Clinical trials of direct thrombin and factor Xa inhibitors in atrial fibrillation.

PURPOSE OF REVIEW: To review the large phase 3 clinical trials that compare direct thrombin or factor Xa inhibitors with dose-adjusted warfarin in patients with atrial fibrillation who have an increased risk of stroke. RECENT FINDINGS: In large clinical trials, the oral direct thrombin inhibitor ximelagatran and the long-acting factor Xa inhibitor idraparinux were effective for reducing the risk of thromboembolic stroke, but were not marketed because of liver toxicity and excessive bleeding, respectively. In separate clinical trials, the oral direct thrombin inhibitor dabigatran etexilate and the short-acting oral factor Xa inhibitor rivaroxaban were noninferior or superior to dose-adjusted warfarin for prevention of thromboembolic stroke and systemic embolism, without increasing the risk of bleeding, and were well tolerated. Apixaban, another oral factor Xa inhibitor, is effective in reducing thromboembolic stroke compared with aspirin alone. Results of a trial comparing apixaban with dose-adjusted warfarin are awaited. SUMMARY: Dabigatran and rivaroxaban are effective, safe alternatives to dose-adjusted warfarin for reducing thromboembolic risk in patients with atrial fibrillation at high risk of stroke.

[Study of thrombin generation in healthy children]. / Étude de la génération de thrombine dans une population pédiatrique.

Exploring coagulation in newborns and children is a challenge due to the low levels of both procoagulant factors and inhibitors. Conventional coagulation tests might be inadequate to explore all of these changes. The aim of the study is to evaluate the thrombin generation assay, a global test to explore coagulation, in a pediatric population (n=586) compared to an adult population (n=166). The thrombin generation assays were performed using Calibrated Automated Thrombography with two different tissue factor concentrations (1 and 5 pM), with and without thrombomodulin (TM). In the absence of TM, the endogenous thrombin potential (ETP) is significantly lower in the pediatric population, reflecting the decrease in procoagulant factors. In the presence of TM, ETP values in pediatric subjects are within the reference range of adult values. The thrombin generation assay demonstrates that coagulation balance is maintained in the pediatric population.

Thrombin Generation in Chinese Pregnant Women and the Effect of Insulin Use on Thrombin Generation in Patients with GDM.

Pregnancy is a hypercoagulable state associated with an increased risk of venous thrombosis. Calibrated automated thrombogram (CAT) is a test to monitor the thrombin generation (TG), a laboratory marker of thrombosis risk, and increases during normal pregnancy, but it is still unclear whether TG is related to the use of insulin in pregnant women with gestational diabetes mellitus (GDM). We performed thrombin generation by CAT on 135 normal pregnant women, including 43 in first trimester, 32 in second trimester, 60 in third trimester, respectively; 68 pregnant women with GDM were also enrolled, 19 patients with GDM using insulin to control blood glucose and 49 patients control their blood glucose through diet and exercise with noninsulin treatment. The overall CAT parameters were calculated using descriptive statistics method with mean ± standard deviation. Mean endogenous thrombin potential, peak thrombin generation, and StartTail time increased significantly with the pregnancy. There was no significant difference in TG test parameters except StartTail time(P = .003) in insulin-treated GDM group when compared to those without insulin in the GDM group. The normal ranges for CAT parameters in pregnant women were determined. Thrombin generation increased significantly in first trimester and remains stable in second and third trimester. The use of insulin in patient with GDM did not affect thrombin generation test. Our study helps to establish the reference range of thrombin generation in Chinese normal pregnant population and provide more basis to predict the risk of thrombus complicating during pregnancy.

Developmental haemostasis. Impact for clinical haemostasis laboratories.

