Diurnal Changes of Fibrinolysis in Patients With Liver Cirrhosis and Esophageal Varices FABIO PISCAGLIA,1 SEBASTIANO SIRINGO,1 RAMON C. HERMIDA,2 CRISTINA LEGNANI,3 MARCO VALGIMIGLI,1 GABRIELE DONATI,1 GUALTIERO PALARETI,3 LAURA GRAMANTIERI,1 STEFANO GAIANI,1 ANDREW K. BURROUGHS,4 AND LUIGI BOLONDI1
Variceal bleeding, whose triggering mechanisms are largely unknown, occurs with a circadian rhythmicity, with 2 peaks, one greater, in the evening, and one smaller, in the early morning. We assessed some clotting and hemodynamic parameters, possibly involved in variceal hemorrhage, over a 24-hour period, at 4-hour intervals, in 16 patients with cirrhosis and esophageal varices and in 9 controls. At each time interval, tissue plasminogen activator (tPA) and tPA inhibitor-1 (PAI-1) antigens and activities and total euglobulin fibrinolytic activity were determined and portal-vein flow velocity, volume, and congestion index were measured by duplex-Doppler. Significant circadian rhythms were searched for by least-squares and cosinor methods. tPA activity showed a circadian rhythm in cirrhosis, with a peak of 2.85 times the trough value, calculated at 18:42, and remained over 2.5-fold until shortly after 22:00. Total fibrinolytic activity showed a similar pattern, which was statistically significant also in controls. tPA and PAI antigens also showed significant circadian rhythm both in controls and cirrhotics, with higher values in the morning. Among the portal hemodynamic parameters only the congestion index showed significant rhythmic changes and only in cirrhosis, with the highest values in the late evening, but with limited diurnal excursion (6 5.5%). In conclusion, we showed the existence of a circadian rhythm of fibrinolysis in cirrhosis, whose temporal distribution might suggest a role of fibrinolysis in variceal hemorrhage on the basis of the comparison to the known chronorisk of variceal bleeding. (HEPATOLOGY 2000;31:349-357.) Several biological processes are driven by rhythms possessing a circadian, infradian, or ultradian frequency.1 Similarly, Abbreviations: HCV, hepatitis C virus; PAI, plasminogen activator inhibitor; PT, prothrombin time; aPTT, activated partial thromboplastin time; t-PA act, tissue plasminogen activity; tPA Ag, tissue plasminogen activator antigen; u-PA act, urokinase plasminogen activator activity; u-PA Ag, urokinase plasminogen activator antigen; PAI act, plasminogen activator inhibitor activator; PAI Ag, plasminogen activator inhibitor antigen; MESOR, midline estimating statistic of rhythm; TEFA, total euglobulin fibrinolytic activity. From the 1Department of Internal Medicine and Gastroenterology, University of Bologna, Bologna, Italy; 2Bioengineering and Chronobiology Laboratories, ETSI Telecomunicacio`n, University of Vigo, Vigo, Spain; 3Department of Angiology and Blood Coagulation, University of Bologna, Bologna, Italy; 4Hepatobiliary and Liver Transplantation Unit, University Department of Medicine, Royal Free Hospital and School of Medicine, London, UK. Supported by funds (60% and 40%) from the Italian Ministry of the University and of Scientific Research (MURST). Address reprint requests to: Prof. Luigi Bolondi, M.D., Divisione di Medicina Interna, Azienda Ospedaliera S.Orsola-Malpighi, via Albertoni 15, 40138 Bologna, Italy. E-mail:
[email protected]; fax: (39) 051 6362725. Copyright r 2000 by the American Association for the Study of Liver Diseases. 0270-9139/99/3102-0015$3.00/0
some clinical manifestations of chronic diseases also appear according to a distinct temporal rhythm.2,3 Discovering the time points of maximal risk favors the identification of the events triggering the onset of clinical manifestations. This was the case for hemostatic, coagulative, and hemodynamic factors for myocardial infarction and cerebrovascular events.2,4,5 Hemorrhage from gastroesophageal varices is one of the most harmful complications of portal hypertension, but the pathophysiology of the acute event is still unclear. In fact, although portal hypertension is the cause for the development of esophageal varices, the relationship between the level of portal pressure, as measured by the hepatic venous pressure gradient and the individual risk of variceal bleeding, has not been definitively established.6-10 In recent years, several authors have shown a similar rhythmic circadian distribution of variceal hemorrhage in cirrhotic patients.11-15 All of them found variceal hemorrhages occurring more frequently in the evening (hours 19:00 to 23:20) and in the early morning (hours 7:00 to 9:00). In the reports that applied a statistical analysis able to consider multiple daily peaks of different amplitude,11,14 the calculated probability of hematemesis was largely greater in the late evening than in the morning, with nearly 40% of the total events taking place between hours 19:00 and 24:00.11,14 One of these studies also reported the circadian behavior of portal pressure,11 that could not, however, convincingly explain the bleeding rhythm, especially considering that a peak of bleeding occurs in the evening when portal pressure is relatively low. Recently, a work hypothesis supposed bacterial infections to suddenly raise portal and variceal pressure via the release of endotoxin and consequently release of the vasoconstrictor endothelins.16 However, in this case it is also unclear how the circadian rhythm of hemorrhage can be explained. The severity of liver failure contributes to determine the risk of variceal bleeding, but by incompletely clarified mechanisms,17 especially if we consider the acute onset of hemorrhage. The possible involvement of the clotting system in the bleeding events of cirrhosis has been invoked by several authors,11,13,14,16 but there is still poor evidence about its participation in variceal hemorrhage. A cross-sectional study showed an increased bleeding risk in cirrhotics with hyperfibrinolysis,18 but no study has yet evaluated such a condition in a diurnal setting. The present study tests the hypothesis that the fibrinolytic activity of portal hypertensive cirrhotic patients shows significant circadian changes. Additionally, it studies the 24-hour profile of some hemodynamic parameters to evaluate other possible factors involved in variceal bleeding.
