Morning Hypercoagulability and Hypofibrinolysis
Diurnal Variations in Circulating Activated Factor VII, Prothrombin Fragment F1+2, and Plasmin–Plasmin Inhibitor Complex
Background Diurnal fluctuations of blood coagulation and fibrinolysis activity are thought to play a role in the observed circadian variation in the frequency of onset of acute cardiovascular events. In the present study, the diurnal variations in blood coagulation and fibrinolysis activity were investigated in 10 young, healthy control subjects by use of specific molecular activation markers.
Methods and Results The plasma levels of activated factor FVII (FVIIa), the active portion of the main coagulation activator, decreased during the day (8 am: 2.03 ng/mL, CI 1.16 to 2.88 ng/mL; 8 pm: 1.16 ng/mL, CI 0.81 to 1.5 ng/mL; P=.005), whereas FVII antigen did not change significantly. In parallel with the diurnal variations of FVIIa, we found a decrease of prothrombin fragment F1+2 (8 am: 0.97 nmol/L, CI 0.79 to 1.15 nmol/L; 8 pm: 0.78 nmol/L, CI 0.64 to 0.93 nmol/L; P=.005), a molecular marker of intravasal thrombin generation. Evidence for a possible functional relevance of circulating FVIIa was found because this parameter was significantly correlated with prothrombin fragment F1+2 in 72 fasting healthy individuals (r=.29, P=.011). Plasminogen activator inhibitor-1 levels decreased (8 am: 9.9 ng/mL, CI 7.7 to 12.1 ng/mL; 8 pm: 5.4 ng/mL, CI 3.8 to 6.9 ng/mL; P<.005), whereas plasmin–plasmin inhibitor complex levels, representing the degree of intravascular plasmin generation, concomitantly increased (8 am: 235 μg/L, CI 198 to 272 μg/L; 8 pm: 449 μg/L, CI 391 to 507 μg/L; P=.008).
Conclusions Our data suggest that the diurnal changes in the plasma levels of activators and inhibitors of coagulation and fibrinolysis lead to corresponding changes in the activity state of these systems, leading to morning hypercoagulability and hypofibrinolysis.
The frequencies of onset of transient myocardial ischemia,1 myocardial infarction,2 sudden cardiac death,3 stroke,4 and pulmonary embolism5 show marked circadian variations, with parallel increases in the period from morning to noon. Because thrombotic vascular obstruction is observed in these diseases, the reported morning hypercoagulability6 and hypofibrinolysis7 may be involved in their pathophysiology. It was our aim to investigate whether the active form of the pivotal coagulation activator factor VII8 is involved in the reported diurnal changes in coagulation activity. Furthermore, it was our intention to look for relations between diurnal variations in the plasma levels of coagulation and fibrinolysis components and the molecular activation markers F1+29 and PPI,10 which represent the activity state of these systems.
Study Design and Subjects
For the determination of normal ranges (5th to 95th percentiles) for coagulation and fibrinolysis parameters, 104 healthy (as previously described11 ) volunteers (group I; aged 19 to 71 years [mean, 35 years]; 48 men, 56 women) were studied. For the calculation of correlations between FVIIa and F1+2, we used a cross-sectional design in 72 healthy volunteers from group I (group II; aged 19 to 40 years [mean, 29 years]; 33 men, 39 women). Blood was drawn between 8 and 10 am after an overnight fasting period. To study the diurnal variation of coagulation and fibrinolysis parameters, we chose a longitudinal study design in 10 representative healthy volunteers (5 men, 5 women) from group II (group III). These subjects were not different in age, body mass index, cholesterol, triglycerides, or fasting blood glucose levels from the 72 individuals in group II. The study protocol was approved by the local Ethics Committee, and written informed consent had been obtained from all volunteers to participate in the study.
