Biological Rhythms in the Physiology and Pharmacology of Blood Coagulation

This article reviews the current knowledge on time-dependent variations in the physiology of blood coagulation and in the anticoagulant effect of heparin and warfarin. Animal data indicated that the shortest blood clotting time and the highest levels of coagulation factors 11, VII, and IX were recorded during the resting period of the animal. These circadian rhythms were not altered by modifications of the lighting regimens. In healthy volunteers, the prothrombin time was longer at the end of the afternoon than early in the morning; the acrophases of activated partial thromboplastin time and thrombin time occurred in the evening or during the night. The acrophases of fibrinogen, factors 11, VII, VIII, and α-1-antitrypsin were obtained in the morning. There is no agreement on the chronobiology of platelet aggregation, and differences can be found in the time of maximal aggregability. The chronopharmacological studies of heparin infused at a constant rate to patients with thromboembolic diseases suggested that maximal effectiveness occurred at 04:00, while it was minimal at 08:00. Animal data indicated that oral administration of warfarin at the end of the activity period of rats produced maximal inhibition of vitamin K-dependent factors. This was the time of day when warfarin interference with the vitamin K cycle of the liver was highest. Further studies are needed to determine the clinical significance of biological rhythms in the physiology and pharmacology of blood coagulation.     

Time-Dependent Variations in the Coagulation Factors II, VII, IX, and X in Young and Elderly Volunteers

The existence of circadian (24-h) rhythms in the coagulation activity of vitamin K-dependent coagulation factors (Factors II, VII, IX, and X) were studied in six healthy young (18-30 years old) and six healthy elderly (69-75 years old) men. Aliquots of 5 ml of blood were obtained from each of the 12 subjects at six different time points over a 24-h period. Factors II, VII, and X were quantified by the prothrombin time test, whereas Factor IX was analyzed by the activated partial thromboplastin time test. Significant circadian variations were found for Factors II and VII in both age groups. The peak and trough values for Factor II were observed at 16: 00 and 00: 00 in young men and at 12: 00 and 16: 00 in elderly men. The amplitude of the rhythmic variation of Factor II was 3.3 ± 1.0 and 4.2 ± 0.9% in young and elderly volunteers, respectively. For Factor VII, the highest values were found during the activity period (08: 00-16: 00), while the lowest values occurred at night (00: 00) for both groups of subjects. The amplitude of the rhythms was twice as large in the young (6.2 ± 2.3%) as in the elderly (3.7 ± 0.8%). The data suggest that age does not alter significantly the chronobiology of Factors II and VII.     

Time-Dependent Variations in the Coagulation Factors II,VII, IX,and X in Young and Elderly Volunteers

The existence of circadian (24-h) rhythms in the coagulation activity of vitamin K-dependent coagulation factors (Factors II, VII, IX, and X) were studied in six healthy young (18–30 years old) and six healthy elderly (69–75 years old) men. Aliquots of 5 ml of blood were obtained from each of the 12 subjects at six different time points over a 24-h period. Factors II, VII, and X were quantified by the prothrombin time test, whereas Factor IX was analyzed by the activated partial thromboplastin time test. Significant circadian variations were found for Factors II and VII in both age groups. The peak and trough values for Factor II were observed at 16: 00 and 00: 00 in young men and at 12: 00 and 16: 00 in elderly men. The amplitude of the rhythmic variation of Factor II was 3.3 ± 1.0 and 4.2 ± 0.9% in young and elderly volunteers, respectively. For Factor VII, the highest values were found during the activity period (08: 00–16: 00), while the lowest values occurred at night (00: 00) for both groups of subjects. The amplitude of the rhythms was twice as large in the young (6.2 ± 2.3%) as in the elderly (3.7 ± 0.8%). The data suggest that age does not alter significantly the chronobiology of Factors II and VII.     

Circadian rhythms of blood clotting time and coagulation factors II, VII, IX and X in rats

The 24-hr variations in clotting times and vitamin K-dependent blood coagulation factors were studied in rats kept on a 12-hr light-dark cycle (light on: 0600-1800 hours). Clotting times were determined under a binocular microscope by measuring the time required for the formation of the first fibrin thread. Factors II, VII and X were analyzed by the prothrombin test while the factor IX was quantified using the activated partial thromboplastin time assay. Results indicated that the clotting times were significantly longer during the dark (activity) period with a peak at 1:00 and a trough at 17:00. Similarly, a variation was found in factor activity levels: prothrombin (II), factor VII and factor X had higher activities during the light span (rest period). The highest activities found at 13:00 and 09:00 were statistically different from the minimum activity levels obtained at 21:00. Factor IX did not show a significant circadian variation.     

