- Email: [email protected]
Influence of gender, age and sampling time on plasma fibrinolytic variables and fibrinogen
Fibrindysir (1993) 7,3lMz3 01993 Longman GroupUK Ltd
Influence of Gender, Age and Sampling Time on Plasma Fibrinolytic Variables and Fibrinogen A Population Study
M. Eliasson, P-E. Evrin, D. Lundblad, K. Asplund, M. Ranby
SUMMARY. Reference data are provided for tissue plasminogen activator (t-PA) activity, t-PA antigen levels, plasmhtogen activator inhibitor type 1 (PAI-1) activity and fibrinogen levels in plasma. The data are from a population-based sample of 1288 healthy 25 to 64-year-old men and women who were not taking any medication. Time of sampling influenced the measurements in that, from 07:OOto 14:00 t-PA activity rose, t-PA antigen levels and PAI- activity declined, whereas plasma fibrinogen levels remained unchanged. Age had little influence on t-PA activity or PAI-1 activity, although t-PA antigen levels correlated with increasing age (r=0.26 and 0.34, in men and women, respectively). Fibrinogen levels also correlated with age (r=0.29 in both sexes). In the 45 to 54-year age group, t-PA activity was significantly lower (p
type 1 (PAI-1) activity strongly predicts reinfarction in survivors of myocardial infarction before the age of 45.5 There is a well-known circadian rhythm in the incidence of myocardial infarction6 and cerebral thrombosis7 with a peak in the morning hours. A parallel trough in fibrinolytic activity and a peak in fibrinolytic inhibition during the late night hours has recently been shown in small groups of healthy volunteers.S” Plasma fibrinogen does not appear to have a diurnal variation. lo The increase in incidence of CVD with age may, in part, be due to decreasing fibrinolysis. Greater PAIactivity and tissue plasminogen activator (t-PA) antigen levels were found in older subjects’2*‘3 but in a recent population study PAI- activity increased with age only in women. I4 Data concerning t-PA activity and age are lacking. Most studies have found an age-related increase in fibrinogen2*‘3*‘5 but this has
The development of cardiovascular disease (CVD) can only partially be explained by traditional risk factors such as smoking, hypertension, hyperlipidaemia and diabetes. Recently, attention has focused on the role of coagulation and fibrinolysis. From the prospective studies in Northwick Park, Framingham and Gothenburg,‘” elevated plasma fibrinogen levels were found to be as strong predictors of stroke and myocardial infarction as serum cholesterol levels in both sexes. Almost all myocardial infarctions involve the formation of an arterial thrombus4 and this has focused interest on the role of the haemostatic system. Increased plasminogen activator inhibitor M. Ehsson, D. Lundblad, Department of Internal Medicine, Lulei-Boden Central Hospital, LuIeP, P-E. Evrin, Department of Clinical Chemistry, Central Hospital, Boden, K. Asplund, Department of Internal Medicine, M. Rhby, Department of Medical Biochemistry, University of Ume& Sweden. 316
Fibrinolysis
been disputed.‘6 We conducted a study on a welldefined, randomly selected population sample free from apparent disease in order to establish relationships and reference values for t-PA activity, t-PA antigen, PAI- activity, plasma fibrinogen in relation to gender, age and time of sampling.
MATERIAL AND METHODS This study was performed within the framework of the Northern Sweden MONICA Project which, in turn, is a part of the WHO MONICA Project (Monitoring of Trends and Determinants in Cardiovascular Disease). In January to April 1990, a population was screened for cardiovascular risk factors. A total of 2000 individuals in the 25 to 64 year range were invited. Within each age group (25-34, 35-44, 45-54, 55-64 years) 250 men and 250 women were randomly selected from continuously updated population registers in Norrbotten and Vasterbotten, the two northernmost provinces of Sweden. They were invited by letter to an examination. If they did not attend, a reminder with a new appointment was sent. People who still did not come, were contacted by telephone to ascertain reasons for reluctance to attend and basic information on social background and risk factors. In all, 1583 subjects participated in the study (79.2% of all invited). It was possible to obtain information on 203 of the 417 non-participants.” In short, participation rate was lower in 25 to 34-yearold men (66.8%) and women (71.1%) than in older age groups (80982.8% in men, 82.4-86.3% in women). The proportion of married or cohabiting people was lower in non-participants than in participants, but the level of education did not differ. Smoking was more common in both male and female non-participants than in participants. The participants were asked to complete a questionnaire with items on, inter alia, social background, smoking habits, medical history and intake of drugs. The questionnaire was returned on the site of the survey, which was performed by two mobile teams in local health centres (or corresponding) throughout the MONICA area. Our aim was to study a healthy population and therefore we excluded 262 participants (126 with treated hypertension, 22 with prior myocardial infarction, 19 with prior stroke, 43 who had diabetes mellitus, 53 who used contraceptive pills and 39 who used oestrogen substitution). Some had two or more concomitant exclusion criteria. No woman was pregnant. Data were incomplete in 33 persons, and thus the study group consisted of 1288 persons. The Northern Sweden MONICA Study has been approved by the Research Ethics Committee of Umei University and the data handling procedures by the National Computer Data Inspection Board.
317
Sampling Examination took place between 7:00 and 15:O0. For purpose of analysis those studied from 07:OOto 07:59 are pooled and allocated to the 7-h time group and so on. Participants sampled before 11:30 had fasted for at least 12 h, those sampled later had fasted for at least 4h. They were instructed not to use tobacco during 1 h before the examination. Venous sampling was done in the sitting position with no special rest and with minimal occlusion. Blood was drawn in 5ml vacuum tubes (Stabilyte, product No. 102080, Biopool, UmeH, Sweden) prefilled with 0.5ml of 0.45moVl citrate buffer pH 4.3. This ensured stability of t-PA activity without causing appreciable haemolysis. Is Tubes were centrifuged at room temperature at 2000xg for 20min in order to remove all platelets and then snap-frozen within an hour and stored in liquid nitrogen. This procedure has been shown not to release PAI- activity from platelets. I9
Methods Reagent kits from Biopool were used for determination of t-PA activity, t-PA antigen and PAIactivity. All absorbance measurements were performed by a Dynatech MR700 microplate reader. t-PA activity was determined using the Spectrolyse/fibrin kit, product number 101101,20 implementing the modifications supplied with the Stabilyte sample collection tubes. ” The conversion of plasminogen to plasmin is catalysed by t-PA in the presence of fibrin. The amount of colour developed by the plasmin-induced cleavage of a chromogenic substrate is proportional to the amount of t-PA in the sample. PAI- activity was analyzed by the microtestplate version of the Spectrolyse/pl kit, product number 101201, but following the modifications supplied with the Stabilyte sample collection tubes,18 with the following exception; one volume of plasma was reacted with two volumes of 40IU/ml of t-PA instead of 20IU/ml. This resulted in an assay range between 0 and 80U/ml. The method was based on the addition of a fixed amount of t-PA in excess to the samples. After binding to PA1 the residual t-PA-activity was measured in a similar way as above.2’ PAI-1 activity values below zero implied that the sample contained less PAI- than t-PA activity. t-PA antigen was determined by the TintElize t-PA kit, product number 101120, which is a double antibody enzyme-linked-immunosorbent-assay.22 Plasma fibrinogen determinations were performed by the reagent kit Fibrinogen Kinetic from Boehringer Mannheim, using a Hitachi 717 analyzer. In short, the sample was mixed with a snake venom, and the fibrin formation is then Batroxobin, measured by turbidimetry.23 The assay was
318 Epidemiology of Fibrinolysis
standardized with standard Scandinorm, Diagnostica Stago (France). There was no difference in fibrinogen concentration when samples obtained with the Stabilyte tubes were compared with samples collected in vacuum tubes containing 0.13 moY1 of sodium citrate (Becton Dickinson) (data not shown).
