Search

SummaryIn fluorogenic thrombin generation (TG) experiments, thrombin concentrations cannot be easily calculated from the rate of the fluorescent signal increase, because the calibration coefficient increases during the experiment, due to substrate consumption and quenching of the fluorescent signal by the product. Continuous, external calibration via an in a parallel sample therefore was hitherto required for an accurate calculation of the TG curve. A technique is presented that allows mathematical transformation of experimental fluorescence intensities into "ideal" data, i.e. in the data that would have been obtained if substrate consumption and quenching by the product would not play a role. The method applies to fluorescence intensities up to 90% of the maximal fluorescent signal corresponding to total substrate conversion and thereby covers the entire region of interest encountered in practice. The first derivative of the transformed signal can then be converted into thrombin concentrations via a conventional, fixed calibration factor. This calibration factor can be obtained from a separate experiment but also by measuring the amidolytic activity of the α2macroglobulin-thrombin complex present in the reaction mixture ("serum") after thrombin generation is over. This method halves the amount of sample required per experiment. Keywordscalibration, thrombin generation, fluorogenic methods

SummaryCardiac surgery with cardiopulmonary bypass (CPB) is associated with blood loss and post-surgery thrombotic complications. The process of thrombin generation is disturbed during surgery with CPB because of haemodilution, coagulation factor consumption and heparin administration. We aimed to investigate the changes in thrombin generation during cardiac surgery and its underlying pro- and anticoagulant processes, and to explore the clinical consequences of these changes using in silico experimentation. Plasma was obtained from 29 patients undergoing surgery with CPB before heparinisation, after heparinisation, after haemodilution, and after protamine administration. Thrombin generation was measured and prothrombin conversion and thrombin inactivation were quantified. In silico experimentation was used to investigate the reaction of patients to the administration of procoagulant factors and/or anticoagulant factors. Surgery with CPB causes significant coagulation factor consumption and a reduction of thrombin generation. The total amount of prothrombin converted and the rate of prothrombin conversion decreased during surgery. As the surgery progressed, the relative contribution of α2-macroglobulin-dependent thrombin inhibition increased, at the expense of antithrombin-dependent inhibition. In silico restoration of post-surgical prothrombin conversion to pre-surgical levels increased thrombin generation excessively, whereas co-administration of antithrombin resulted in the normalisation of post-surgical thrombin generation. Thrombin generation is reduced during surgery with cardiopulmonary bypass because of a balance shift between prothrombin conversion and thrombin inactivation. According to in silico predictions of thrombin generation, this new balance increases the risk of thrombotic complications with prothrombin complex concentrate administration, but not if antithrombin is co-administered.

SummaryBy the use of a fluorogenic thrombin substrate and continuous calibration of each individual sample, it is now possible to obtain a thrombin generation (TG) curve (or thrombogram) in plasma, with or without platelets, in an easy routine procedure at high throughput and with an acceptable experimental error (< 5%). Evidence is growing that the parameters of the thrombogram, and notably the area under the curve (endogenous thrombin potential, ETP), are useful in assessing bleeding-or thrombotic risk and its modification by antithrombotic-or haemostatic treatment. Available data strongly suggest that conditions (congenital, acquired, drug-induced) that increase TG all cause a thrombotic tendency and that conditions that decrease TG prevent thrombosis but, beyond a limit, cause bleeding. Diminution of TG is a common denominator of all antithrombotic treatment, including anti-platelet drugs. The thrombogram can also be used as a tool in the search for new antithrombotics and reflects the haemorrhagic or thrombotic side effects of other drugs (e.g. oral contraceptives).The thrombogram thus is a promising new approach to clinical management of bleeding and thrombotic disease as well as a tool in drug research and epidemiology. Our experience at this moment is insufficient, however, to already clearly define its limits.