Battery of tests for profiling abnormalities of vitamin K-dependent coagulation factors in drug-toxicity studies in rats.

A battery of simple tests for profiling abnormalities of vitamin K-dependent coagulation factors encountered in drug-toxicity studies was verified in rats treated with warfarin (3 and 10 mg/kg, p.o). The thrombotest, or hepaplastin-test, is useful as a follow-up test after routine screening tests for coagulation abnormalities based on PT and APTT, to rule out other coagulation-factor abnormalities. Measurement of coagulation factor activities (factors II, VII, IX and X) using factor-deficient human plasmas provides direct evidence of decreased activities of vitamin K-dependent factors. Furthermore, Echis carinatus venom coagulation time, together with factor II activity, allows us to confirm the generation of PIVKA-II.     

Circadian changes in anticoagulant effect of heparin infused at a constant rate.

Six patients with venous thromboembolism were treated with heparin, administered intravenously by a constant infusion pump. The initial daily dose of heparin was adjusted to keep the activated partial thromboplastin time, sampled at 0800, between 1.5 and 2.5 times the control level. Once that level was obtained, this dose was kept constant. Anticoagulation was thereafter measured, every four hours for 48 hours, by activated partial thromboplastin time, thrombin time, and coagulation factor Xa inhibition assay. The results of all three coagulation tests showed a circadian variation in the six patients. Maximum values were achieved at night and minimum values in the morning. These circadian variations were reproduced for two consecutive days. Differences between night and morning values reached almost 50% for activated partial thromboplastin time, 60% for thrombin time, and 40% for factor Xa inhibition assay. This circadian variation resulted from two rhythms, a circadian rhythm lasting 24 hours and an ultradian rhythm lasting 12 hours, which were detected by cosinor analysis for each coagulation test (p less than 0.01). A circadian rhythm was detected individually in most of the patients for each coagulation test (p less than 0.05). All patients had a nocturnal peak in activated partial thromboplastin time on both days. In four patients this peak exceeded the upper desired limit of activated partial thromboplastin time. These rhythms should be taken into account when evaluating the dosage of heparin to be administered.     

Circannual changes in blood coagulation factors and the effect of warfarin on the hedgehog Erinaceus europaeus

Levels of blood coagulation factors were measured monthly in hedgehogs kept at 20 degrees C under a natural-light schedule for 1 yr using prothrombin time and the P & P method. Hedgehogs display a circannual cycle of blood coagulation factors in the absence of temperature cues, with high levels corresponding to the active period and low levels corresponding to the hibernation period. Ten hedgehogs and ten guinea pigs were treated with clinical doses of warfarin and blood coagulation factors measured. Warfarin caused a similar decrease in vitamin K-dependent blood coagulation factors in both species.     

Circadian and seasonal variations of physiological and biochemical determinants of acute myocardial infarction

Almost all cardiovascular events occur according to a circadian rhythm with a greater frequency in the morning on waking and when resuming activity, the mechanism and precise triggering events for myocardial infarction (MI) are not yet fully known. Multiple biologic functions show a diurnal and/or seasonal variation that may contribute to adverse cardiac outomes. Exogenous factors may also modulates these variations. The MI peak usually occurs between 07:00 and 12:00 h. This timing corresponds to the concurrent increase in platelet aggregability, blood concentration of cortisol, catecholamines, angiotensin II, myocardial oxygen demand and coagulation activity, while fibrinolytic activity is decreased. In this review paper we will point out the biological rhythms of a number of functions involved in acute myocardial infarction e.g. blood pressure, hormonal determinants, cholesterol, among others.     

Circadian Variations in Blood Coagulation Parameters, Alpha-Antitrypsin Antigen and Platelet Aggregation and Retention in Clinically Healthy Subjects

Ten clinically healthy subjects (5 men and 5 women), 31 11 yrs of age, were studied at six timepoints (0800, 1200, 1600, 2000, 0000, 0400) distributed over a 1-week span. Circadian rhythms in platelet aggregation in response to adenosine diphosphate (ADP) and adrenalin (A), platelet adhesiveness measured as retention in a glass bead column, prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen, Factor VIII activity and alpha-1-antitrypsin antigen showed circadian rhythms. The plasma concentrations of plasminogen, alpha-2-macroglobulin, and antithrombin III (AT III) antigen, Factor V and fibrinogen degradation products showed no circadian rhythm by ANOVA or cosinor analysis. The phase relations of the rhythms of different coagulation parameters are of interest in the physiology and pathobiology of the coagulation-fibrinolytic system. The extent of the circadian rhythm (range of change) described is not of a magnitude to lead to diagnostic problems in the clinical laboratory. The timing of these rhythms, however, may determine transient risk states for thromboembolic phenomena, including myocardial infarction and stroke. Several but not all coagulation parameters suggest a transient state of hypercoagulability during the morning hours. The recognition of these rhythmic, and thus in the time of the occurrence predictable temporary risk states for thromboembolic phenomena, may lead to timed treatment and/or effective prevention.     

