Giorgio Mombelli

The influence of a prolonged sitting position on the biochemical markers of coagulation activation in healthy subjects: evidence of reduced thrombin generation

Dear Sir, Long trips, especially air flights, are considered a risk for venous thromboembolism [1–5]. The frequency of symptomatic thromboembolic events increases with the distance flown, but appears to be low [3]. In a recent study, however, Scurr et al. found symptomless calf vein thrombosis detected by ultrasonography in up to 10% of long-haul travelers [4]. Given the limited sensitivity of the method [6], the true incidence of travel-associated distal thrombi may even be higher. Taken together, these data suggest that subclinical thrombosis may be frequent during air travel but usually resolves once mobility is resumed. Accordingly, symptomatic thromboembolic events would reflect a rare situation where activation of coagulation escapes the natural control systems. The prolonged sitting position during an air trip is associated with venous stasis, decreased blood flow and hemoconcentration, and is widely assumed to be the main causal factor for thrombus formation in this setting. If this assumption is correct, prolonged immobility in a sitting position by itself would be sufficient to activate coagulation. Subclinical activation of coagulation a so-called prethrombotic state [7] can be assessed by sensitive biochemical markers of thrombin generation (prothrombin fragment 1þ 2, F1þ 2), of ongoing fibrin formation (fibrinopeptide A, FPA) and lysis (D-dimer, DD). The aim of this study was to test the hypothesis that prolonged immobility in a sitting position similar to the one occurring during air traveling activates coagulation. Forty healthy subjects of either sex (median age 29.5 years, range 19–58) with a negative personal and family history and no known risk factor for thromboembolism were kept seated for 6 h on normal chairs, with their knees bent; during this period they were allowed to drink, eat, read or watch television. At baseline and after 3 and 6 h of immobilization, blood was collected in CTADPPACK anticoagulant from sitting probands with a clean venepuncture from an antecubital vein under controlled venous stasis for measurement of F1þ 2 (Enzygnost F1þ 2 Behring), FPA (RIA reagents supplied by Imco), and DD (mini-VIDAS, BioMérieux). Hemoglobin concentration and hematocrit were determined as well. In order to control for the circadian variations of the hemostatic system [8], we used the same protocol for blood sampling in a subgroup of 18 volunteers who were allowed to move freely (control group). Statistical probability was assessed with repeated measures ANOVA on ranks followed by pairwise multiple comparison procedures (Student–Newman–Keuls method) for non-normally distributed data. A linear regression was done for correlation between data at baseline and after 6 h. A difference was considered significant when P <0.05. Over the 6 h of sitting position there was no change in the plasma levels of FPA and DD, whereas F1þ 2 levels decreased from a median of 0.49 to 0.40 nmol L 1 (P< 0.0001, Table 1). The F1þ 2 decrease was both relevant in quantitative terms ( 20% of the baseline values) and consistent among the volunteers (being present in 80% of them). For the three markers, there was a significant correlation between the values at baseline and those measured after 6 h (F1þ 2: r1⁄4 0.63, P< 0.0001; FPA: r1⁄4 0.52, P< 0.0007; DD: r1⁄4 0.83, P< 0.0001). Plasma volume did not change over the 6 h as indicated by stable values for hemoglobin and hematocrit. In the control group, values for F1þ 2 significantly decreased in the sitting position but did not change under ambulant conditions (Table 2). Overall, the quality of blood sampling and processing was very high, as reflected by individual values for FPA which were within the normal range in 168/176 samples (96%), were slightly increased in seven samples, and were above 5.5 nmol L 1 (and thus likely to represent a faulty venepuncture) in one sample (0.6%). Using the biochemical markers of thrombin generation and of ongoing fibrin formation and lysis, we were not able to confirm the hypothesis of activation of coagulation in subjects immobilized in a sitting position. While the failed increase in FPA and DD would not by itself definitively reject the study hypothesis (small quantities of fibrin deposited in the lower limbs could have escaped detection due to the effect of dilution of these markers in circulating blood), the significant decrease of F1þ 2 indicates a reduced rate of thrombin generation during sitting. The decrease in F1þ 2 cannot be explained solely by circadian variations and was the consequence of the prolonged immobilization in a sitting position. It is tempting to speculate that the reduction in thrombin generation reflects a protective down-regulation of the hemostatic system to counter the effect of venous stasis. This suggestion, however, has to be tempered by the fact that thrombin has both procoagulant and anticoagulant effects [9], and that the protein C system mediated anticoagulant effect may even be prevalent under physiologic conditions and in the presence of an intact vascular bed. Journal of Thrombosis and Haemostasis, 1: 380–399

Adherence to the medical regime in patients with heart failure

To assess adherence to medical treatment in patients with heart failure (HF) using a specific questionnaire and measurement of the serum concentration of digoxin.