Study Protocol and Blood Sampling
For the longitudinal study, all 10 overnight-fasting, healthy subjects arrived at our department at 7:45 am on the study day. Blood was drawn at 8 am, noon, 4 pm, and 8 pm into siliconized glass tubes (Vacutainer, Becton Dickinson) with sodium citrate (final concentration of 0.013 mol/L) or without additive (for determination of lipids). Platelet-poor plasma was prepared from citrated blood by centrifugation at 3000g for 20 minutes at room temperature. Plasma samples were kept frozen at −80°C for ≤4 weeks before analysis except for factor VII:c and FVIIa, which were analyzed immediately. To avoid hypoglycemia, volunteers received a fat-free meal (24 g complex carbohydrates) after each blood sample was drawn.
FVIIa was determined by use of Staclot VIIa-rTF (Diagnostica Stago) measured with a fully automated STA analyzer (Diagnostica Stago) (normal range, 0.85 to 3.23 ng/mL). This clotting assay was performed according to Morrissey et al8 with slight modifications with the use of a recombinant soluble tissue-factor mutant that abolishes activation of factor VII but not cofactor function for FVIIa in the activation of factor X. All assays were performed in duplicate. Factor VII clotting activity was determined by use of a one-step clotting assay (factor VII–deficient plasma and prothrombin time reagent Thromborel S [Behringwerke]) and a KC-10 coagulometer (Amelung) (normal range, 75% to 130%). FVII:Ag was determined by use of an ELISA (Asserachrom VII:Ag; Diagnostica Stago) (normal range, 76% to 123%); F1+2 by an ELISA (Enzygnost F1+2; Behringwerke) (normal value, <1.9 nmol/L); active PAI-1 antigen by an ELISA (Technoclone) (normal value, <40 ng/mL); and PPI by an ELISA (Behringwerke) (normal range, 95 to 410 μg/L). Cholesterol and triglycerides were determined enzymatically with the use of a fully automated Hitachi 747 (Boehringer Mannheim) (normal ranges, 3.1 to 5.2 mmol/L and 0.8 to 2.3 mmol/L, respectively).
Data are presented as absolute values (means and the 95% CIs) or percent change from baseline (means and the 95% CI). Because data were nonnormally distributed, nonparametric tests were used. To test diurnal variations of measured end points for significance, the Friedman ANOVA and the Wilcoxon signed rank test for post hoc comparisons were used. Correlations were calculated by Spearman’s rank correlation test. To correct for multiple comparisons, the two-tailed probability value was set at P=.016.
FVIIa decreased by −39% (CI, −48% to −31%) from 2.03 ng/mL (CI, 1.16 to 2.88 ng/mL) at 8 am (P=.005). FVII:c decreased by −12% (CI, 6% to 18%) from 105% (CI, 87% to 124%) at 8 am (P=.005). FVII:Ag did not change (P>.05). F1+2 also decreased by 19% (CI, −24% to −14%), from 0.97 μg/L (CI, 0.79 to 1.15 μg/L) at 8 am (P=.005). The participants’ individual data (FVIIa and F1+2) are depicted in Fig 1⇓. A significant correlation between FVIIa and F1+2 was found in fasting healthy individuals (n=72; r=.29, P=.011) (Fig 2⇓).
Active PAI-1 antigen decreased by 46% (CI, −60% to −32%) during the day from 9.9 ng/mL (CI, 7.7 to 12.1 ng/mL) at 8 am (P=.005). PPI measured at 8 pm was 105% (CI, 75% to 135%) higher than that measured at 8 am (235 μg/L; CI, 198 to 272 μg/L; P=.008). The individual data (PAI-1 and PPI) of the 10 volunteers investigated are depicted in Fig 1⇑.
Total serum cholesterol increased minimally (3%; CI, 2% to 4%) from 8 am to 8 pm from 4.48 mmol/L (CI, 4.02 to 4.95 mmol/L) (P=.012). Triglyceride serum levels did not change during the time period observed (P>.05).