349
350 PISCAGLIA ET AL. MATERIALS AND METHODS Patients. The study was performed in 16 hospitalized patients (mean age 6 SD; 56.2 years 6 8.9 years; 12 males, 4 females) with liver cirrhosis (Child-Pugh class: 6 5 A, 9 5 B, and 1 5 C) and esophageal varices (variceal size: 9 5 F1, 4 5 F2, and 3 5 F3), without a history of variceal bleeding in the previous 6 months. The diagnosis of cirrhosis had been confirmed by liver biopsy in 12 and by unequivocal clinical, ultrasonographical, and biochemical features in the remaining 4, in whom liver biopsy was contraindicated. The etiology of cirrhosis was hepatitis C virus (HCV) in 7 cases, alcoholic abuse in 4, hepatitis B virus in 3, HCV plus alcoholic abuse in 1, and association of HBV and HCV in 1. At the time of the study, all patients had abstained from alcohol for at least 1 month. Any vasoactive or diuretic drug was suspended at least 3 days before the study. The patients were admitted to the hospital at least 3 days before the beginning of the study and consumed a controlled hyposodic diet (220 mmol/day of natrium-chloride). As a control group for clotting and fibrinolytic tests, 40 healthy subjects, mainly hospital staff, were evaluated (age range: 24 to 64 years, mean 40.5 years, 24 males). In these subjects blood samples were withdrawn, between hours 8:00 and 10:00, and processed as described for cirrhotic patients. In a subgroup of 9 (mean age 50.2 years), the complete circadian study was performed, with all time points as in patients. On the day of the study, these 9 subjects attained to the protocol as described for patients. Four additional time points to the regular ones were done in 5 control subjects, to assess whether the use of more frequent sampling compares well with the 4-hour interval. In 8 control subjects the complete circadian hemodynamic assessment was also performed (in the 9th healthy subject the hemodynamic assessment could not be completed, due to the loss of the feasibility to Doppler ultrasonography during the day). The hemodynamic data of 40 additional healthy subjects (mean age 46.8 years, range 22 to 70 years) in a fasting state was derived from the control groups of previous studies19,20 and, together with the 8 healthy subjects enrolled for the circadian study, were used as reference for the baseline duplex-Doppler study. All subjects gave their informed consent to participate in the study, whose design was in accordance with the principles of the Declaration of Helsinki and was approved by the local ethical committee. Study Protocol. Starting from hour 8:00 of the day of the study to hour 8:00 of the subsequent day, blood withdrawals and hemodynamic evaluations of cirrhotic patients and controls were repeated at intervals of 4 hours. The subjects lay supine for at least 15 minutes before each examination. Artificial illumination was permitted, if needed, only from hours 7:00 to 22:30, whereas in the remaining period a very dim light (less than 15 lux) was turned on only for the time of the measurements at hours 24:00 and 4:00. Subjects were asked to lie in their beds from hours 22:30 to 7:00. Smoking and consumption of caffeine-containing substances were prohibited from midnight of the day preceding the study. Breakfast, lunch, and dinner were consumed just after the examinations of hours 8:00, 12:00, and 20:00; in the intervals subjects were permitted to ingest water freely, but not any caloric-containing solid or liquid, to avoid interference with the splanchnic hemodynamics. Coagulation Study. Sample collection. Blood samples were drawn from an antecubital vein with minimal stasis and clean venipuncture, using a 21-gauge butterfly needle immediately after the hemodynamic measurements, in order not to affect these later. Separate venipunctures were performed at each time point with subjects lying supine. Venous blood was immediately anticoagulated with trisodium citrate (0.129 mol/L, 1/10), except for plasminogen activator inhibitor (PAI) measurements, in which tubes of Diatube Htm (sodium citrate, citric acid, theophylline, adenosine, and dipyridamole, Stago) were used to avoid platelet activation and secretion of PAI. Blood samples were kept in melting ice until
HEPATOLOGY February 2000