In fluorogenic thrombin generation (TG) experiments, thrombin concentrations cannot be easily calculated from the rate of the fluorescent signal increase, because the calibration coefficient increases during the experiment, due to substrate consumption and quenching of the fluorescent signal by the product. Continuous, external calibration via an in a parallel sample therefore was hitherto required for an accurate calculation of the TG curve. A technique is presented that allows mathematical transformation of experimental fluorescence intensities into "ideal" data, i.e. in the data that would have been obtained if substrate consumption and quenching by the product would not play a role. The method applies to fluorescence intensities up to 90% of the maximal fluorescent signal corresponding to total substrate conversion and thereby covers the entire region of interest encountered in practice. The first derivative of the transformed signal can then be converted into thrombin concentrations via a conventional, fixed calibration factor. This calibration factor can be obtained from a separate experiment but also by measuring the amidolytic activity of the alpha(2)macroglobulin-thrombin complex present in the reaction mixture ("serum") after thrombin generation is over. This method halves the amount of sample required per experiment.

SummaryIn fluorogenic thrombin generation (TG) measurement the concentrations of thrombin are obtained from the course of fluorescence intensity. Because of fluorescence quenching, in one series of normal plasmas (n=60), the rate of fluorescence increase at fixed thrombin activity was 70 ± 13% of that in buffer, in another (n= 139) 75 ± 8%. Using a calibration factor (CF) measured in buffer therefore underestimates thrombin concentrations in plasma and introduces a source of error. A fixed CF also neglects the 25 – 35% increase of CF during the experiment and thus distorts the form of the TG curve so that the ETP cannot be determined. Continuous individual calibration (CIC), in which CF is determined continuously in a parallel sample, avoids such systematic errors but adds random error because the thrombin course is calculated from two different measurements. We determined the intra-individual coefficients of variation (CV) of the peak-height and ETP as obtained with CIC to those obtained with a fixed CF measured in buffer. With the fixed CF, the CVs varied between 18% and 49%; with CIC they lowered to 4–7% (n=5x12), i.e. in a range allowing clinical application. It is shown that CIC can be discarded for the measurement of peak thrombin values and replaced by comparison to a reference plasma only if quenching is not a systematic confounder. This was shown to be the case in the set of 139 normal plasmas but not in the set used for determining the intraindividual CVs, a difference that may depend upon preanalytical conditions.

By the use of a fluorogenic thrombin substrate and continuous calibration of each individual sample, it is now possible to obtain a thrombin generation (TG) curve (or thrombogram) in plasma, with or without platelets, in an easy routine procedure at high throughput and with an acceptable experimental error (5%). Evidence is growing that the parameters of the thrombogram, and notably the area under the curve (endogenous thrombin potential, ETP), are useful in assessing bleeding- or thrombotic risk and its modification by antithrombotic- or haemostatic treatment. Available data strongly suggest that conditions (congenital, acquired, drug-induced) that increase TG all cause a thrombotic tendency and that conditions that decrease TG prevent thrombosis but, beyond a limit, cause bleeding. Diminution of TG is a common denominator of all antithrombotic treatment, including anti-platelet drugs. The thrombogram can also be used as a tool in the search for new antithrombotics and reflects the haemorrhagic or thrombotic side effects of other drugs (e.g. oral contraceptives). The thrombogram thus is a promising new approach to clinical management of bleeding and thrombotic disease as well as a tool in drug research and epidemiology. Our experience at this moment is insufficient, however, to already clearly define its limits.

SummaryIn haemophilia patients, a relationship is usually observed between the clinical expression of the disease and plasmatic factor VIII/factor IX (FVIII/FIX) activity. However, it is known from clinical experience, that some haemophilia patients, despite similar FVIII/FIX plasma levels, could exhibit different bleeding phenotype. After determining preanalytical test conditions, we evaluated the thrombin generation capacity from haemophilia plasma samples in various conditions and the potential usefulness of thrombin generation test (TGT) in haemophilia patients. In a series of 46 haemophilia patients (34 haemophilia A and 12 haemophilia B patients), we found a significant correlation between plasmatic FVIII/FIX levels and endogenous thrombin potential (ETP), peak and time to peak obtained by thrombin generation measurement. In addition, a correlation was found between severe clinical bleeding phenotype and ETP. Our results suggest that TGT could be a promising tool to evaluate haemostasis capacity in patients with haemophilia. Our ex vivo results, obtained 24 hours after FVIII concentrate administration, showed that in patients presenting similar plasmatic FVIIII levels, thrombin generation capacity may be significantly different. These results suggest that in patients with haemophilia, TGT could be useful for individually tailoring prophylactic regimens as well as for adapting clotting factors infusions in surgical situations, in addition to FVIII/FIX plasma clotting activities.