Assay Imprecision and Analytical Sensitivity Determinations of t-PA and PAI- activity were performed in duplicate. For each method, control samples were analyzed repeatedly on different days to allow estimates of assay imprecision. Control samples were prepared as pellets in liquid nitrogen, stored at -70°C and thawed just before analysis. The coefficient of variation (CV) for t-PA activity at 0.45 and 2.0IU/ml was 11.1 and 9.6% respectively (n=44). For PAI- activity, CV at 5.8 and 29.4U/ml was 27.3 and 5.8%, respectively (n=33). For t-PA antigen CV at 1.9 and 12.Ot~g/l was 13.3 and 11.2%, respectively (n=16). For plasma fibrinogen a freezedried control plasma, Control Plasma Normal, from Nycomed A/S, Norway, was used. The CV was 4.0% at a mean level of 2.72g/l (n=42). The analytical sensitivity of the method was determined by analyzing zero standard containing PAI-1 and t-PA depleted plasma for t-PA and PA1 activity and the PET-buffer for t-PA-antigen. The sensitivity was calculated as mean+3 S.D. of the absorbance values for the t-PA activity and t-PA antigen and as mean -3 S.D. for PAI- activity. The analytical sensitivity obtained in this way was O.O5IU/ml, 2.4U/ml and 0.3@1 for t-PA activity, PAI- activity and t-PA antigen, respectively.
RESULTS A sample of 1288 persons was studied, 671 men and 617 women. Relevant background variables are shown in Table 1. Influence of Time of Sampling The number and sex of subjects in each time-group are shown in Table 2.40.2% of the men and 47.2% of the women were sampled before 10:OO. Age, BMI, blood pressure, lipid levels and fasting glucose were independent of time groups (data not shown). t-PA activity rose steadily during the day from a median value of 0.48 to 1.09IIJml (an increase of 127%) p
Influence of Age Due to the strong influence of time of sampling, age-related values for t-PA activity, PAI- activity and t-PA antigen are shown for a ‘morning’ group sampled between 07:OOand 09:59 which may resemble a clinical setting. This group includes 40% of the men and 47% of the women. For plasma fibrinogen all participants were included. Correlations between Table 1 Background variables for the study population. Means (SD)
Statistics As the distributions for all four dependent variables were skewed we report medians (i.e the 50th percentile) and percentiles (2.5,5, 95, 97.5). PAI- activity values below zero were found in 13.2% of the samples (6.8% in samples at 07:OO and 27% at 14:OO). We therefore report the tenth and the twenty-fifth percentile for PAI-1 activity instead of the two-and-ahalf and fifth percentile. Comparisons between groups were made with the non-parametric KruskalWallis one-way analysis of variance (ANOVA). Two-tailed significance was used and due to the multiple comparisons only p values below 0.01 were considered significant. Pearson’s correlation coefficients were calculated with values for fibrinogen, t-PA activity and t-PA antigen transformed by the natural logarithm. The computer program SPSS/PC+4.01 was used for the analyses.
Variable
Males
Females
BMI (kg/m*) Cholesterol (mmol/I) HDL-cholesterol (mmoVI) Systolic BP (mm Hg) Diastolic BP (mm Hg) Smokers Postmenopausal
25.5 (3.2) 6.3 (1.3) 1.29 (0.30) 129 (16) 82 (11) 22.8%
24.7 (4.2) 6.0 (1.3) 1.57 (0.33) 124 (18) 77 (11) 27.4% 29.9%
Table 2 Numbers and gender in each time-group
Time-group
Men
Women
07 08 09 10 11 12 13 14
57 97 116 145 60 50 92 54
46 129 116 118 46 39 81 42
Fibrinolysis
A
319
19% (p~O.001) in women. The correlation r=0.29 in both sexes (p
tPA activity - males
tPA activitv
was
Influence of Gender
OL
I
1
’
0730
0830
B
0930
I
I
1030 1130 Time of sampling
I
I
I
1230
1330
1430
tPA activity - females
. . . . . . . . . . . . . I2 tPA activity ( IUlml )
_I
2
In the 45 to 54-year age group, women had significantly greater levels of t-PA activity than men, whereas the levels were similar in the oldest age group (Table 3). A difference was also noted for PAI- activity in the 45 to 54-year age group with significantly greater activity among men (Table 4). t-PA antigen levels were consistently greater in men than in women in all but the oldest age group (Table 3). Plasma fibrinogen levels were greater in women than in men but the difference was significant (p
.
. . . . . . . . . . .
Interrelationship Between Fibrinolytic Variables In both sexes t-PA activity showed a strong, inverse relation with PAI- activity and a weak, negative
. .
PAI-
A
activity
- males
PAI- activity ( U/ml )
40[ .....&
.............................::.............
”
0730
0830
0930
1030 1130 Time of sampling
1230
1330
1430
Fig. 1 t-PA activity according to time of sampling (A: males, B:females). 2.5,5,95 and 97.5 percentiles with thin lines, medians and thick line.
30 ~~$__.&.~ .............................
20
..............................
10
”
. . ........
E
fibrinolytic parameters and age were calculated for the entire group as age was evenly distributed during the day. As shown in Table 3, median levels of t-PA activity remained essentially unchanged in both men and women in the 25 to 64-year range. There was a weak positive univariate correlation between t-PA activity and age in men (r=O. 14, p~O.001) but not in women (r=0.09, p>O.Ol). Median levels increased with 9.6% among men from the youngest to the oldest age group (p=O.4). PAI-1 activity showed no correlation with age but in women median levels were 53% greater (p=O.O3) in the oldest group compared to the youngest group (Table 4). Median levels of t-PA antigen increased by 56% (p<0.0001) in men and 90% (p
0730
0830
PAI-
B
0930
1030 1130 Time of sampling
activitv ”
1230
1330
1430
- females
PAL1 activity (U/ml)
0730
0830
0930
1030 1130 Time of sampling
1230
1330
1430
Fig. 2 PAL1 activity according to time of sampling (A: males,
B:females). 10, 25, 95 and 97.5 percentiles in thin lines, medians in thick line.
320
Epidemiology
of Fibrinolysis
tPA antigen - males
A tPA antigen 30W)
I
correlation with t-PA antigen (Table 6). t-PA activity correlated with plasma fibrinogen levels in men but not in women. PAI- activity showed a strong positive relation with t-PA-antigen in both men and women. PAI-1 activity correlated with plasma fibrinogen only among women. t-PA antigen also correlated with plasma fibrinogen, more strongly in women than in men.