Variation in coagulation inhibitors during prolonged sitting: possible pathogenetic mechanisms for travel-associated thrombosis

Epidemiological studies have shown an increased risk of venous thrombosis after (air) travel [1,2]. Blood stasis is one of the mechanisms involved in deep vein thrombosis, but so far limited information is available on the pathophysiologic relationship between stasis and thrombosis. We [3] and others [4,5] have found a decrease in thrombin generation during sitting in an experimental setting or during a long-haul flight. This finding is intriguing, as it does not fit the conventional concept of a prethrombotic state defined by an increased level of prothrombin fragment 1 + 2 (F1+2) but a normal concentration of fibrinopeptide A (FPA) [6]. On the other hand, less thrombin may be a hazard in an intact vascular bed; in fact, thrombin on a molecular basis has a pivotal influence on hemostasis by its action as a procoagulant in damaged vessels, and as an anticoagulant in an intact vascular bed by binding thrombomodulin (TM) and thus activating protein C, which in concert with protein S inactivates factor (F) Va and FVIIIa [7]. With this background we tried to elucidate the mechanism(s) of the decrease in thombin generation during sitting and to answer the question whether this decrease may represent a hypercoagulable state. To this end, we measured biological markers of thrombin formation and fibrinolysis, concentrations of the three main inhibitors of coagulation, activity of different coagulation factors and an inhibitor of fibrinolysis (thrombin activatable fibrinolysis inhibitor, TAFI) in a in vivo model of venous stasis. Twenty healthy subjects (11 men, mean age 29 ± 6 years) with no known personal or familial risk factors for venous thromboembolic disease were kept seated on a chair, as described [3]. At baseline (08.00 hours 1⁄4 T0), and after 3 (T3) and 6 (T6) hours, blood was drawn with a clean venipuncture from an antecubital vein under manometrically controlled venous stasis of 50 mmHg into a 10 mL plastic syringe (Monovette, Sarstedt, Nümbrecht, Germany) containing 1 mL 0.106 M trisodium citrate, and into a 5 mL syringe (Monovette) containing 1 mL of CTADPPACK and immediately put on melting ice. Plasma was separated by cold centrifugation, and aliquots were stored at )70 C until processing. All analyses for every single parameterwere donewithin a single working session, using the same reagent batches, and the technicians performing the tests were blinded as to the origin of the samples and other results. The study was approved by the local ethics committee, and all subjects gave informed consent. F1+2 was measured with a commercial ELISA (Enzygnost by Dade Behring, Marburg, Germany), FPA with RIA (reagents supplied by Imco, Stockholm, Sweden), and Ddimers (DD) with ELISA (miniVidas by bioMérieux, Lyon, France). Plasma concentrations of TM were measured with micro-ELISA (Asserachrom Thrombomodulin, Diagnostica Stago, Asnières, France). Total tissue factor pathway inhibitor (TFPI) was measured by ELISA (Asserachrom Diagnostica Stago). The quantitative determination of the heparin cofactor activity of antithrombin (AT) was performed using a commercial kit (Coamatic LR Antithrombin, Chromogenix, Mölndal, Sweden). Conventional one-stage assays detected activity of FV (FV:C); the respective FVIII:C was measured by an activated partial thromboplastin based assay using Pathrombin SL (Dade Behring) and a factor-deficient plasma. VonWillebrand factor antigen and ristocetin cofactor activity levels (vWF:AG and vWF:RF) were measured using an ELISA kit from Dako (Glostrup, Denmark), and BC von Willebrand Reagent from Dade Behring, respectively. Antigen determination of TAFI was performed with a commercially available kit from Milan Analytica (LaRoche, Switzerland). Other investigations (at baseline and after 6 h) included hematocrit, creatinine, global coagulation tests, and fibrinogen. Statistical probability was assessed with Friedman repeated measures analysis of variance on ranks (ANOVA) followed by pairwise multiple comparison procedures (Student-NewmanKeuls method) for non-normally distributed data, otherwise a one way repeated measures ANOVA was used. For single comparison a paired t-test/a Wilcoxon signed rank test for normally/not normally distributed data was used. A linear Correspondence: Hans Stricker, Author Ospedale La Carità, Department of Medicine, Via all’Ospedale, CH-6600 Locarno, Switzerland. Tel.: +4191 8114549; fax: +4191 8114783; e-mail: hans.stricker@ eoc.ch