subjected to 20-minute centrifugation at 3500 rpm and at 4°C. Platelet-poor plasma was distributed in coded plastic tubes, snap frozen in liquid nitrogen, and stored at 270°C until further processing. Before use, plasma was thawed rapidly in a water bath at 37°C and put on ice. The following clotting/fibrinolytic tests were performed: prothrombin time (PT; RecombiPlasTin, Ortho-Clinical Diagnostics, Milan, Italy); activated partial thromboplastin time (aPTT; Organon Teknika, Turnhout, Belgium); clottable fibrinogen (according to Clauss,21 Hemolab Fibrinomat, bioMerieux, Marcylı´Etoile, France); antithrombin III activity (Coatest, Ortho-Clinical Diagnostics); tissue Plasminogen Activator activity (t-PA act) as described elsewhere22 and antigen (t-PA Ag; TintElize tPA, Biopool, Umea, Sweden); urokinase Plasminogen Activator activity (u-PA act; Chromolyze uPA, Biopool) and antigen (u-PA Ag; TintElize uPA, Biopool); Plasminogen Activator Inhibitor activity (PAI act; Coatest, Ortho-Clinical Diagnostics) and antigen (PAI Ag; TintElize PAI-1, Biopool). The total fibrinolytic activity was measured in the euglobulin fraction using a chromogenic substrate as reported in detail elsewhere.22 Prothrombin time, aPTT, antithrombin III, and fibrinogen were measured only at baseline. Hemodynamic Study. The hemodynamic evaluation was performed by duplex-Doppler equipment, an Esaote-Hitachi AU590 Asynchronous, with a 3.5 MHz convex probe. A preliminary examination was performed on one of the days preceding the study to allow the subjects to familiarize themselves with the examination. Ultrasound measurements were performed by 2 skilled operators (F.P. and S.S.), with each subject examined serially by the same operator. Splanchnic vascular examinations were performed according to accepted guidelines, so that the inter- and intraoperator variations were reduced to nonsignificant levels.23,24 Changes between the first and the last examination of each subject, which were taken at the same time (8:00) of 2 subsequent days, were used for calculating the intraobserver variation of Doppler parameters. The following parameters were measured: (1) portal-vein flow velocity, measured as the time-averaged maximal velocities, according to the guidelines described by Sabba` et al.23 (2) Portal-vein flow volume, calculated as portal-vein cross-sectional area multiplied by mean flow velocity. Cross-sectional area was calculated from the vein diameter assuming a circular shape of the vessel and mean velocity by multiplying the mean of maximal velocities 3 0.57.25 (3) Portal-vein congestion index,25 calculated as: cross-sectional area (cm2)/mean velocity (cm/s). (4) Superior mesenteric artery pulsatility index, calculated according to the formula:pulsatility index 5 (peak velocity 2 min velocity)/mean of maximal velocities. Technical details have been described elsewhere.19,24 Heart rate and arterial pressure were measured at each time point. Mean arterial pressure was calculated as: diastolic pressure 1 (pulse pressure/3). Statistical Analysis. Baseline hemodynamic and coagulation data are reported as mean 6 SD or median and range, according to the skewness of the data. Comparison of baseline data between patients and controls was performed by the Mann-Whitney U test. Data were analyzed on a microcomputer Power Macintosh 9500 (Apple Computer, Cupertino, CA) using Chronolab,26 a software package for biologic time series analysis by linear and nonlinear least-squares estimation that also includes the single and population-mean cosinor methods,27-29 as well as the fit of multiple components.30 Linear least-squares rhythmometry was designed for the detection of periodic components in short and noisy time series (because they are usually present in clinical situations involving patients). This approach is based on regression techniques.28,29 In particular, circadian rhythm parameters were first computed for each individual series by the single cosinor fit27 of a 24-hour cosine curve (only as a first approximation and for descriptive purposes, given the nonsinusoidal circadian waveform of most of the analyzed variables so that only very few individuals showed diurnal variations reaching a statistical significance). The individual parameter esti-
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PISCAGLIA ET AL.