DISCUSSION ”
0730
0830
0930
1130 Time.of sampling 1030
1230
1330
1430
tPA antigen - females
B tPA antigen
(Id)
AND CONCLUSIONS
This is the first large population-based study of fibrinolytic variables that is restricted to subjects without concomitant disease or medication and includes measurements of t-PA activity. We found a gradual increase in t-PA activity during the day. t-PA antigen and PAI- activity decreased. These results confirm those from small experimental studies in which individuals were sampled frequently during 24 h 7s” which reported a peak in t-PA activity and a
Plasma fibrinogen - males PhIIa fibrinogen
A
pm
-
tu
I
I
4 0730
0830
0930
1130 1030 Time of sampling
1230
1330
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..f................ C
1430 t
Fig. 3 t-PA antigen levels according to time of sampling (A:males, B:females). 2.5,5, 95 and 97.5 and 97.5 percentiles thin lines, medians in thick line.
I
in
0730
0830
0930
1030
1130
1230
1330
1430
Time of sampling Table 3 t-PA activity (W/ml) and t-PA antigen (&I), age and gender. Percentiles. Pooled values from 07:oO until 09:59
Plasma fibrinogen - females
B Age
n
t-PA activity 2.5% 50%
97.5%
t-PA antigen 2.5% 50%
2534 M F
66 72
0.18 0.21
1.77 2.56
0.63 0.77
2.0 0.4
5.5 4.0
2.6 1.9
6.6 4.8
13.1 22.2
74 80
0.19 0.23
1.89 1.71
0.64 0.68
f...i
4
***
***
*** 78 74
0.19 0.28
0.57 0.80
1.66 2.38
3.0 2.2
8.1 5.5
17.7 34.1
................................................................ 1
28.6 12.3 2
45-54 M F
1
I-
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0730
55-64 M F
I
***
35-44 M F
Plasma fibrinogen (gn)
97.5%
0830
0930
1030
1130
1230
1330
1430
Time of sampling 52 65
0.17 0.19
0.69 0.69
4.40 1.75
***=p
2.9 2.0
8.6 7.6
31.4 29.3
Fig. 4 Plasma fibrinogen levels according to time of sampling (A: males, B: females). 2.5,5, 95 and 97.5 percentiles in thin lines, medians in thick line.
Fibrinolysis
gender. Percentiles.
Table 4 PAL1 activity (U/ml), age and
Pooled values from 07:OOuntil 0959 n
10%
25%
50%
F
66 72
1.4 0.6
4.0 3.4
8.1 6.4
40.6 48.0
35-44 M F
74 80
0.4 0.1
3.0 3.2
7.4 6.8
34.6 35.8
78 74
0.6 0.4
4.0 2.8
10.6 6.0
48.0 28.0
52 65
0 1.3
3.2 5.2
7.6 9.8
44.4 40.0
Age 25-34 M
4S54 M F 55-64 M
F
97.5%
**
**=p
Table 5 Plasma fibrinogen (g/l), age and gender. Percentiles. Pooled values from 07:OOuntil 14:59
n
2.5%
50%
M F
161 144
2.0 2.0
2.9 3.2
4.8 5.7
35-44 M F
190 174
1.9 2.2
3.1 3.2
5.2 5.1
45-54 M F
175 161
2.3 2.4
3.5 3.5
5.1 6.1
153 143
2.5 2.7
3.5 3.8
Age 25-34
97.5%
***
55-64
***
M F
5.3 6.2
***=p
Table 6 Pearson’s correlation coefficients for fibrinolytic
parameters and plasma fibrinogen. Men (n=677, upper), women (n=622, lower) PAL1 activity
t-PA antigen
*** t-PA activity
-0.73 *** -0.59
Fibrinogen
*** -0.21
***
-0.19
** 0.11 -0.01
*** PAL1 activity
0.40 0.37
***
-0.01
**
0.14 ***
t-PA antigen
0.16 0.34
**=p
***=p
***
321
trough in PAI-1 activity and t-PA antigen around 18$JO . .M’ Least t-PA activity was found between midnight and 06:00, when PAI- activity and t-PA antigen levels peaked. High levels of PAI- in the morning are thought to inactivate t-PA and lead to low t-PA activity and high levels of t-PA antigen” as this mainly measures the inactive t-PA-PAIcomplex. “.** If so, circadian rhythms in fibrinolysis are mainly due to variations in PAI- and not to fluctuating t-PA release. Our data show that the circadian rhythm in fibrinolytic variables previously observed under experimental-like conditions is also found in population-based samples. The nadir in t-PA activity in the morning may contribute to the well established peak in incidence of myocardial infarction6 and ischaemic stroke7 in the morning hours. Chandler searched for hormonal regulators in the diurnal rhythm of fibrinolysis but found no relations with insulin, cortisol or cathecholamines. ” As has previously been noted,” we found no circadian change in plasma fibrinogen. We noted that the upper reference vaue (i.e. 97.5% percentile) for PAI- activity is the high in the morning, falls during the forenoon and then rises after 12:00 t-PA activity showed no changes at this time. We cannot suggest any explanation for this. We found no evidence that t-PA activity decreases with age, at least in subjects below 65 years. This agrees with the finding of unchanged euglobulin fibrinolytic activity between 20-year-olds and 80year-olds, I3 but not with the finding of a considerable decrease in t-PA activity in the euglobulin fraction of plasma with age. ‘* It is worth noting that the present study is the first large population study which employs methods for t-PA activity that are reasonably free from sample collection artifacts and assay non-specificites. ‘s We found no distinct age effect on PAI- activity except for an increase in women between 55 and 64 years. The two cross-sectional studies which addressed this issue included 260 and 367 subjects, of both sexes, aged 20 to 60 years, respectively.‘4*24 Sundell et al found no influence of age on PAIactivity in men but a significant increase occurred in women 50 to 60 years of age.14 Our median values correlate well with those reported in a population study from the same region24 but our upper reference limits (97.5 percentiles) are somewhat higher in the younger age groups. Two smaller studies have noted increased PAI- activity with age.‘*,13 The method used for assaying PAI- activity, addition of fixed amount of t-PA activity to the sample and determination of residual t-PA activity is biased by the endogenous t-PA activity of the sample. Thus, a sample containing lOU/ml of PAI- activity and lIU/ml of t-PA activity will be assayed as containing 9U/ml of PAIactivity. The PAIactivity results presented in this study, as in Table 4, have not been corrected for the effect of endogenous t-PA activity. Inaccuracy due to this effect is larger in
322 Epidemiology of Fibrinolysis
samples with low PAI-1 activity because these, on averge, contain more t-PA activity. If the results had been corrected, the mean and median PAI- activity values would have been about lU/ml higher, the values for the 95 and 97.5 percentiles would have been about 0.4 and 0.3IU/ml higher, respectively. About 13% of the samples tested yielded negative values for PAI- activity. This percentage would have been reduced to about 7% by correction for the effect of endogenous t-PA activity. We assume that PAIactivity in this group is close to zero and have yielded a negative PAI-1 activity value due to the precision of the assay. We suggest that results below 3 SD for a PAI-1 depleted plasma should, in a clinical setting, be reported as being below this value. With our procedure this value was 2.4U/ml. Thus we would report such results from the clinical samples as ‘<2.4U/ml’. Our study shows an age-related increase in t-PA antigen, which has been reported before. ‘2-‘4,22We found correlations with age similar to those reported by Sundell et a1,14 and a distinct increase in women between the 45 to 54-year age group and the 55 to 64-year age group, supposedly caused by the menopause. The concept that fibrinogen levels increase with has been interpreted as an effect of conage **13*15 comitant disease.16 In our study we excluded all subjects with any kind of known vascular disease, diabetes or treated hypertension. However, we still noted a strong age-dependence for plasma fibrinogen levels in both sexes. As smoking, obesity and hypercholesterolaemia increase fibrinogen levels25 we compared these variables in different age groups (data not shown). Both body mass index and cholesterol levels increased with age but smoking did not. In a multiple regression analysis age still remained an independent determinant for fibrinogen levels. In general, women had greater t-PA activity and lower PAI- activity up till the age of 55 where the differences levelled off and for PAI- activity even reversed. A similar pattern was noted for t-PA antigen and PAI-1 activity in another study.14 A decrease in t-PA activity among men aged 45 to 54 seems to be due to a distinct increase in PAIactivity. The reason for this is unclear. We found significantly greater fibrinogen levels in women in the youngest and oldest age groups. Higher levels have been reported in women,* but this has not been explained. Plasma fibrinogen levels increase after the menopause and probably contribute to the increase in CVD in post-menopausal women. l5 We conclude that fibrinolytic variables vary sufficiently between different groups of healthy individuals to make age- and gender-specific reference data necessary. The time of blood sampling must be standardized. The close interrelations between t-PA activity and PAI- activity and the modest negative associations between t-PA activity and t-PA antigen levels support the contention that PAI- is the major regulator of t-PA activity. Measurements of t-PA
antigen levels poorly reflect fibrinolytic function and, as has been previously noted,” cannot be a substitute for t-PA activity determinations.