Individual dosage of digoxin in patients with heart failure

BACKGROUND After the publication of DIG trial, the therapeutic target of serum digoxin concentration (SDC) for the treatment of heart failure (HF) has been lowered (0.40-1.00 ng/ml). However, the majority of equations to calculate digoxin dosages were developed for higher SDCs. Recently, a new equation was validated in Asian population for low SDCs by Konishi et al., but results in Caucasians are unknown. AIM This study was aimed to test the Konishi equation in Caucasians specifically targeting low SDCs. Furthermore, the Konishi equation was compared with other frequently used equations. DESIGN This was a prospective, multicenter study. METHODS Clinically indicated digoxin was given in 40 HF patients. The dosage was calculated with the Konishi equation. The SDC was measured at 1 and 6 months after starting digoxin. Adherence to digoxin was monitored with a specific questionnaire. RESULTS After exclusion of patients admitting poor adherence, we found a reasonable correlation between predicted and measured SDC (r=0.48; P<0.01) by the Konishi equation. Excluding patients with poor adherence and relevant worsening of renal function, the measured SDC (n=54 measurements) was within the pre-defined therapeutic range in 95% of the cases. The mean, maximal and minimal measured SDC were 0.69±0.19, 1.00 and 0.32 ng/ml, respectively. The correlation was weaker for the Jelliffe, the Koup and Jusko, and the Bauman equations. CONCLUSION This study supports the clinical validity of the Konishi equation for calculating individual digoxin dosage in Caucasians, targeting SDCs according to current HF guidelines.

Venous stasis and thrombin generation

Central Hematology Laboratory, Inselspital, University Hospital, Bern, Switzerland; *Department of Internal Medicine, Ospedale Regionale diLocarno, Switzerland; and Thrombosis Research Laboratory, Inselspital, University Hospital, Bern, SwitzerlandTo cite this article: Colucci G, Stricker H, Roggiani W, Haeberli A, Mombelli G. Venous stasis and thrombin generation. J Thromb Haemost 2004;2: 1008–9.

Acute effect of smoking and of 2 weeks of folate substitution on hemostasis and homocysteine in healthy chronic smokers

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Effect of prolonged sitting on thrombin generation: not evidenced yet: reply to a rebuttal

We appreciate the comment by Dr Schreijer and colleagues [1] with regard to our study published in the Journal [2]. The authors contest our conclusion of a reduced thrombin generation in a group of 40 volunteers after 6 h of sitting with the argument that comparison has not been done at the same time points between a subgroup of 18 probands analyzed after a 3and 6-h sitting period and the same group in ambulant conditions (i.e. at 08.00 and 11.00 h and at 14.00 h), claiming that variations in the sitting position could still have been induced by circadian variations. We agree that the direct comparison at the single time points would offer the most stringent criterion to confirm our conclusions. Unfortunately, confronting the two groups is not applicable from a statistical point of view due to intra-individual variations in prothrombin fragment 1+2 (F1+2) concentration. Sakkinen et al. showed in their study on 26 healthy individuals that the intra-individual coefficient of variation for 3-weekly measurements of F1+2 over 24 weeks was 24.2% [3], which is more than twice the difference in the median measured in our study. In our experiment, R for correlation of intra-individual values after logarithmic transformation at different time points varied from 0.29 to 0.95 in the sitting condition and from 0.59 to 0.89 in the ambulant situation, but were clearly lower when comparing the volunteers at the same time points (of different days) but under different conditions (0.15–0.19). Remarkably, the values at 08.00 h where the probands were in a comparable situation R was rather low (0.19). This premise clearly precludes the utilization of the statistics suggested by Schreijer et al. in a study designed to demonstrate small changes in the hemostatic factors where the experimental and control studies have to be done on different days. Only a high number of test persons would have permitted to avoid a type II error. In fact, the cited study by Crosby et al. was aimed to confute a previously reported large effect by hypoxia on coagulation activation, and the authors acknowledge that the number of participants was insufficient to exclude more minor effects [4]. Our results do not exclude a circadian variation of F1+2, although it is not correct to state from our data that such an effect was present. The finding that the F1+2 concentration significantly decreased during the sitting experiment (P < 0.0001) while it did not change in the ambulatory situation (P 1⁄4 0.3) strongly suggests that it was the prolonged sitting position causing the decrease in F1+2. A significant difference was already present after 3 h of sitting, too short a time for circadian variations to be manifest. In spite of the theoretical restrictions mentioned above, we compared our data at the same time points finding with three different statistical methods (paired t-test, P 1⁄4 0.033; Wilcoxon-signed rank test, P 1⁄4 0.011; and multiple linear regression, P 1⁄4 0.01) a significant difference between the decrease of F1+2 after the first 3 h in the sitting vs. the ambulant group, whereas this difference was not significant for the whole study duration. Apart from statistical considerations, we again found a significant decrease of F1+2 concentrations in another study where in 10 healthy volunteers venous stasis was induced for 30 min by an externally applied compression of the thighs [5]. In conclusion, we are convinced that the reduced generation of thrombin demonstrated by a decrease in F1+2 concentration was due to venous stasis and not to circadian variation.