level of free endogeneous heparin (7). AT III was determined by commercial Berichrom AT III test (kinetic method Code No OUBP). AT III is transformed by heparin into an inhibitor which inactivates the alfa-thrombin added in abundance. The remaining quantity of thrombin is assessed by kinetic test when absorption is measured at 405 nm in the presence of D-Cvclohexvlalanil -2- aminobutyrylarginyl -p- nitroanilid (HD-CHA~But-Arg-PHA).-
RESULTS -The findings of some parameters at various intervals of the day, from 08,00h, till 18.00h, are presented in table 1 and the results of PA and ELT studies at the same intervals and at mindight as well are presented in table 2.
Table 1 Time (h)
AT (set)
ELT (min)
~ a
laW-13.9
z&.8);
11
lla+la
< lO(14); ,lO(ll)
1 13
7.8+/-0.4
lO4+/-X3.0
15
l2.1+/-1.4*
17 18
7lO(l2);
AT III (:
~.
8,6+/-0.9 --___ 7.1+/-0.4*
.o
HTT (set)
FDP (ng/ml) >lO(7)
7loQ3)
l52.5+15.5
0.74t/-O.l8
lU+/-24.1*
0.8l+/-Q.l5*
2ll.4t/-35.5 0.9WO.7
62.8+/-l2.5* '1002);
>lo(v)
3l2.8+/-32.5* 0.82+/-0.12+
9.4+/-1.6
W-19.4
~1002);
'lo(V)
26C.7+/-37.8 0.8f%/-o.l4
9.8+/-1.0
92+/-14.3
QO(l2); =O(l3)
222.0+?2.6
0.@3+O.u3
Circadian variation of PA, ELT, FDP, KIT and AT III All values are shown as mean +/- standard deviation *=
Statistically significant difference to previous term ( p < 0.005
CIRCADIAN VARIATIONS...
68
Vol. 62, Nos. 112
Tn none of all these groups any variabilities whatsoever were observable in levels of other parameters studied. Not one of the parameters studied revealed any variabilities in those subjects who worked night shifts and after that stayed awake during the day compared to the subjects who worked by day after their night's sleep. No significant variations were observed in the degree of any parameter's prominence between those subjects who, during the investigation, were at bed rest and those who were doing their regular jobs on one hand, nor between those who were working night shifts and those who worked by day on the other. Figure 1 presents the PA and ELT Z&hour
rhythm.
FIGURE 1. Circadian variation of PA and ELT - statistically significant difrerence to previous term (p