ACKNOWLEDGEMENTS This study was supported by grants from the Swedish Medical Research Council (27X-07192) The Heart and Chest Fund, King Gustaf V’s 80th Anniversary Fund, King Gustaf V’s and Queen Victoria’s Foundation, the 1987 Stroke Fund, the Joint Committee of the Northern Sweden Health Care Region, Norrbotten Local Authority Research Fund and the National Association for Heart and Lung Patients. Sven-Ola Fredriksson provided excellent technical assistance.
REFERENCES 1. Meade T W, Brozovic M, Chakrabarti R R et al. Haemostatic function and ischaemic heart disease: principal results of the Northwick Park Heart Study. Lancet 1986; ii: 533-537. 2. Kannel W B, Wolf P A, Castelli W P, D’Agostino R B. Fibrinogen and risk of cardiovascular disease. J A M A 1987; 258: 1183-1186. 3. Wilhelmsen L, Svlrdsudd K, Korsan-Bengtsen K, Larsson B, Welin L, Tibblin G. Fibrinogen as a risk factor for stroke and myocardial infarction. N Engl J Med 1984; 311: 501505. 4. DeWood M, Spores J, Notske R, Mouser L T, Burroughs R, Golden M S, Lang H T. Prevalence of total coronary occlusion during the early hours of transmural myocardial infarction. N Engl J Med 1980; 303: 897-902. 5. Hamsten A, Walldius G, Szamosi A et al. Plasminogen activator inhibitor in plasma: risk factor for recurrent myocardial infarction. Lancet 1987; ii: 3-8. 6. Muller J E, Stone P H, Turi Z G et al. Circadian variation in the frequency of onset of acute myocardial infarction. N Engl J Med 1985; 313: 15-22. 7. Wroe S J, Sandercock P, Bamford J, Dennis M, Slattery J, Warlow C. Diurnal variation in incidence of stroke: Oxford community stroke project. Br Med J 1992; 304: 155-157. 8. Andreotti F, Davies G J, Hacket D R et al. Major circadian fluctuations in fibrinolytic factors and possible relevance to time of onset of myocardial infarction, sudden cardiac death and stroke. Am J Cardiology 1988; 62: 635-637. 9. Angleton P, Chandler W L, Schmer G. Diurnal variation of tissue-type plasminogen activator and its rapid inhibitor (PAI-1). Circulation 1989: 79: 101-106. 10. kkiyama Y, Kazama M, Tahara C et al. Reference values of hemostasis related factors of healthy Japanese adults I: circadian fluctuations. Thromb Res 1990; 60: 281-289. 11. Chandler W L, Mornin D, Whitten R 0 et al. Insulin, cortisol and catecholamines do not regulate circadian variations in fibrinolytic activity. Thromb Res 1990; 58: 1-12. 12. Hashimoto Y, Kobayashi A, Yamazaki N, Sugarawa Y, Takada Y, Takada A. Relationship between age and plasma t-PA, PA-inhibitor and PA activity. Thromb Res 1987; 46: 625-633. 13. Aillaud M F, Pignol F, Alessi M C et al. Increase in plasma concentrations of plasminogen activator inhibitor, fibrinogen, von Willebrand factor, factor VIII:C and in erythrocyte sedimentation rate with age. Thromb Haemost 1986; 55: 33&332. 14. Sundell I B, Nilsson T K, Ranby M, Hallmans G, Hellsten G. Fibrinolytic variables are related to age, sex, blood pressure, and body build measurements: a cross-sectional study in Norsjo, Sweden. J Clin Epidemiol 1989; 42: 719723.
Fibrinolysis
15. Balleisen L, Bailey J, Epping P H, Schulte H, van de Loo. J. Epidemiological study on factor VII, factor VIII and fibrinogen in an industrial population. I. Baseline data on the relation to age, gender, body weight, smoking, alcohol, pill-using and menopause. Thromb Haemost 1985; 54: 475479. 16. Ernst E, Koenig W, Matrai A, Keil U. Hlmorheologische Variablen bei manifesten arteriellen Geftisserkrankungen. VASA 1986; 15: 365-372. 17. Aplund K, Huhtasaari F, Lundberg V, Stegmayr B, Wester P 0. Trends in cardiovascular risk factors in the northern Sweden MONICA study: Who are the winners? Cardiovascular Risk Factors (in press). 18. RBnby M, Sundell I B, Nilsson T K. Blood collection in strong acidic citrate anticoagulant used in a study of dietary influence on basal t-PA activity. Thromb Haemost 1989; 62: 917-922. 19. Eriksson E, Tengborn L, Risberg B. The effect of various anticoagulant/antiplatelet mixtures on determination of plasminogen activator inhibitor, platelet proteins and hemostasis parameters. Thromb Haemost 1989; 61: 511-516.
Received: 9 September 1992 Accepted after revision: 29 December 1992 Offprint orders to: Mats Eliasson MD, Department Medicine, Lulel Hospital, S-951 28 Lulel, Sweden. Fax: 46 (0)920-71083.
of Internal
323
20. Wiman B, Mellbring G, Ranby M. Plasminogen activator release during venous stasis and exercise as demonstrated by a new specific assay. Clin Chim Acta 1983; 127: 279. 21. Chmielewska J, RBnby M, Wiman B. Evidence for a rapid inhibitor to tissue plasminogen activator in plasma. Thromb Res 1983; 31: 427-436. 22. Rgnby M, Bergsdorf N, Nilsson T, Mellbring G, Winblad B, Bucht G. Age dependence of tissue plasminogen activator concentrations in plasma as studied by an improved enzymelinked immunosorbent assay. Clin Chem 1986; 32: 21602165. 23. Becker U, Bartl K, Wahlefeld W. A functional photometric assay for plasma fibrinogen. Thromb Res 1984; 35: 475-484. 24. Ranby M, Sundell I B, Johnsson 0, Ledin M-C, DahlCn G. Activity of plasminogen activator inhibitor-l (PAI-1) in a population of Northern Sweden. Fibrinolysis 1990; 4 (suppl 2): 54-55. 25. Ernst E. Plasma fibrinogen - an independent cardiovascular risk factor. J Int Med 1990; 227: 365-372.