mates derived from this were used as imputations or first-order statistics for assessing the circadian rhythm characteristics for all subjects using the population-mean cosinor method (a procedure describing rhythmic variation in hybrid time series, in data sampled from a group of subjects, in this case the cirrhotics as a group). With this method, the parameter estimates are based on the means of estimates obtained from individuals in the sample; their confidence intervals depend on the variability amongst individual parameter estimates.29 Thus, for the period under consideration one obtains an estimate of: (1) the rhythm-adjusted mean or MESOR (midline estimating statistic of rhythm), defined as the average value of the rhythmic function (e.g., cosine curve) fitted to the data; (2) amplitude, defined as half the extent of rhythmic change in a cycle approximated by the fitted cosine curve (difference between the maximum and the MESOR of the fitted curve); and (3) acrophase, the lag from a defined reference time point (usually midnight of the first day of measurement when the fitted period is 24 hours) of the crest time in the cosine curve fitted to the data. Even though the use of the population-mean cosinor method, which applies only the fit of a single sinusoidal waveform, might be suboptimal in case the variable under study has a nonsinusoidal daily profile, it is the best method for the first approach to data like the present ones, when assessing whether statistically significant diurnal changes are present or not.30 In fact, it is able to identify significant diurnal variations even of variables without a sinusoidal profile, two examples of which are the rhythms of growth hormone and blood pressure. Few single individuals will show a statistically significant sinusoidal profile for the former, but the concurrence of the peaks of all individuals in a short time span (around 1 AM) will make more than probable a significant sinusoidal 24-hour rhythm with the populationmean method. For the latter, on the contrary, most individuals will show a significant sinusoidal rhythm, even though the variable does not have a sinusoidal profile and day and night are not of the same length (as expected in a sinusoidal variable). Hence, this method is the best way to model the variation of the variables from the present study, although it might be considered as a first approximation to establish whether diurnal changes exist or not and in which part of the day they occur. In fact, it does not provide the definite optimal description of the circadian profile for the variables that do not follow an exact sinusoidal circadian pattern. Rhythm detection is sought by testing the null hypothesis of zero amplitude with an F-test.29 The null hypothesis was rejected for P , .05. RESULTS Baseline Measurements. At baseline, practically all coagulative and fibrinolytic parameters were altered in patients with liver cirrhosis (Table 1A), indicating an impairment of
TABLE 1A. Clotting and Fibrinolytic Baseline Data in Cirrhotic Patients and Controls Median (range min-max) Cirrhosis
(1000/mm3)
Platlet count PT ratio aPTT ratio Fibrinogen (mg/dL) Antitrombin III (%) t-PA antigen (ng/mL) t-PA activity (mU/mL) u-PA antigen (ng/mL) u-PA activity (mU/mL) PAI-1 antigen (ng/mL) PAI-1 activity (mU/mL) TEFA (mU/mL)
Controls
67 (30-143) 162 (139-210) 1.38 (1.20-1.57) 1.03 (0.98-1.12) 1.30 (0.96-1.75) 1.12 (0.96-1.25) 191 (130-332) 248 (165-345) 63 (35-94) 97 (81-115) 10.3 (1.6-35.0) 4.03 (1.8-8.3) 373 (0-771) 63 (37-109) 0.73 (0.00-1.75) 0.185 (0-0.36) 1.01 (0.55-1.84) 0.27 (0-0.77) 12.1 (5.7-38.4) 18.9 (3.9-40.9) 0.5 (0.0-9.0) 9.8 (1.4-26.1) 372 (0-696) 245 (0-454)
MannWhitney U Test: P Value
,.0001 .009 .036 .008 ,.001 .007 ,.001 ,.001 ,.001 n.s. ,.001 ,.009
351
TABLE 1B. Hemodynamic Baseline Data in Cirrhotic Patients and Controls
Portal velocity (cm/sec) Portal-flow volume (ml/min) Portal-vein congestion index SMA-PI Mean arterial pressure (mmHg) Heart rate (beats/min)
Cirrhosis
Controls
t-test: P Values
23.8 6 6.6 950 6 336 0.093 6 0.034 2.55 6 0.61 94.8 6 10.2 66.1 6 6.9
27.9 6 7.7 754 6 312 0.045 6 0.017 3.34 6 0.81 97.7 6 14.3 62.1 6 10.1
.009 n.s. .01 .01 n.s. n.s.
NOTE: Congestion index is calculated as cross sectional area divided by mean flow velocity. Abbreviation: SMA-PI, superior mesenteric artery pulsatility index.