SEASONAL VARIATION IN THE EFFECT OF A FIXED DOSE OF HEPARIN ON ACTIVATED CLOTTING TIME IN PATIENTS PREPARED FOR OPEN-HEART SURGERY *

We investigated the effect of an injected bolus of 5 mg kg^- heparin at one circadian stage (08:30 to 11:00) on blood coagulation during different months of the year. Activated clotting times (ACTs) were assessed before and 5 min after heparin dosing to ensure extracorporeal circulation during open-heart surgery. The ACT data of 1083 presumably day-active Turkish patients (816 men and 267 women, mostly older than 46 years) who underwent coronary bypass surgery between 08:30 and 11:00 in the years from 1994 to 1997 were analyzed for annual rhythmicity. The ACT values obtained just before and 5 min after heparinization were subjected to cosinor analysis using a 365.25-day period to assess seasonality in basal ACT level and heparin effect. A small-amplitude annual rhythm with a wintertime peak was documented in the morning ACT in the group of 1083 patients. Rhythms of similar magnitude and staging were also detected in heparin effect on ACT in the 1083 patients and in subgroups categorized by gender. Circannual rhythmicity in the heparin effect on ACT was also documented in the elderly (≥ 45 years old), but not young (18-45 years old) patients. The annual mean effect of heparin on the ACT was statistically significantly greater in younger than older patients. The relatively low-amplitude circannual rhythm in heparin effect on ACT (∼10% of the annual mean) is not viewed as being meaningful in patient preparation for bypass surgery for the 5 mg kg^-1 level of heparin dosing. (Chronobiology International, 18(5), 865-873, 2001)     

Factors V and VII anticoagulant activities in the salivary glands of feeding Dermacentor andersoni ticks

The salivary glands of Dermacentor andersoni ticks possess anticoagulant activities that can alter the clotting time of rabbit whole blood. Salivary gland extracts from female ticks inhibit both the intrinsic and extrinsic coagulation systems, and maximal activities against both pathways occur when the ticks attain about 250 mg feeding weight. These anticoagulants are directed against both coagulation factors V and VII, but they do not affect factors II or X. Despite this salivary anticoagulant activity, heavily tick-infested rabbits suffer no visible alteration of their peripheral blood coagulability and have no detectable circulating fibrin degradation products, suggesting that the ticks do not secrete a factor with fibrinolytic activity.     

SEASONAL VARIATION IN THE EFFECT OF A FIXED DOSE OF HEPARIN ON ACTIVATED CLOTTING TIME IN PATIENTS PREPARED FOR OPEN-HEART SURGERY*

We investigated the effect of an injected bolus of 5 mg kg^? heparin at one circadian stage (08:30 to 11:00) on blood coagulation during different months of the year. Activated clotting times (ACTs) were assessed before and 5 min after heparin dosing to ensure extracorporeal circulation during open-heart surgery. The ACT data of 1083 presumably day-active Turkish patients (816 men and 267 women, mostly older than 46 years) who underwent coronary bypass surgery between 08:30 and 11:00 in the years from 1994 to 1997 were analyzed for annual rhythmicity. The ACT values obtained just before and 5 min after heparinization were subjected to cosinor analysis using a 365.25-day period to assess seasonality in basal ACT level and heparin effect. A small-amplitude annual rhythm with a wintertime peak was documented in the morning ACT in the group of 1083 patients. Rhythms of similar magnitude and staging were also detected in heparin effect on ACT in the 1083 patients and in subgroups categorized by gender. Circannual rhythmicity in the heparin effect on ACT was also documented in the elderly (≥ 45 years old), but not young (18–45 years old) patients. The annual mean effect of heparin on the ACT was statistically significantly greater in younger than older patients. The relatively low-amplitude circannual rhythm in heparin effect on ACT (～10% of the annual mean) is not viewed as being meaningful in patient preparation for bypass surgery for the 5 mg kg^?1 level of heparin dosing. (Chronobiology International, 18(5), 865–873, 2001)     