Influence of Gender, Age and Sampling Time on Plasma Fibrinolytic Variables and Fibrinogen A Population Study
M. Eliasson, P-E. Evrin, D. Lundblad, K. Asplund, M. Ranby
SUMMARY. Reference data are provided for tissue plasminogen activator (t-PA) activity, t-PA antigen levels, plasmhtogen activator inhibitor type 1 (PAI-1) activity and fibrinogen levels in plasma. The data are from a population-based sample of 1288 healthy 25 to 64-year-old men and women who were not taking any medication. Time of sampling influenced the measurements in that, from 07:OOto 14:00 t-PA activity rose, t-PA antigen levels and PAI- activity declined, whereas plasma fibrinogen levels remained unchanged. Age had little influence on t-PA activity or PAI-1 activity, although t-PA antigen levels correlated with increasing age (r=0.26 and 0.34, in men and women, respectively). Fibrinogen levels also correlated with age (r=0.29 in both sexes). In the 45 to 54-year age group, t-PA activity was significantly lower (p
type 1 (PAI-1) activity strongly predicts reinfarction in survivors of myocardial infarction before the age of 45.5 There is a well-known circadian rhythm in the incidence of myocardial infarction6 and cerebral thrombosis7 with a peak in the morning hours. A parallel trough in fibrinolytic activity and a peak in fibrinolytic inhibition during the late night hours has recently been shown in small groups of healthy volunteers.S” Plasma fibrinogen does not appear to have a diurnal variation. lo The increase in incidence of CVD with age may, in part, be due to decreasing fibrinolysis. Greater PAIactivity and tissue plasminogen activator (t-PA) antigen levels were found in older subjects’2*‘3 but in a recent population study PAI- activity increased with age only in women. I4 Data concerning t-PA activity and age are lacking. Most studies have found an age-related increase in fibrinogen2*‘3*‘5 but this has
The development of cardiovascular disease (CVD) can only partially be explained by traditional risk factors such as smoking, hypertension, hyperlipidaemia and diabetes. Recently, attention has focused on the role of coagulation and fibrinolysis. From the prospective studies in Northwick Park, Framingham and Gothenburg,‘” elevated plasma fibrinogen levels were found to be as strong predictors of stroke and myocardial infarction as serum cholesterol levels in both sexes. Almost all myocardial infarctions involve the formation of an arterial thrombus4 and this has focused interest on the role of the haemostatic system. Increased plasminogen activator inhibitor M. Ehsson, D. Lundblad, Department of Internal Medicine, Lulei-Boden Central Hospital, LuIeP, P-E. Evrin, Department of Clinical Chemistry, Central Hospital, Boden, K. Asplund, Department of Internal Medicine, M. Rhby, Department of Medical Biochemistry, University of Ume& Sweden. 316
Fibrinolysis
been disputed.‘6 We conducted a study on a welldefined, randomly selected population sample free from apparent disease in order to establish relationships and reference values for t-PA activity, t-PA antigen, PAI- activity, plasma fibrinogen in relation to gender, age and time of sampling.
MATERIAL AND METHODS This study was performed within the framework of the Northern Sweden MONICA Project which, in turn, is a part of the WHO MONICA Project (Monitoring of Trends and Determinants in Cardiovascular Disease). In January to April 1990, a population was screened for cardiovascular risk factors. A total of 2000 individuals in the 25 to 64 year range were invited. Within each age group (25-34, 35-44, 45-54, 55-64 years) 250 men and 250 women were randomly selected from continuously updated population registers in Norrbotten and Vasterbotten, the two northernmost provinces of Sweden. They were invited by letter to an examination. If they did not attend, a reminder with a new appointment was sent. People who still did not come, were contacted by telephone to ascertain reasons for reluctance to attend and basic information on social background and risk factors. In all, 1583 subjects participated in the study (79.2% of all invited). It was possible to obtain information on 203 of the 417 non-participants.” In short, participation rate was lower in 25 to 34-yearold men (66.8%) and women (71.1%) than in older age groups (80982.8% in men, 82.4-86.3% in women). The proportion of married or cohabiting people was lower in non-participants than in participants, but the level of education did not differ. Smoking was more common in both male and female non-participants than in participants. The participants were asked to complete a questionnaire with items on, inter alia, social background, smoking habits, medical history and intake of drugs. The questionnaire was returned on the site of the survey, which was performed by two mobile teams in local health centres (or corresponding) throughout the MONICA area. Our aim was to study a healthy population and therefore we excluded 262 participants (126 with treated hypertension, 22 with prior myocardial infarction, 19 with prior stroke, 43 who had diabetes mellitus, 53 who used contraceptive pills and 39 who used oestrogen substitution). Some had two or more concomitant exclusion criteria. No woman was pregnant. Data were incomplete in 33 persons, and thus the study group consisted of 1288 persons. The Northern Sweden MONICA Study has been approved by the Research Ethics Committee of Umei University and the data handling procedures by the National Computer Data Inspection Board.
317
Sampling Examination took place between 7:00 and 15:O0. For purpose of analysis those studied from 07:OOto 07:59 are pooled and allocated to the 7-h time group and so on. Participants sampled before 11:30 had fasted for at least 12 h, those sampled later had fasted for at least 4h. They were instructed not to use tobacco during 1 h before the examination. Venous sampling was done in the sitting position with no special rest and with minimal occlusion. Blood was drawn in 5ml vacuum tubes (Stabilyte, product No. 102080, Biopool, UmeH, Sweden) prefilled with 0.5ml of 0.45moVl citrate buffer pH 4.3. This ensured stability of t-PA activity without causing appreciable haemolysis. Is Tubes were centrifuged at room temperature at 2000xg for 20min in order to remove all platelets and then snap-frozen within an hour and stored in liquid nitrogen. This procedure has been shown not to release PAI- activity from platelets. I9
Methods Reagent kits from Biopool were used for determination of t-PA activity, t-PA antigen and PAIactivity. All absorbance measurements were performed by a Dynatech MR700 microplate reader. t-PA activity was determined using the Spectrolyse/fibrin kit, product number 101101,20 implementing the modifications supplied with the Stabilyte sample collection tubes. ” The conversion of plasminogen to plasmin is catalysed by t-PA in the presence of fibrin. The amount of colour developed by the plasmin-induced cleavage of a chromogenic substrate is proportional to the amount of t-PA in the sample. PAI- activity was analyzed by the microtestplate version of the Spectrolyse/pl kit, product number 101201, but following the modifications supplied with the Stabilyte sample collection tubes,18 with the following exception; one volume of plasma was reacted with two volumes of 40IU/ml of t-PA instead of 20IU/ml. This resulted in an assay range between 0 and 80U/ml. The method was based on the addition of a fixed amount of t-PA in excess to the samples. After binding to PA1 the residual t-PA-activity was measured in a similar way as above.2’ PAI-1 activity values below zero implied that the sample contained less PAI- than t-PA activity. t-PA antigen was determined by the TintElize t-PA kit, product number 101120, which is a double antibody enzyme-linked-immunosorbent-assay.22 Plasma fibrinogen determinations were performed by the reagent kit Fibrinogen Kinetic from Boehringer Mannheim, using a Hitachi 717 analyzer. In short, the sample was mixed with a snake venom, and the fibrin formation is then Batroxobin, measured by turbidimetry.23 The assay was
318 Epidemiology of Fibrinolysis
standardized with standard Scandinorm, Diagnostica Stago (France). There was no difference in fibrinogen concentration when samples obtained with the Stabilyte tubes were compared with samples collected in vacuum tubes containing 0.13 moY1 of sodium citrate (Becton Dickinson) (data not shown).