hemostasis and coagulation and a hyperfibrinolytic state, as expected at this stage of liver disease. Portal-vein velocity and superior mesenteric artery pulsatility index were low and the congestion index high in cirrhotics in comparison to control subjects (Table 1B).19,20,25,31,32 The coefficient of variation for the Doppler parameters in cirrhosis was fairly good (median and range for portal-vein velocity were respectively 4.2% and 0% to 11.9%; for superior mesenteric artery pulsatility index 6.6% and 0.1% to 23.0%; for portal-flow volume 9.7% and 1.4% to 28.0%; for congestion index 10.7% and 0.7% to 23.6%), suggesting a satisfactory reliability of the measurements in patients. These variations are similar to previously published studies and reflect in part the variations intrinsic to the physiological status of the patients (no previous study showed an absolute stability of these parameters over time). Circadian Measurements. A significant sinusoidal circadian rhythm was detected for t-PA Ag, PAI-1 Ag, and TEFA in patients and controls and additionally also for t-PA act in cirrhosis (Fig 1). Values are reported in Tables 2 and 3 as percentages of the 24-hour mean. Reporting data as mean of percent allows, to overcome interference in recognizing a circadian rhythm due to interindividual differences and to better compare the extent of the daily changes of the various parameters. The changes of u-PA Ag, u-PA act, and PAI-1 act had similar trends towards circadian variations, but did not reach statistical significance neither in patients nor in controls, also due to their limited extent of variations. In fact, u-PA Ag was higher in the morning and lowest in the evening in both groups, but variations ranged only 6 4.2% for patients and 6 5.0% for controls around the MESOR. In patients and in controls u-PA act was maximal in evening, but also in this case the modifications around the MESOR were rather strict (6 3.8% and 6 7.3%, respectively). t-PA Ag had a peak in the morning and its lowest level in the evening in both groups. However, due to the concomitant greater decrease of PAI-1 Ag and act (with troughs occurring around late afternoon in both groups), t-PA act increased during the day and showed its maximal predicted value at hour 18:42 in patients. In controls the maximal t-PA values were also reached at the time point of the early evening (hour 20:00), but the circadian pattern could not be adequately described by a single sinusoidal curve so that our present analysis did not show a statistically significant rhythm. t-PA act appeared to be responsible practically for the totality of TEFA in cirrhosis at all time points (Tables 1 and 2), whereas it is only a limited component of TEFA (around 15%) in healthy subjects throughout the day (Table 3), except for a relative increase at the time of t-PA act peak, in which it becomes
12.47 ng/mL 781.0 mU/mL 0.78 ng/mL 1.09 mU/mL 14.31 ng/mL 2.84 U/mL 786.9 mU/mL
98.5% 97.6% 99.5% 100.3% 97.3% 92.8% 103.9%
MESOR as % of Mean h 12
105.1 6 30.5% 91.3 6 47.3% 97.8 6 16.3% 99.5 6 9.7% 99.6 6 19.0% 81.9 6 81.0% 103.2 6 44.2%
h 08
108.8 6 17.0% 51.5 6 27.2% 104.6 6 13.7% 98.4 6 4.3% 127.6 6 26.3% 130.5 6 68.0% 64.3 6 38.7%
91.1 6 21.9% 132.0 6 42.6% 96.2 6 19.8% 101.2 6 14.1% 83.5 6 11.3% 33.5 6 45.8% 140.6 6 58.7%
h 16
98.7 6 19.2% 147.0 6 56.7% 97.1 6 13.8% 100.9 6 10.6% 80.5 6 15.3% 102.7 6 151.3% 156.9 6 67.4%
h 20
86.8 6 12.2% 104.9 6 39.7% 95.6 6 24.6% 104.4 6 29.2% 79.1 6 24.7% 68.7 6 63.0% 107.4 6 13.1%
h 00
100.2 6 26.4% 78.1 6 43.4% 106.1 6 17.5% 96.8 6 6.0% 108.0 6 19.9% 149.5 6 134.9% 51.6 6 12.9%
h 04
.003 .006 n.s. n.s. ,.001 n.s. .002
P
9:20 18:42
7:45 18:14
24.6% 51.8%
Acro (time)
8.8% 49.3%
Ampl
3.52 ng/mL 63.5 mU/mL 0.162 ng/mL 0.535 mU/mL 9.70 ng/mL 3.58 U/mL 420.1 mU/mL
98.1% 105.5% 100.1% 98.6% 96.2% 94.3% 102.9%
h 12
107.6 6 8.0% 145.8 6 260.5% 102.5 6 11.7% 97.5 6 9.9% 101.1 6 22.4% 110.0 6 121.1% 124.1 6 53.2%
h 08
115.3 6 9.6% 82.2 6 119.4% 100.9 6 11.7% 101.4 6 6.2% 124.4 6 23.9% 110.0 6 49.6% 61.9 6 17.0%
95.1 6 16.9% 45.4 6 70.2% 102.5 6 14.3% 101.6 6 15.4% 88.3 6 36.6% 73.6 6 63.3% 142.4 6 45.9%
h 16
90.9 6 12.5% 217.8 6 246.2% 99.6 6 15.7% 102.8 6 11.6% 80.1 6 41.6% 41.3 6 2.9% 101.8 6 40.8%
h 20
83.2 6 14.6% 108.7 6 171.2% 97.5 6 14.7% 95.5 6 22.1% 64.4 6 8.9% 87.5 6 98.1% 95.7 6 44.5%
h 00
TABLE 3. Diurnal Fibrinolytic Parameters in Control Subjects
92.5 6 13.9% 17.9 6 53.6% 99.7 6 19.1% 97.1 6 10.0% 117.3 6 37.5% 167.7 6 87.3% 112.3 6 37.1%
h 04
.007 n.s. n.s. n.s. ,.002 n.s. .05
P
10:03
8:20 17:22
26.4% 26.3%
Acro (time)
15.2%
Ampl
NOTE: Values are expressed as percent 6 s.e. of the 24-hour absolute mean value, considered as 100%. Abbreviations: Acro, acrophase, time at which the peak value is reached; Ampl, amplitude, half the extent of rhythmic change of the cosine curve; C.I. Acro, confidence interval of the acrophase.
tPA:Ag tPA:Act uPA:Ag uPA:Act PAI-1:Ag PAI-1:Act TEFA
Mean
MESOR as % of Mean
NOTE: Values are expressed as percent 6 s.e. of the 24-hour absolute mean value, considered as 100%. Abbreviations: Acro, acrophase, time at which the peak value is reached; Ampl, amplitude, half the extent of rhythmic change of the cosine curve; C.I. Acro, confidence interval of the acrophase.