Congenital deficiency of factor VII in a canine family

Prolonged prothrombin time in the blood coagulation test was seen in some beagle dogs whose activated partial prothrombin times were distributed within the normal range. This phenomenon suggested possible abnormalities in coagulation factors II, V, VII, and/or X. Therefore, a revised cross-matching test was given and a determination of coagulation factors related to the extrinsic system was performed. We also determined whether or not factor VII inhibitor was present. The results were as follows: 1) In the revised cross-matching test, the prolonged prothrombin times were revised when normal canine serum was added to the plasma that showed prolongation of prothrombin time, but not when pooled normal canine plasma absorbed with BaSO4 was added to it. 2) The level of factor VII in the plasma with prolonged prothrombin time was 5 approximately 10% of the level in normal canine plasma. 3) Factor VII inhibitor was not detected in the plasma with prolonged prothrombin time or in normal plasma. Consequently, the prolongation of prothrombin time was attributed to a deficiency in factor VII. This abnormality was confirmed to be congenital.     

The effects of sleep deprivation and time of day on cognitive performance

The extent to which the diurnal fluctuations of different cognitive processes could be affected by sleep loss may be explored to predict performance decrements observed in the real world. Twenty healthy male subjects voluntarily took part in 8 test sessions at 06:00, 10:00, 14:00, and 18:00 h, following either a night with or without sleep in random order. Measurements included oral temperature, simple reaction time, sign cancelation, Go/NoGo, and the Purdue pegboard test. The results indicate that simple reaction time and motor coordination had morning–afternoon variations closely following the rhythms of temperature and vigilance. Inhibitory attention (Go/NoGo) presented no morning–afternoon variations. Sleep deprivation may affect the profiles of cognitive performance depending on the processes solicited. Sustained and inhibitory attention are particularly affected in the morning (after 24 and 28 waking hours), while a complex task (visuo-motor coordination) would be affected after 32 waking hours only.     

Diurnal variations of serum GM-CSF levels

We aimed to investigate the daily variations of serum granulocyte-macrophage colony-stimulating factor (GM-CSF) levels and to correlate them with peripheral blood cells counts. Venous blood samples from eleven healthy volunteers were taken four times a day, being at 08:00, 14:00, 20:00 and 02:00h and serum GM-CSF levels measured by ELISA. We could not find a significant overall difference among GM-CSF levels at four different times of the day using the Friedman test. On the other hand, serum GM-CSF levels at night (20:00h) were found to be significantly increased when compared to the morning levels (08:00h) using the Wilcoxon test (P=0. 022). The levels of lymphocytes and white blood cells (WBCs) at 20:00h were also higher than the morning levels (08:00h) as expected. While there was a strong relationship between the morning levels of GM-CSF (08:00h) and all measurements of peripheral blood cells during the day, the levels of GM-CSF measured at 02:00, 14:00 and 20:00h were found to be significantly correlated with only the WBC levels. It was concluded that there may be a significant difference between morning and night levels of GM-CSF and morning levels of GM-CSF may be more important in the regulation of WBC counts during the day. These variations warrant further studies about diurnal rhythms of haematopoiesis chronotherapy with CSFs.     

A new vitamin K-dependent protein. Purification from bovine plasma and preliminary characterization.

Four proteins active in blood coagulation have long been known to require vitamin K for their proper biosynthesis: factors II, VII, IX, and X. This paper describes the purification of a hitherto unrecognized vitamin K-dependent glycoprotein from bovine plasma. The biosynthesis of this protein is interfered with by the vitamin K antagonist Dicoumarol. The molecular weight of the protein is approximately 56,000 and, like factor X, it has two polypeptide chains. The light chain binds Ca2+. Its NH2-terminal amino acid sequence is homologous to the NH2-terminal sequences of the other vitamin K-dependent proteins and it contains vitamin K-dependent gamma-carboxyglutamic acid residues. The biological function of this protein is unknown.     

TIME-OF-DAY EFFECTS ON THE INTERNAL SIMULATION OF MOTOR ACTIONS: PSYCHOPHYSICAL EVIDENCE FROM POINTING MOVEMENTS WITH THE DOMINANT AND NON-DOMINANT ARM