The diurnal variations of hemostatic/fibrinolytic variables, pointing to morning hypercoagulability and hypofibrinolysis, are thought to play a pathogenetic role in the higher frequency of arterial1 2 3 4 and venous5 thrombotic events observed between morning and noon compared with the rest of the day. Circadian changes in the activity of plasma coagulation, estimated by the prothrombin time and activated partial thromboplastin time, and the plasma levels of fibrinogen and factor V have been reported previously.6 There is evidence for morning hypofibrinolysis caused by a decrease of the pivotal fibrinolysis inhibitor PAI-1 during the day, which is accompanied by an increase in plasma TPA activity.7 In addition, morning platelet hyperreactivity12 and increased blood viscosity13 may also be of pathophysiological importance for the morning peaks of thrombotic disease. To obtain more detailed information on the circadian variations in coagulation activity, we studied for the first time the diurnal change of factor VII activity (FVIIa) and its antigen (FVII:Ag), the enzyme component of the main coagulation activator (the tissue factor–factor VII complex), in healthy subjects between 8 am and 8 pm. The FVIIa assay used in the present study specifically measures FVIIa, a mediator that is of biological importance,14 particularly at sites where blood may come into contact with tissue factor, eg, atherosclerotic plaques15 or prothrombotically activated endothelium.16 We demonstrate that FVIIa peaks in the morning. Concomitant with the circadian changes of FVIIa, we found a decrease in F1+2, a molecular marker of intravascular thrombin formation. From these results, one might conclude that high morning FVIIa may increase basal thrombin formation, particularly because we found a significant correlation between FVIIa and F1+2 in 72 fasting healthy subjects in the present study and previously in 60 women taking oral contraceptives.17 The triggering mechanisms causing enhanced FVIIa in the morning remain to be determined, because FVII:Ag does not change significantly during the day. FVIIa correlated with total cholesterol in our trial (data not shown) and in previous studies and increased after fat intake.18 The volunteers of the present study were kept on a low-calorie, fat-free diet, and we measured only a minimal increase in total cholesterol from morning to evening of 3%, whereas triglycerides did not change significantly. Thus, in the fasting state, the circadian changes of total cholesterol do not seem to play a role in the diurnal variation of FVIIa. Thus far, it is not clear whether low morning plasma PAI-1 and increased TPA activity7 cause an enhanced systemic activation of plasminogen into plasmin. We measured plasma levels of complexes of plasmin with its specific inhibitor.10 In accordance with diurnal changes in TPA and PAI-1, we demonstrated higher levels of PPI in the evening than in the morning, pointing to low basal systemic plasminogen activation in the morning. It is very likely that diurnal changes in PPI levels reflect variations in systemic plasminogen activation, because the two other components of these complexes, plasminogen and antiplasmin, do not show diurnal variations19 ; however, the present study did not rule out the possibility that changes in the clearance half-lives of PPI contribute to the phenomena observed. Our data indicate that diurnal variations in the plasma levels of activators and inhibitors of blood coagulation and fibrinolysis affect the activity state of these systems, as shown by corresponding changes in molecular activation markers. There is indirect evidence of the clinical importance of hypercoagulability for thrombotic vessel disease, as Miller et al20 found a clear, positive gradient between the extent of hemostasis activation and the risk of fatal coronary heart disease; furthermore, Miller et al found a highly significant positive association between FVIIa and F1+2 and fibrinopeptide A. Because data obtained from prospective studies providing firm evidence of an association between elevated FVIIa and thrombotic events in patients at risk are lacking, one can only speculate that morning hypercoagulability and hypofibrinolysis, together with other triggering factors21 that show a morning surge, such as elevated arterial blood pressure, enhanced plasma epinephrine, and platelet hyperaggregability,12 may contribute considerably to the circadian variation in the onset of these diseases. These diurnal changes may also be of importance for the observed morning resistance to anticoagulant22 23 and thrombolytic therapy.24
Selected Abbreviations and Acronyms
|ELISA||=||enzyme-linked immunosorbent assay|
|F1+2||=||prothrombin fragment F1+2|
|FVII:Ag||=||factor VII antigen|
|FVIIa||=||activated factor VII|
|PAI-1||=||plasminogen activator inhibitor-1|
|PPI||=||plasmin–plasmin inhibitor complex|
|TPA||=||tissue plasminogen activator|
- Received February 3, 1997.
- Revision received May 8, 1997.
- Accepted May 9, 1997.
- Copyright © 1997 by American Heart Association
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