tPA:Ag tPA:Act uPA:Ag uPA:Act PAI-1:Ag PAI-1:Act TEFA
Mean
TABLE 2. Diurnal Fibrinolytic Parameters in Cirrhosis
15:21-19:42
5:12-11:48
6:40-12:56
C.I. Acro
17:08-19:56
6:36-8:44
6:44-12:36 17:44-20:00
C.I. Acro
352 PISCAGLIA ET AL. HEPATOLOGY February 2000
23:11 5.5% n.s. , 0.008 n.s. n.s. n.s. n.s. 94.4 6 6.9% 107.5 6 6.3% 96.9 6 10.7% 103.6 6 14.8% 96.1 6 5.7% 99.1 6 7.3% 99.8 6 8.2% 101.5 6 10.9% 101.2 6 9.7% 94.7 6 14.9% 97.5 6 7.3% 100.5 6 4.9% 98.2 6 6.6% 104.7 6 7.8% 100.7 6 8.9% 104.0 6 11.5% 101.7 6 8.6% 100.5 6 8.1% 101.9 6 8.7% 100.7 6 10.2% 104.1 6 13.1% 90.7 6 13.2% 101.9 6 6.1% 102.9 6 6.4% 102.1 6 8.4% 94.8 6 7.0% 100.5 6 11.0% 99.8 6 11.9% 100.2 6 5.1% 98.3 6 5.4% Portal vel Cong Index Portal flow SMA-PI MAP Heart rate
23.3 cm/sec 0.099 cm p sec 972 ml/min 2.40 93.05 mmHg 67.8 bpm
99.9% 100.7% 100% 99.1% 100% 100.2%
101.6 6 4.7% 95.8 6 6.6% 98.1 6 7.6% 104.0 6 9.3% 101.3 6 3.6% 99.2 6 5.4%
h 16 h 12 h 08 Mean
TABLE 4. Diurnal Hemodynamics in Cirrhosis
The present findings show the existence of a statistically significant circadian rhythm of fibrinolysis in cirrhosis, with maximal capacity predicted between hours 18 and 19. In particular we focused our attention on t-PA, u-PA, and on PAI-1, the fast-acting inhibitor of PAs, because they are the individual components responsible for the circadian variation of the plasma fibrinolytic activity in healthy subjects33 and no data about their circadian behavior were available in cirrhosis.33 In the last decades the identification of circadian rhythms proved to be of great value to understand the pathophysiology of acute manifestations. For instance in the mid 1980’s it was proved that events potentially favoring vascular occlusion, namely hypercoagulability and hypofibrinolysis show their peaks at the time of the maximal incidence of myocardial infarction both in normal subjects and in patients with ischemic heart diseases.5,33-37 A chronorisk-based evidence for the determination of the acute events was thereby produced. In the case of portal hypertension, previous studies showed that large varices,38 variceal cherry red spots,38 hepatic functional status,17 hyperfibrinolysis,18 and portal-vein congestion index39 are predictive factors for variceal hemorrhage, but what acutely determines variceal bleeding remains unclear and is possibly the result of several concurrent factors. Recently, a biphasic circadian rhythm of variceal hemorrhage was identified, with 2 distinct peaks in the early morning and in the evening11-14 reproducible either in Southern11,12,14 and Northern European countries13 and in the United States,15 with the evening peak greater than the other.11,14 Meal ingestion determines an increase of portal pressure, evaluated as hepatic venous pressure gradient, of about 13.4% after 30 minutes,40 but no temporal relationship between bleeding incidence and consumption of lunch, which is the main daily meal in Italy, was found.12,14 In addition to the postprandial changes, portal pressure shows also a significant sinusoidal circadian rhythm, with peak calculated at hour 8:32 and trough at hour 20:32, differing from each other by 6.9%.11 Altogether it might be speculated that portal pressure has some role in determining the morning peak of variceal
MESOR as % of Mean
DISCUSSION
h 20
h 00
h 04
P
Ampl
Acro
about one third of TEFA (Table 3). This was due to the fact that, differently from all other parameters, t-PA showed no activity at all in many control subjects at most time points, afternoon and evening excluded. Amongst all the splanchnic and systemic hemodynamic parameters, only the congestion index presented variations after a statistically significant rhythm (P 5 .008) in patients (Table 4), with the peak reached at hour 23:11 (Fig 2), mainly due to lower portalflow velocities during nighttime. However, the extent of diurnal variations of all hemodynamic parameters, including the congestion index, was rather small in patients (Table 4). Superior mesenteric artery pulsatility index would be best described by a sinusoidal cycle of only 12 hours, with 2 cycles in the day, but statistical significance was not reached. In controls no splanchnic hemodynamic parameter showed any statistically significant rhythm (Table 5), even though mean estimated showed an increase of the congestion index in the evening (at the time point of hour 20:00). Also in controls SMA-PI could probably be best described as a sinusoidal cycle of only 12 hours, because the lowest values were found at hours 16:00 and 24:00.
NOTES: Values are expressed as percent 6 s.e. of the 24-hour absolute mean value, considered as 100%. Abbreviations: Acro, acrophase, time at which the peak value is reached; Ampl, amplitude, half the extent of rhythmic change of the cosine curve; Portal vel, flow velocity in the portal vein; Cong Index, congestion index, calculated as reported in the Methods section.