It is well known that circadian rhythms modulate human physiology and behavior at various levels. However, chronobiological data concerning mental and sensorimotor states of motor actions are still lacking in the literature. In the present study, we examined the effects of time-of-day on two important aspects of the human motor behavior: prediction and laterality. Motor prediction was experimentally investigated by means of imagined movements and laterality by comparing the difference in temporal performance between right and left arm movements. Ten healthy participants had to actually perform or to imagine performing arm-pointing movements between two targets at different hours of the day (i.e., 08:00, 11:00, 14:00, 17:00, 20:00, and 23:00?h). Executed and imagined movements were accomplished with both the right and left arm. We found that both imagined and executed arm pointing movements significantly fluctuated through the day. Furthermore, the accuracy of motor prediction, investigated by the temporal discrepancy between executed and imagined movements, was significantly better in the afternoon (i.e., 14:00, 17:00, and 20:00?h) than morning (08:00 and 11:00?h) and evening (23:00?h). Our results also revealed that laterality was not stable throughout the day. Indeed, the smallest temporal differences between the two arms appeared at 08:00 and 23:00?h, whereas the largest ones occurred at the end of the morning (11:00?h). The daily variation of motor imagery may suggest that internal predictive models are flexible entities that are continuously updated throughout the day. Likewise, the variations in temporal performance between the right and the left arm during the day may indicate a relative independence of the two body sides in terms of circadian rhythms. In general, our findings suggest that cognitive (i.e., mental imagery) and motor (i.e., laterality) states of human behavior are modulated by circadian rhythms. (Author correspondence: charalambos.papaxanthis@u-bourgogne.fr)     

Ingestion of superwarfarin leading to coagulopathy: a case report and review of the literature

Superwarfarins are found in many pesticides, including D-con, Prufe I and II, Ramik, Talon-G, Ratak, and Contrac. Ingestion of can lead to significant morbidity and even mortality. Physicians need to consider this diagnosis in any patient presenting with coagulopathy of unclear etiology. We present a patient with superwarfarin-induced coagulopathy and review previous cases in adults in the literature. The patient is a 60-year-old man who presented to our medical center with painless hematuria. Laboratory studies revealed an elevated prothrombin time (PT) (42.5 seconds), partial thromboplastin time (PTT) (64.6 seconds), and international normalized ratio (INR) of 7. Liver-associated enzymes were normal, and complete blood cell count (CBC) showed no evidence of disseminated intravascular coagulation. Subsequent work-up included the absence of an inhibitor by mixing study and deficiencies of vitamin K-dependent coagulation factors. The patient's warfarin level was negative. A brodifacoum level was positive, confirming superwarfarin-induced coagulopathy. The patient is currently doing well with normal coagulation studies after receiving high doses of vitamin K for several weeks. The cause of his exposure to superwarfarin remains uncertain. Physicians need to be cognizant of this unusual cause of coagulopathy in adults. The appropriate diagnostic work-up and unique features of therapy are discussed.     

Automated synthetic substrate assays for coagulopathies of dogs

Automated synthetic substrate assays for factors II and VII in the extrinsic coagulation pathway have been developed in our laboratory. In both kinetic assays, p-nitroaniline (pNA) was cleaved from the substrate and measured spectrophotometrically at 405 nm. Plasma samples from 5-8 month old beagle dogs were analyzed for factors II and VII, and also for prothrombin times. A subpopulation considered abnormal (prothrombin times greater than 8 seconds) had slightly elevated factor II levels, but extremely low factor VII levels, relative to the normal group. Therefore, the prolonged prothrombin times of this abnormal group can now be confidently attributed to their decreased factor VII levels. The known genetic predisposition of beagle dogs to factor VII deficiency supports this conclusion. Thus, synthetic substrates are useful for the measurement of coagulation factors in dogs, and permit ready automation of the assay.     

Effect of Time of Day and Partial Sleep Deprivation on Short‐Term,High‐Power Output

The purpose of this study was to determine whether delaying bedtime or advancing rising time by 4 h affects anaerobic performance of individuals the following day in the morning and afternoon. Eleven subjects participated in the study, during which we measured the maximal, peak, and mean powers (i.e., P_max [force‐velocity test], P_peak, and P_mean [Wingate test], respectively). Measurements were performed twice daily, at 07∶00 and 18∶00 h, following a reference normal sleep night (RN), a partial sleep deprivation timed at the beginning of the night (SDB), and a partial sleep deprivation timed at the end of the night (SDE), and oral temperature was measured every 4 h. Each of the three experimental conditions was separated by a one‐week period. Our results showed a circadian rhythm in oral temperature, and analysis of variance revealed a significant sleep×test‐time effect on peak power (P_peak), mean power (P_mean), and maximal power (P_max). These variables improved significantly from the morning to the afternoon for all three experimental conditions. Whereas the morning‐afternoon improvement in the measures was similar after the RN and SDB conditions, it was smaller following the SDE condition. There was no significant difference in the effect of the two sleep‐deprivation conditions on anaerobic performances at 07∶00 and at 18∶00 h under the SDB condition in comparison with the post‐reference night. However, the performance variables were significantly lower at 18∶00 h after the SDE condition. In conclusion, a 4 h partial sleep deprivation at the end of the night appears to be more disturbing than partial sleep deprivation at the beginning of the night.