Developmental haemostasis is a concept, now universally accepted, introduced by Andrew et al. in the late 1980's. However, coagulation analysers and reagents have changed significantly over the past 15 years. Coagulation testing is known to be sensitive to changes in individual reagents and analysers. We hypothesised that the reference ranges developed by Andrew et al. may not be appropriate for use in a modern coagulation laboratory. Our study was designed to determine whether a current day coagulation testing system (STA Compact analyser and Diagnostica Stago reagent system) was sensitive to age-related changes in coagulation assays. This is the first large scale study since Andrew et al. to determine the age associated numerical changes in coagulation proteins. Our results confirm the concepts of developmental haemostasis elucidated by Andrew et al. However, our results clearly demonstrate that the absolute values of reference ranges for coagulation assays in neonates and children vary with analyser and reagent systems. The results confirm the need for coagulation laboratories to develop age-related reference ranges specific to their own testing systems. Without this, accurate diagnosis and management of neonates and children with suspected bleeding or clotting disorders is not possible. Finally we present age related reference ranges for D-dimers, TFPI, and endogenous thrombin potential, previously not described.

Specific determination of plasmatic thrombin activity.

Thrombin is the key enzyme of coagulation. Its activity can be determined via fibrinogen Ø fibrin conversion or via cleavage of a chromogenic substrate. The latter method is easier than the first one, but in plasma it is hampered due to unspecific cleavage of the chromogenic substrate by thrombin-like enzymes of hemostasis, especially those of the contact phase. The concentration of the thrombin substrate (HD-CHG-Ala-Arg-pNA) was optimized, using final substrate concentrations of 0 to 5 mM, a final arginine concentration of 1.13 M, and samples of 10 mIU/mL purified thrombin in 7% human albumin or pooled normal citrated plasma without and with EDTA. Twenty microliters pooled normal citrated plasma (frozen/thawed) or factor II-deficient plasma (lyophilized) were incubated with 10 microL 0% to 0.5% Thromborel S (100% = 162 ng/mL tissue factor [TF]) in 6% BSA or with 10 microL 0% (physiol. NaCl) to 50% Pathromtin SL and with 20 microL 25 mM CaCl(2). After 0 to 22 minutes (37 degrees C), 20 microL 1.7 M arginine, pH 8.7 were added. Fifteen microliters 0.9 mM HD-CHGAla-Arg-pNA in 2.3 M arginine, pH 8.6, were added and the increase in absorbance (deltaA) at 405 nm was determined. Thrombin activity was standardized against the (3)A measured for 1 IU/mL thrombin in 7% human albumin (8.8 mA/min RT). The optimal final chromogenic substrate concentration to detect thrombin in this assay system is less than 0.6 mM. Higher substrate concentrations in a plasma milieu result in unspecific cleavage of the substrate. Using final concentrations of chromogenic substrate less than 0.4 mM (the approximate Km- value for thrombin) and final concentrations of arginine greater than 800 mM, in factor II-depleted plasma, when activated either by TF or by the contact phase, there is no significant thrombin generation. The circulating thrombin activity measured in EDTA plasma of 39 healthy donors is 100 +/- 20% of norm (mean value +/- 1 SD; 100% = 5.5 mIU/mL thrombin). This chromogenic assay detects thrombin activity independent of clotting seconds or fibrin mediated turbidity increases. This technique allows to standardize the thrombin activity generated in any biologic system in international thrombin units.

A reunification of the US ("NIH") and International Unit into a single standard for Thrombin.

The existence of two different units for Thrombin in widespread international use has caused confusion for many years. The holders of the WHO International Standard (IS) for Alpha Thrombin and the US Standard (also known as the "NIH Standard") now report on a collaboration to reunite the International Unit (IU) and the US unit ("NIH unit"). A study was organised involving 25 laboratories in 15 countries to investigate the possibility of preparing a common Standard with a common unit and to investigate aspects of methodology that cause divergence of results using the IS and US Standard. Laboratories were asked to measure the potency of two candidate replacement standards (C, 01/578 and D, 01/580), and potencies were calculated relative to both the existing US Standard (lot J) and the IS (89/588). Data analysis of a total of 128 assays indicated that sample D would make an ideal replacement joint Standard with a potency of 110 IU/ampoule (equivalent to 110 US units per ampoule) based on data from clotting assays. No significant differences in results were observed using fibrinogen of human or bovine origin, or using human plasma. Comparisons of chromogenic and clotting assays indicated that sample D had a similar high proportion of alpha thrombin to the current IS for Alpha Thrombin (89/588). Sample D was adopted as the IS for Thrombin (01/580) and the US Standard (lot K) with a potency of 110 IU/ampoule.