PISCAGLIA ET AL. 21:16-02:08
HEPATOLOGY Vol. 31, No. 2, 2000
353
FIG. 2. Circadian variations of the portal-vein congestion index, expressed as percent changes respective to the MESOR value. ‘‘A’’ dots 5 observed mean values 6 S.E.; sinusoidal line 5 calculated values. The distribution presents a significant circadian rhythmicity (P , .008). 99.9% 102.7% 100.6% 99.1% 100.1% 100.4%
h 12
98.5 6 18.9% 96.4 6 37.7% 92.1 6 17.1% 108.3 6 11.6% 104.4 6 3.4% 96.4 6 7.8%
h 08
100.4 6 8.0% 87.8 6 22.1% 97.5 6 12.5% 111.1 6 16.9% 101.8 6 4.4% 99.1 6 3.4%
108.8 6 14.5% 87.9 6 25.3% 114.5 6 15.9% 74.2 6 19.3% 99.1 6 2.4% 104.3 6 4.4%
h 16
88.9 6 11.4% 160.0 6 131.0% 93.4 6 13.7% 125.0 6 34.5% 105.6 6 4.0% 99.8 6 6.0%
h 20
107.0 6 9.7% 83.7 6 23.4% 108.3 6 21.1% 74.6 6 23.2% 95.6 6 4.2% 105.1 6 9.3%
h 00
96.1 6 14.6% 96.3 6 30.2% 96.6 6 15.8% 95.6 6 19.5% 91.6 6 6.3% 96.2 6 7.8%
h 04
n.s. , n.s. n.s. n.s. , 0.02 n.s.
P
4.7%
Ampl
14:17
Acro
12:36-19:16
NOTES: Values are expressed as percent 6 s.e. of the 24-hour absolute mean value, considered as 100%. Abbreviations: Acro, acrophase, time at which the peak value is reached; Ampl, amplitude, half the extent of rhythmic change of the cosine curve; Portal vel, flow velocity in the portal vein; Cong Index, congestion index, calculated as reported in the Methods section.
26.9 cm/sec 0.060 cm p sec 760 ml/min 3.41 85.9 mmHg 58.4 bpm
bleeding; at this time, in fact, the postprandial (postbreakfast) and the endogenous circadian pressure increases are additive and may theoretically raise portal pressure to 20.3% over the trough level. However, the mechanism of the evening bleedTABLE 5. Diurnal Hemodynamics in Controls
FIG. 1. Circadian variations of t-PA activity. t-PA activity is reported as percent changes respective to the MESOR value (5762 mU/mL). ‘‘A’’ dots 5 observed mean values 6 S.E.; sinusoidal line 5 calculated values. The distribution presents a significant circadian rhythmicity (P , .01).
Portal vel Cong Index Portal flow SMA-PI MAP Heart rate
Mean
MESOR as % of Mean
354 PISCAGLIA ET AL. HEPATOLOGY February 2000
HEPATOLOGY Vol. 31, No. 2, 2000
ing peak, which seems to occur at the time when the portal pressure starts to raise from the trough level, but is still relatively low, remains obscure. Interestingly, the period of highest fibrinolytic capacity reported in the present study overlapped or just preceded the reported peak time of hematemesis.11-15 This temporal observation alone does not establish a cause-effect relation with variceal hemorrhage and experimental observations are warranted. However, some arguments that may suggest a possible role of hyperfibrinolysis in facilitating variceal bleeding should be considered. A role for local fibrinolysis of the esophagus as a cause of hemorrhage in cirrhosis was suggested about 20 years ago41 and subsequently plasma hyperfibrinolysis was clinically found to be a risk factor for bleeding in patients with cirrhosis.18,42-44 Indeed patients with an impaired liver function have hyperfibrinolysis45 and this may be a possible explanation for the contribution of the liver function estimate in predicting variceal bleeding.17 In particular, the t-PA activity level in cirrhosis was reported to be 0.75, 2.75, and 6.81 times that of control, respectively, in patients in ChildPugh class A, B, and C46 who also show a progression of the risk of variceal bleeding from class A to C.15,17 The present study shows that the mean ratio between the peak and trough levels of t-PA activity and TEFA is as high as 2.85 and 3.04, respectively, and that at the peak time t-PA act and TEFA in patients are 10 times and 4 to 5 times higher than those of the morning in controls, respectively, suggesting that fibrinolytic changes during the day might be great enough to effectively influence the risk of bleeding. The calculation of an exact time of the fibrinolytic peak (18:42 for t-PA act) is a first approximation, so that more correctly it should be said that the peak occurs in late afternoon/early evening. Moreover, the calculation of the time was made considering the time of the start of the assessments. However, according to the protocol, the blood withdrawal was made at the end of each hemodynamic assessment, which means about 30 minutes later. Therefore, the calculated peak should be considered to occur about 30 minutes later than what was reported from the mathematical analysis. In summary, after reaching its peak, t-PA act seems to remain over 2.5 times the trough level until later than hour 22:30, which means over nearly the whole period of maximal bleeding risk. A similar temporal distribution was found also for TEFA, of which tPA seems to be the main determinant in cirrhosis. The TEFA data of patients appear to be superimposable to those of controls (the extent of the daily changes are 92.6% and 80.5%, respectively, in the 2 groups and the calculated time of peak, hours 18:14 and 17:22), making even more reliable the result of the peak of fibrinolysis in the early evening. t-PA activity seemed to take over a much more relevant role in cirrhosis than it had in controls, although also in these latter it was a main determinant for the presence of a circadian rhythm of TEFA.33 The relative contribution of t-PA act to TEFA in cirrhosis with respect to that in controls has been reported by very few studies, with data consistent with our present ones.47 A link between hemostasis and clinically evident variceal bleeding might also be suggested by the practical experience in measuring intravariceal pressure by needle devices in cirrhotic patients. The puncture of the varix produces an opening in the vessel, which is usually efficiently plugged at the withdrawal of the needle after a minimal bleeding. Hence, a small rupture of a varix does not appear to be sufficient to cause a significant bleeding, because it is usually adequately