Assay Imprecision and Analytical Sensitivity Determinations of t-PA and PAI- activity were performed in duplicate. For each method, control samples were analyzed repeatedly on different days to allow estimates of assay imprecision. Control samples were prepared as pellets in liquid nitrogen, stored at -70°C and thawed just before analysis. The coefficient of variation (CV) for t-PA activity at 0.45 and 2.0IU/ml was 11.1 and 9.6% respectively (n=44). For PAI- activity, CV at 5.8 and 29.4U/ml was 27.3 and 5.8%, respectively (n=33). For t-PA antigen CV at 1.9 and 12.Ot~g/l was 13.3 and 11.2%, respectively (n=16). For plasma fibrinogen a freezedried control plasma, Control Plasma Normal, from Nycomed A/S, Norway, was used. The CV was 4.0% at a mean level of 2.72g/l (n=42). The analytical sensitivity of the method was determined by analyzing zero standard containing PAI-1 and t-PA depleted plasma for t-PA and PA1 activity and the PET-buffer for t-PA-antigen. The sensitivity was calculated as mean+3 S.D. of the absorbance values for the t-PA activity and t-PA antigen and as mean -3 S.D. for PAI- activity. The analytical sensitivity obtained in this way was O.O5IU/ml, 2.4U/ml and 0.3@1 for t-PA activity, PAI- activity and t-PA antigen, respectively.
RESULTS A sample of 1288 persons was studied, 671 men and 617 women. Relevant background variables are shown in Table 1. Influence of Time of Sampling The number and sex of subjects in each time-group are shown in Table 2.40.2% of the men and 47.2% of the women were sampled before 10:OO. Age, BMI, blood pressure, lipid levels and fasting glucose were independent of time groups (data not shown). t-PA activity rose steadily during the day from a median value of 0.48 to 1.09IIJml (an increase of 127%) p
Influence of Age Due to the strong influence of time of sampling, age-related values for t-PA activity, PAI- activity and t-PA antigen are shown for a ‘morning’ group sampled between 07:OOand 09:59 which may resemble a clinical setting. This group includes 40% of the men and 47% of the women. For plasma fibrinogen all participants were included. Correlations between Table 1 Background variables for the study population. Means (SD)
Statistics As the distributions for all four dependent variables were skewed we report medians (i.e the 50th percentile) and percentiles (2.5,5, 95, 97.5). PAI- activity values below zero were found in 13.2% of the samples (6.8% in samples at 07:OO and 27% at 14:OO). We therefore report the tenth and the twenty-fifth percentile for PAI-1 activity instead of the two-and-ahalf and fifth percentile. Comparisons between groups were made with the non-parametric KruskalWallis one-way analysis of variance (ANOVA). Two-tailed significance was used and due to the multiple comparisons only p values below 0.01 were considered significant. Pearson’s correlation coefficients were calculated with values for fibrinogen, t-PA activity and t-PA antigen transformed by the natural logarithm. The computer program SPSS/PC+4.01 was used for the analyses.
Variable
Males
Females
BMI (kg/m*) Cholesterol (mmol/I) HDL-cholesterol (mmoVI) Systolic BP (mm Hg) Diastolic BP (mm Hg) Smokers Postmenopausal
25.5 (3.2) 6.3 (1.3) 1.29 (0.30) 129 (16) 82 (11) 22.8%
24.7 (4.2) 6.0 (1.3) 1.57 (0.33) 124 (18) 77 (11) 27.4% 29.9%
Table 2 Numbers and gender in each time-group
Time-group
Men
Women
07 08 09 10 11 12 13 14
57 97 116 145 60 50 92 54
46 129 116 118 46 39 81 42
Fibrinolysis
A
319
19% (p~O.001) in women. The correlation r=0.29 in both sexes (p
tPA activity - males
tPA activitv
was
Influence of Gender
OL
I
1
’
0730
0830
B
0930
I
I
1030 1130 Time of sampling
I
I
I
1230
1330
1430
tPA activity - females
. . . . . . . . . . . . . I2 tPA activity ( IUlml )
_I
2
In the 45 to 54-year age group, women had significantly greater levels of t-PA activity than men, whereas the levels were similar in the oldest age group (Table 3). A difference was also noted for PAI- activity in the 45 to 54-year age group with significantly greater activity among men (Table 4). t-PA antigen levels were consistently greater in men than in women in all but the oldest age group (Table 3). Plasma fibrinogen levels were greater in women than in men but the difference was significant (p
.
. . . . . . . . . . .
Interrelationship Between Fibrinolytic Variables In both sexes t-PA activity showed a strong, inverse relation with PAI- activity and a weak, negative
. .
PAI-
A
activity
- males
PAI- activity ( U/ml )
40[ .....&
.............................::.............
”
0730
0830
0930
1030 1130 Time of sampling
1230
1330
1430
Fig. 1 t-PA activity according to time of sampling (A: males, B:females). 2.5,5,95 and 97.5 percentiles with thin lines, medians and thick line.
30 ~~$__.&.~ .............................
20
..............................
10
”
. . ........
E
fibrinolytic parameters and age were calculated for the entire group as age was evenly distributed during the day. As shown in Table 3, median levels of t-PA activity remained essentially unchanged in both men and women in the 25 to 64-year range. There was a weak positive univariate correlation between t-PA activity and age in men (r=O. 14, p~O.001) but not in women (r=0.09, p>O.Ol). Median levels increased with 9.6% among men from the youngest to the oldest age group (p=O.4). PAI-1 activity showed no correlation with age but in women median levels were 53% greater (p=O.O3) in the oldest group compared to the youngest group (Table 4). Median levels of t-PA antigen increased by 56% (p<0.0001) in men and 90% (p
0730
0830
PAI-
B
0930
1030 1130 Time of sampling
activitv ”
1230
1330
1430
- females
PAL1 activity (U/ml)
0730
0830
0930
1030 1130 Time of sampling
1230
1330
1430
Fig. 2 PAL1 activity according to time of sampling (A: males,
B:females). 10, 25, 95 and 97.5 percentiles in thin lines, medians in thick line.
320
Epidemiology
of Fibrinolysis
tPA antigen - males
A tPA antigen 30W)
I
correlation with t-PA antigen (Table 6). t-PA activity correlated with plasma fibrinogen levels in men but not in women. PAI- activity showed a strong positive relation with t-PA-antigen in both men and women. PAI-1 activity correlated with plasma fibrinogen only among women. t-PA antigen also correlated with plasma fibrinogen, more strongly in women than in men.
DISCUSSION ”
0730
0830
0930
1130 Time.of sampling 1030
1230
1330
1430
tPA antigen - females
B tPA antigen
(Id)
AND CONCLUSIONS
This is the first large population-based study of fibrinolytic variables that is restricted to subjects without concomitant disease or medication and includes measurements of t-PA activity. We found a gradual increase in t-PA activity during the day. t-PA antigen and PAI- activity decreased. These results confirm those from small experimental studies in which individuals were sampled frequently during 24 h 7s” which reported a peak in t-PA activity and a
Plasma fibrinogen - males PhIIa fibrinogen
A
pm
-
tu
I
I
4 0730
0830
0930
1130 1030 Time of sampling
1230
1330
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..f................ C
1430 t
Fig. 3 t-PA antigen levels according to time of sampling (A:males, B:females). 2.5,5, 95 and 97.5 and 97.5 percentiles thin lines, medians in thick line.