Thromb Haemost 2006; 95: 756–7 The paper “Age dependency measured by calibrated automated thrombinography (CAT)” by Haidl et al. (1) is one in a series of investigations from the Graz group that significantly contributes to the understanding of paediatric haemostasis. More or less as a by-product of these studies they now found that adults of over 35 years of age have a significantly higher thrombin generation potential (ETP) than those aged from 18 – 35. The figure in this publication suggests an apparently linear increase with age that is as high as ~30% between the ages of 20 and 50. This is an important observation because it shows that, also in a group of apparently healthy volunteers without known prothrombotic traits, the age group that is more likely to get thrombosis is also the one that generates more thrombin. In a series of congenital conditions, such as antithrombin deficiency (2), congenital hyperprothrombinaemia (G20210A mutation) (3), and dysfunction of the protein C-system (unpublished observation), it is evident that the genetic defect primarily causes higher thrombin generation, and one concludes that higher thrombin generation leads to a higher risk of venous thrombosis. If we observeAPC-resistance in the presence of the lupus anti(!)-coagulant (4) or in women using oral contraceptives (5), it is not a long shot to surmise that in these cases, as in congenital conditions, higher thrombin generation is the cause of the thrombotic tendency. Taken together, the body of circumstantial evidence for high thrombin generation potential being the cause of a prothrombotic condition becomes rather impressive. It is reinforced again by the inverse observation, i.e. the fact that all antithrombotic drugs decrease thrombin generation [see further (6)]. The observation by Haidl et al. carries the subject a step further. It has been observed that thrombin generation is rather variable in a normal population. Where the intraindividual variation of the ETP is ~5%, the interindividual variation is >15% (7). This means that 99% of all normal individuals have a thrombin generating capacity of between 55 and 145 % of the mean. In other words: some people in the normal population generate twice as much thrombin as others. In congenital conditions such as antithrombin deficiency or the prothrombin A20210G mutation, such large variations can be attributed to one single cause. Apparently, in the normal population they can also be caused by a combination of circumstances. “Mr. Low” who, by chance, has a low normal set of procoagulant factors and a high normal set of anticoagulant factors may produce half as much thrombin when his plasma is triggered compared to “Mrs. High”, a person with high procoagulant and low anticoagulant factors. The question now is whether, as a consequence, he has a much smaller risk of developing thrombosis. Now that automated calibrated measurement of thrombin generation allows such variations to be measured, we feel that the time is ripe for larger epidemiological studies relating the parameters of thrombin generation to thrombotic risk in those populations where no recognised risk factors can be found. Before embarking upon such studies, epidemiologist may well ask why thrombin generation might do better than the time honoured clotting times, which have essentially failed as predictors of thrombotic disease. The answer can be given on different levels: i) Clotting times are insensitive to variations in clotting function in the region of interest, i.e. in a large area around the normal mean. If the thromboplastin time, as defined by Armand Quick, is invariably 12 seconds in the normal, this is because it does not distinguish between the clotting system of Mr. Low and Mrs. High. This again is caused by the fact that the clotting time already approaches its minimum when the concentration of prothrombin (or of any other clotting factor) exceeds the ~25% level (8). ii) At the moment that the clotting time is recorded there is only very little thrombin formed. The large majority of all prothrombin (> 98%) still has to be converted. So, not enough thrombin is formed for the protein C system to start, and the plasma antithrombins are not yet of significant influence. Contrary to the clotting time, thrombin generation is very sensitive to variations of prothrombin and antithrombins around the normal mean as well as to the activity of the APC system. iii) The conditions of a thrombin generation experiment can be obtained reproducibly at almost any concentration of tissue factor (TF). For clotting times we have essentially the choice between an unphysiologically large excess of TF, as in the thromboplastin time, or an equally unphysiological absence of TF as in the activated partial thromboplastin time (aPTT). Measuring clotting times at lower TF concentrations is possible and has been practiced in the