The International and "NIH" units for thrombin--how do they compare?

The current International Standard (IS) for alpha-thrombin was established in 1991. It was related in unitage with the 1st IS for Thrombin established in 1975 and contains 100 IU of thrombin activity. The National Institute of Health (NIH) standard (Lot J) is in common use for the calibration of commercial thrombin reagents and this study reports on a comparison of the thrombin units defined by these 2 standards. This study has indicated that one "NIH" unit is equivalent to 1.1 to 1.3 IU of thrombin, depending on the influence of PEG in the assay. A unitage ratio figure of 1.15 was recommended following analysis of data obtained in the presence and absence of PEG in the comparative assays. This was confirmed by amidolytic assay data.

A collaborative study to establish an international standard for alpha-thrombin.

Since 1975 an International Standard for Thrombin of low purity has been used. While this standard was stable and of value for calibrating thrombins of unknown potency the need for a pure alpha-thrombin standard arose both for accurate calibration and for precise measurement of thrombin inhibitors, notably hirudin. An international collaborative study was undertaken to establish the potency and stability of an ampouled pure alpha-thrombin preparation. A potency of 97.5 international units (95% confidence limits 86.5-98.5) was established for the new alpha-thrombin standard (89/588) using a clotting-assay procedure. Stability data at various elevated temperatures indicated that the standard could be transported and stored with no significant loss of potency. Ampoules of lyophilised alpha-thrombin (coded 89/588) have been recommended as an International Standard for alpha-thrombin with an assigned potency of 100 international units per ampoule by the International Society for Thrombosis and Haemostasis (Thrombin and its Inhibitors Sub-Committee) in Barcelona, Spain in July 1990 while the Expert Committee on Biological Standardisation and Control of the World Health Organisation will consider its status at its next meeting in Geneva in 1991.

[Thrombin Reference Standard (Control 961) of National Institute of Health Sciences].

The "Thrombin Reference Standard (Control 961)" of National Institute of Health Sciences was prepared. The precision of filling into ampoule was about 1% as C.V. The content of a-thrombin was about 87%. The thrombin potency of the standard material was assayed against the Thrombin Reference Standard (Control 8710) according to the method of JP XIII and the potency was 1033 +/- 59 unit/ampoule. From the results, the potency of the proposed material for Thrombin Reference Standard was defined as 1,030 units per ampoule.

[Thrombin Reference Standard (Control 031) of National Institute of Health Sciences].

The "Thrombin Reference Standard (Control 031", of National Institute of Health Sciences was prepared. The precision of filling into ampoule was about 11.5% as C.V. The content of alpha-thrombin was about 89%. The thrombin potency of the standard material was assayed against the Thrombin Reference Standard (Control 961) according to the method of JP-X IV and the potency was 692 +/- 35 units/ampoule. From the results, the potency of the proposed material for Thrombin Reference Standard was defined as 690 units per ampoule.

International Normalized Ratio (INR), coagulation factor activities and calibrated automated thrombin generation - influence of 24 h storage at ambient temperature.

International Normalized Ratio (INR) measurements are used to monitor oral anticoagulation therapy with coumarins. Single coagulation factor activities and calibrated automated thrombin (CAT) generation are considered as more advanced methods for evaluating overall haemostatic capacity. The aims were to assess the variability of INR, coagulation factor activities, and CAT, during 24 h of storage of blood samples at ambient temperature. A total of 24 patients on stable coumarin treatment were followed prospectively for 6 weeks. INR was analyzed at 0, 6 and 24 h after blood sampling and 1-stage clotting activity of coagulation factors II, VII, IX, and X as well as CAT generation was recorded after 0 and 24 h respectively. Statistical analyses included Bland-Altman plot, 95% limits of agreement, and a variability test using a mixed effect model. The level of INR remained statistically unchanged from 0 to 6 and 24 h of storage. Coagulation factor activities and CAT revealed no significant difference induced by 24 h of storage, although the limits of agreement were wide. Patients' individual INR, coagulation factor activities, and CAT generation were not significantly influenced by 24 h storage of blood samples, but for the CAT generation analyses a trend toward time dependency was detected.