I
in
0730
0830
0930
1030
1130
1230
1330
1430
Time of sampling Table 3 t-PA activity (W/ml) and t-PA antigen (&I), age and gender. Percentiles. Pooled values from 07:oO until 09:59
Plasma fibrinogen - females
B Age
n
t-PA activity 2.5% 50%
97.5%
t-PA antigen 2.5% 50%
2534 M F
66 72
0.18 0.21
1.77 2.56
0.63 0.77
2.0 0.4
5.5 4.0
2.6 1.9
6.6 4.8
13.1 22.2
74 80
0.19 0.23
1.89 1.71
0.64 0.68
f...i
4
***
***
*** 78 74
0.19 0.28
0.57 0.80
1.66 2.38
3.0 2.2
8.1 5.5
17.7 34.1
................................................................ 1
28.6 12.3 2
45-54 M F
1
I-
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0730
55-64 M F
I
***
35-44 M F
Plasma fibrinogen (gn)
97.5%
0830
0930
1030
1130
1230
1330
1430
Time of sampling 52 65
0.17 0.19
0.69 0.69
4.40 1.75
***=p
2.9 2.0
8.6 7.6
31.4 29.3
Fig. 4 Plasma fibrinogen levels according to time of sampling (A: males, B: females). 2.5,5, 95 and 97.5 percentiles in thin lines, medians in thick line.
Fibrinolysis
gender. Percentiles.
Table 4 PAL1 activity (U/ml), age and
Pooled values from 07:OOuntil 0959 n
10%
25%
50%
F
66 72
1.4 0.6
4.0 3.4
8.1 6.4
40.6 48.0
35-44 M F
74 80
0.4 0.1
3.0 3.2
7.4 6.8
34.6 35.8
78 74
0.6 0.4
4.0 2.8
10.6 6.0
48.0 28.0
52 65
0 1.3
3.2 5.2
7.6 9.8
44.4 40.0
Age 25-34 M
4S54 M F 55-64 M
F
97.5%
**
**=p
Table 5 Plasma fibrinogen (g/l), age and gender. Percentiles. Pooled values from 07:OOuntil 14:59
n
2.5%
50%
M F
161 144
2.0 2.0
2.9 3.2
4.8 5.7
35-44 M F
190 174
1.9 2.2
3.1 3.2
5.2 5.1
45-54 M F
175 161
2.3 2.4
3.5 3.5
5.1 6.1
153 143
2.5 2.7
3.5 3.8
Age 25-34
97.5%
***
55-64
***
M F
5.3 6.2
***=p
Table 6 Pearson’s correlation coefficients for fibrinolytic
parameters and plasma fibrinogen. Men (n=677, upper), women (n=622, lower) PAL1 activity
t-PA antigen
*** t-PA activity
-0.73 *** -0.59
Fibrinogen
*** -0.21
***
-0.19
** 0.11 -0.01
*** PAL1 activity
0.40 0.37
***
-0.01
**
0.14 ***
t-PA antigen
0.16 0.34
**=p
***=p
***
321
trough in PAI-1 activity and t-PA antigen around 18$JO . .M’ Least t-PA activity was found between midnight and 06:00, when PAI- activity and t-PA antigen levels peaked. High levels of PAI- in the morning are thought to inactivate t-PA and lead to low t-PA activity and high levels of t-PA antigen” as this mainly measures the inactive t-PA-PAIcomplex. “.** If so, circadian rhythms in fibrinolysis are mainly due to variations in PAI- and not to fluctuating t-PA release. Our data show that the circadian rhythm in fibrinolytic variables previously observed under experimental-like conditions is also found in population-based samples. The nadir in t-PA activity in the morning may contribute to the well established peak in incidence of myocardial infarction6 and ischaemic stroke7 in the morning hours. Chandler searched for hormonal regulators in the diurnal rhythm of fibrinolysis but found no relations with insulin, cortisol or cathecholamines. ” As has previously been noted,” we found no circadian change in plasma fibrinogen. We noted that the upper reference vaue (i.e. 97.5% percentile) for PAI- activity is the high in the morning, falls during the forenoon and then rises after 12:00 t-PA activity showed no changes at this time. We cannot suggest any explanation for this. We found no evidence that t-PA activity decreases with age, at least in subjects below 65 years. This agrees with the finding of unchanged euglobulin fibrinolytic activity between 20-year-olds and 80year-olds, I3 but not with the finding of a considerable decrease in t-PA activity in the euglobulin fraction of plasma with age. ‘* It is worth noting that the present study is the first large population study which employs methods for t-PA activity that are reasonably free from sample collection artifacts and assay non-specificites. ‘s We found no distinct age effect on PAI- activity except for an increase in women between 55 and 64 years. The two cross-sectional studies which addressed this issue included 260 and 367 subjects, of both sexes, aged 20 to 60 years, respectively.‘4*24 Sundell et al found no influence of age on PAIactivity in men but a significant increase occurred in women 50 to 60 years of age.14 Our median values correlate well with those reported in a population study from the same region24 but our upper reference limits (97.5 percentiles) are somewhat higher in the younger age groups. Two smaller studies have noted increased PAI- activity with age.‘*,13 The method used for assaying PAI- activity, addition of fixed amount of t-PA activity to the sample and determination of residual t-PA activity is biased by the endogenous t-PA activity of the sample. Thus, a sample containing lOU/ml of PAI- activity and lIU/ml of t-PA activity will be assayed as containing 9U/ml of PAIactivity. The PAIactivity results presented in this study, as in Table 4, have not been corrected for the effect of endogenous t-PA activity. Inaccuracy due to this effect is larger in
322 Epidemiology of Fibrinolysis
samples with low PAI-1 activity because these, on averge, contain more t-PA activity. If the results had been corrected, the mean and median PAI- activity values would have been about lU/ml higher, the values for the 95 and 97.5 percentiles would have been about 0.4 and 0.3IU/ml higher, respectively. About 13% of the samples tested yielded negative values for PAI- activity. This percentage would have been reduced to about 7% by correction for the effect of endogenous t-PA activity. We assume that PAIactivity in this group is close to zero and have yielded a negative PAI-1 activity value due to the precision of the assay. We suggest that results below 3 SD for a PAI-1 depleted plasma should, in a clinical setting, be reported as being below this value. With our procedure this value was 2.4U/ml. Thus we would report such results from the clinical samples as ‘<2.4U/ml’. Our study shows an age-related increase in t-PA antigen, which has been reported before. ‘2-‘4,22We found correlations with age similar to those reported by Sundell et a1,14 and a distinct increase in women between the 45 to 54-year age group and the 55 to 64-year age group, supposedly caused by the menopause. The concept that fibrinogen levels increase with has been interpreted as an effect of conage **13*15 comitant disease.16 In our study we excluded all subjects with any kind of known vascular disease, diabetes or treated hypertension. However, we still noted a strong age-dependence for plasma fibrinogen levels in both sexes. As smoking, obesity and hypercholesterolaemia increase fibrinogen levels25 we compared these variables in different age groups (data not shown). Both body mass index and cholesterol levels increased with age but smoking did not. In a multiple regression analysis age still remained an independent determinant for fibrinogen levels. In general, women had greater t-PA activity and lower PAI- activity up till the age of 55 where the differences levelled off and for PAI- activity even reversed. A similar pattern was noted for t-PA antigen and PAI-1 activity in another study.14 A decrease in t-PA activity among men aged 45 to 54 seems to be due to a distinct increase in PAIactivity. The reason for this is unclear. We found significantly greater fibrinogen levels in women in the youngest and oldest age groups. Higher levels have been reported in women,* but this has not been explained. Plasma fibrinogen levels increase after the menopause and probably contribute to the increase in CVD in post-menopausal women. l5 We conclude that fibrinolytic variables vary sufficiently between different groups of healthy individuals to make age- and gender-specific reference data necessary. The time of blood sampling must be standardized. The close interrelations between t-PA activity and PAI- activity and the modest negative associations between t-PA activity and t-PA antigen levels support the contention that PAI- is the major regulator of t-PA activity. Measurements of t-PA
antigen levels poorly reflect fibrinolytic function and, as has been previously noted,” cannot be a substitute for t-PA activity determinations.