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plugged. Some further arguments for this hypothesis are that it happens to see no active bleeding at urgent endoscopy in patients who clinically will not stop bleeding, suggesting that hemorrhage is a ‘‘stop and go’’ process in which hemostatic and bleeding components are dynamically challenged. Additionally, red color signs noted at endoscopy in cirrhotic patients are believed to represent small intramucosal hemorrhages outside of the varix proper or small hemorrhages without a clinical manifestation, because of the effectiveness of primary hemostasis, a so-called leak and seal phenomenon.16 Hence, it is conceivable that a relatively impaired hemostatic/clotting activity, as that occurring in the evening, may render ‘‘small’’ hemorrhages more prone to proceed to clinically evident events. Unfortunately, it is very difficult to plan experimental studies to show any hypothesis on variceal bleeding, because it occurs unpredictably. Based on the present findings, a new work hypothesis would be a study in which antifibrinolytic treatments are focused only at the time of maximal chronorisk in comparison to placebo. However, even though the fibrinolytic system is likely a major factor affecting a susceptibility to bleeding, other hemostatic and coagulative factors could contribute, particularly if the studies on healthy subjects could be reproduced in cirrhosis, in which no data are yet available. In fact, in healthy subjects, PT and aPTT have circadian rhythms of limited amplitude, but statistically significant, with the highest values, respectively, at hours 16:00 and 20:00 to 24:00.37 Interestingly, also platelet adhesiveness37 and aggregation34 are relatively lower in the late evening, theoretically making primary hemostasis less effective, although the extent of variation of all these parameters in healthy subjects is much more limited than that of fibrinolysis. The confirmation of these rhythms in cirrhosis would further detail and strengthen the hypothesis of a contribution of the hemostatic and clotting systems in facilitating the evening peak of hemorrhage. Although the hemostatic-clotting system likely facilitates a clinical manifestation of variceal bleeding, the hemodynamic factors are to be analyzed to understand why varices rupture. In connection to this aspect, no major advances have been produced by the present work. Portal-vein velocity and superior mesenteric artery pulsatility index were reduced and congestion index increased in patients with respect to the normal range of control subjects. This is in accordance with presence of high-portal resistance and of a splanchnic hyperkinetic circulation in cirrhosis with portal hypertension. The congestion index takes into account that, due to the increased portal resistance, flow velocity tends to decrease and portal caliber to increase. It was shown to correlate well with portal resistance,31 the size of the varices,32 and to improve the predictability of early bleedings.39 In our series of patients, this parameter evaluated for the first time in a circadian setting, was the only hemodynamic one to show a significant circadian rhythm, with the highest predicted value shortly after hour 23:00. Therefore, the congestion index remains in the quartile of the highest values during the time period from hour 20:00 to 02:00, which covers the time of the evening peak of bleeding. However, this parameter does not correspond with any single hemodynamic event and the extent of its variations was rather limited (65.5% around the daily mean), hence no direct conclusion can be drawn about its effect on the mechanism of variceal rupture, although in general a hemodynamic contribution is suggested. The pattern of the congestion index is not in contradiction to the
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previously reported variations of portal pressure, because it correlates well with portal resistance, but poorly with portal pressure in cirrhosis,31 and probably its levels are influenced by several other hemodynamic conditions. All other hemodynamic parameters showed even more limited daily changes, and none reached statistical significance in cirrhosis nor in controls. In particular, the blood flow volume and velocity of the portal vein did not present any significant circadian variation, in agreement with a previous work in Italy48 and in contrast with the data from a South American unit, which found an isolated portal-flow increase at midnight.49 Such differences remain to be clarified and might be due to the patient populations, the study protocol, or to environmental conditions. We also measured the superior mesenteric artery pulsatility index, because it indirectly reflects the downstream arterial vascular impedance.50 This measurement was aimed at assessing any change of the mesenteric venous inflow, possibly improving the circadian evaluation of portal hemodynamics with respect to the portal trunk alone. Our findings, however, did not show any significant circadian rhythm, suggesting that changes of mesenteric inflow are not among the main determinants of variceal rupture. This is in agreement with the absence of an increased bleeding risk after meals, a time of marked splanchnic arterial overflow.51 In conclusion, the demonstration of a temporal association between a relative hyperfibrinolytic state and the previously reported chronorisk of variceal bleeding is not sufficient to establish a direct cause-effect relation, but may be enough to suggest a role of fibrinolysis in the event and may aid in the design of new studies. Acknowledgment: The authors thank Dr. Claudio Gaia for his work in the collection of data.
12. 13. 14. 15. 16. 17. 18. 19.
20.
21. 22.
23.
24.
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