ACKNOWLEDGEMENTS This study was supported by grants from the Swedish Medical Research Council (27X-07192) The Heart and Chest Fund, King Gustaf V’s 80th Anniversary Fund, King Gustaf V’s and Queen Victoria’s Foundation, the 1987 Stroke Fund, the Joint Committee of the Northern Sweden Health Care Region, Norrbotten Local Authority Research Fund and the National Association for Heart and Lung Patients. Sven-Ola Fredriksson provided excellent technical assistance.
REFERENCES 1. Meade T W, Brozovic M, Chakrabarti R R et al. Haemostatic function and ischaemic heart disease: principal results of the Northwick Park Heart Study. Lancet 1986; ii: 533-537. 2. Kannel W B, Wolf P A, Castelli W P, D’Agostino R B. Fibrinogen and risk of cardiovascular disease. J A M A 1987; 258: 1183-1186. 3. Wilhelmsen L, Svlrdsudd K, Korsan-Bengtsen K, Larsson B, Welin L, Tibblin G. Fibrinogen as a risk factor for stroke and myocardial infarction. N Engl J Med 1984; 311: 501505. 4. DeWood M, Spores J, Notske R, Mouser L T, Burroughs R, Golden M S, Lang H T. Prevalence of total coronary occlusion during the early hours of transmural myocardial infarction. N Engl J Med 1980; 303: 897-902. 5. Hamsten A, Walldius G, Szamosi A et al. Plasminogen activator inhibitor in plasma: risk factor for recurrent myocardial infarction. Lancet 1987; ii: 3-8. 6. Muller J E, Stone P H, Turi Z G et al. Circadian variation in the frequency of onset of acute myocardial infarction. N Engl J Med 1985; 313: 15-22. 7. Wroe S J, Sandercock P, Bamford J, Dennis M, Slattery J, Warlow C. Diurnal variation in incidence of stroke: Oxford community stroke project. Br Med J 1992; 304: 155-157. 8. Andreotti F, Davies G J, Hacket D R et al. Major circadian fluctuations in fibrinolytic factors and possible relevance to time of onset of myocardial infarction, sudden cardiac death and stroke. Am J Cardiology 1988; 62: 635-637. 9. Angleton P, Chandler W L, Schmer G. Diurnal variation of tissue-type plasminogen activator and its rapid inhibitor (PAI-1). Circulation 1989: 79: 101-106. 10. kkiyama Y, Kazama M, Tahara C et al. Reference values of hemostasis related factors of healthy Japanese adults I: circadian fluctuations. Thromb Res 1990; 60: 281-289. 11. Chandler W L, Mornin D, Whitten R 0 et al. Insulin, cortisol and catecholamines do not regulate circadian variations in fibrinolytic activity. Thromb Res 1990; 58: 1-12. 12. Hashimoto Y, Kobayashi A, Yamazaki N, Sugarawa Y, Takada Y, Takada A. Relationship between age and plasma t-PA, PA-inhibitor and PA activity. Thromb Res 1987; 46: 625-633. 13. Aillaud M F, Pignol F, Alessi M C et al. Increase in plasma concentrations of plasminogen activator inhibitor, fibrinogen, von Willebrand factor, factor VIII:C and in erythrocyte sedimentation rate with age. Thromb Haemost 1986; 55: 33&332. 14. Sundell I B, Nilsson T K, Ranby M, Hallmans G, Hellsten G. Fibrinolytic variables are related to age, sex, blood pressure, and body build measurements: a cross-sectional study in Norsjo, Sweden. J Clin Epidemiol 1989; 42: 719723.
Fibrinolysis
15. Balleisen L, Bailey J, Epping P H, Schulte H, van de Loo. J. Epidemiological study on factor VII, factor VIII and fibrinogen in an industrial population. I. Baseline data on the relation to age, gender, body weight, smoking, alcohol, pill-using and menopause. Thromb Haemost 1985; 54: 475479. 16. Ernst E, Koenig W, Matrai A, Keil U. Hlmorheologische Variablen bei manifesten arteriellen Geftisserkrankungen. VASA 1986; 15: 365-372. 17. Aplund K, Huhtasaari F, Lundberg V, Stegmayr B, Wester P 0. Trends in cardiovascular risk factors in the northern Sweden MONICA study: Who are the winners? Cardiovascular Risk Factors (in press). 18. RBnby M, Sundell I B, Nilsson T K. Blood collection in strong acidic citrate anticoagulant used in a study of dietary influence on basal t-PA activity. Thromb Haemost 1989; 62: 917-922. 19. Eriksson E, Tengborn L, Risberg B. The effect of various anticoagulant/antiplatelet mixtures on determination of plasminogen activator inhibitor, platelet proteins and hemostasis parameters. Thromb Haemost 1989; 61: 511-516.
Received: 9 September 1992 Accepted after revision: 29 December 1992 Offprint orders to: Mats Eliasson MD, Department Medicine, Lulel Hospital, S-951 28 Lulel, Sweden. Fax: 46 (0)920-71083.
of Internal
323
20. Wiman B, Mellbring G, Ranby M. Plasminogen activator release during venous stasis and exercise as demonstrated by a new specific assay. Clin Chim Acta 1983; 127: 279. 21. Chmielewska J, RBnby M, Wiman B. Evidence for a rapid inhibitor to tissue plasminogen activator in plasma. Thromb Res 1983; 31: 427-436. 22. Rgnby M, Bergsdorf N, Nilsson T, Mellbring G, Winblad B, Bucht G. Age dependence of tissue plasminogen activator concentrations in plasma as studied by an improved enzymelinked immunosorbent assay. Clin Chem 1986; 32: 21602165. 23. Becker U, Bartl K, Wahlefeld W. A functional photometric assay for plasma fibrinogen. Thromb Res 1984; 35: 475-484. 24. Ranby M, Sundell I B, Johnsson 0, Ledin M-C, DahlCn G. Activity of plasminogen activator inhibitor-l (PAI-1) in a population of Northern Sweden. Fibrinolysis 1990; 4 (suppl 2): 54-55. 25. Ernst E. Plasma fibrinogen - an independent cardiovascular risk factor. J Int Med 1990; 227: 365-372.