SummaryAn enzyme-linked immunosorbent assay (ELISA) was developed for the determination of thrombin-antithrombin III complex (TAT) in human plasma. The test system follows the sandwich principle and uses two different antibodies directed against human thrombin and human antithrombin III, respectively. The antibodies bind selectively to the corresponding antigen moieties of TAT. The assay was calibrated with definite concentrations of preformed purified TAT added to TAT-poor plasma. The lower limit of sensitivity of the assay was 0.5 μg/1. Mean coefficients of variation of 4.2% (intraassay) and 3.5% (interassay) were found for TAT concentrations between 2 and 60 μg/1. A reference range from 0.85 to 3.2 μg/1 was calculated from TAT concentration in plasma samples from 88 healthy donors (mean value ± SD: 1.45 ± 0.4 μg/I). In plasma samples from patients with pulmonary embolism (n = 17), TAT concentrations between 3 and 25 μg/1 were measured. In 15 patients with deep vein thrombosis, TAT was found up to 3 to 25 μg/1. From these data we conclude that measurement of TAT can be a sensitive parameter for specific detection of a latent activation of the clotting pathway.

SummaryThe present investigation describes a novel approach to prepare a specific antibody against prothrombin activation fragment 1+2 (F 1+2). The antibody discriminates between native prothrombin and F 1+2 in plasma. A synthetic peptide from the negatively charged region of F 1+2, which becomes the carboxyterminal sequence after cleavage of prothrombin by factor Xa, was used for immunization of rabbits. Obtained antiserum was immunopurified and an enzyme-linked immunosorbent assay (ELISA) was constructed for determination of F 1+2. The test system follows the sandwich principle and uses two different antibodies directed against F 1+2 and prothrombin, respectively. The ELISA was calibrated with purified F 1+2 added to F 1+2-poor plasma. The lower limit of sensitivity of the assay was 0.02 nmol/1. Coefficients of variation of 6.9 to 10.4% (intraassay) and 6.7 to 11% (interassay) were found for F 1+2 concentrations between 0.08 and 4.9 nmol/1. A reference range from 0.32 to 1.2 nmol/l was calculated from 95 healthy donors (mean value ± SD: 0.67 ± 0.19 nmol/l). In patients with deep vein thrombosis (n = 7) confirmed by phlebography and in patients with pulmonary embolism (n = 8) confirmed by lung scan, F 1+2levels were found up to 1.5 to 9.5 nmol/l. In plasma samples of patients under oral anticoagulant therapy in the stable state F 1+2 concentrations were found to be in the range of 0.08 to 0.5 nmol/l. The results indicate that the antibody is specific and highly sensitive for quantification of F 1+2 in plasma. It can be supposed that the ELISA we have developed is a valuable tool for detection of both hypercoagulable as well as hypocoagulable states.

Key Points• GlycoPEGylated demonstrates the same efficacy and prolonged effect in animal models as native FVIII.• Circulatory half-life of glycoPEGylated FVIII (N8-GP) is prolonged by approximately twofold in several species.Frequent infusions of intravenous factor VIII (FVIII) are required to prevent bleeding associated with hemophilia A. To reduce the treatment burden, recombinant FVIII with a longer half-life was developed without changing the protein structure. FVIII-polyethylene glycol (PEG) conjugates were prepared using an enzymatic process coupling PEG (ranging from 10 to 80 kDa) selectively to a unique O-linked glycan in the FVIII B-domain. Binding to von Willebrand factor (VWF) was maintained for all conjugates. Upon cleavage by thrombin, the B-domain and the associated PEG were released, generating activated FVIII (FVIIIa) with the same primary structure and specific activity as native FVIIIa. In both FVIII-and VWF-deficient mice, the half-life was found to increase with the size of PEG. In vivo potency and efficacy of FVIII conjugated with a 40-kDa PEG (N8-GP) and unmodified FVIII were not different. N8-GP had a longer duration of effect in FVIII-deficient mouse models, approximately a twofold prolonged half-life in mice, rabbits, and cynomolgus monkeys; however, the prolongation was less pronounced in rats. Binding capacity of N8-GP on human monocyte-derived dendritic cells was reduced compared with unmodified FVIII, resulting in several-fold reduced cellular uptake. In conclusion, N8-GP has the potential to offer efficacious prevention and treatment of bleeds in hemophilia A at reduced dosing frequency. (Blood. 2013;121(11):2108-2116

SummaryThis study was performed to determine the accuracy of D-Dimer fibrin derivatives, thrombin-antithrombin III (TAT) complexes and prothrombin fragments 1 + 2 (F 1 + 2) determinations for the diagnosis of deep vein thrombosis (DVT). One hundred and sixteen consecutive patients referred to the angiology unit of our hospital for a clinically suspected DVT were investigated. They were submitted to mercury strain gauge plethysmography and to ultrasonic duplex scanning examination; in cases of inconclusive results or of proximal DVT (n = 35), an ascending phlebography was performed. After these investigations were completed, the diagnosis of DVT was confirmed in 34 and excluded in 82. One half of the patients were already under anticoagulant therapy at the time of investigation. The 3 biological markers were assayed using commercially available ELISA techniques and the D-Dimer was also assayed with a fast latex method. The normal distribution of these markers was established in 40 healthy blood donors. The most accurate assay for the diagnosis of DVT was the D-Dimer ELISA which had both a high sensitivity (94%) and a high negative predictive value (95%). The D-Dirner latex, TAT complexes and F 1 + 2 were far less sensitive and provided negative predictive values which ranged between 78 and 85%. In spite of positive and significant correlations between the levels of ihe 3 markers, their association did not improve their overall accuracy for detecting D\/L Therefore, with the exception of the D-Dimer ELISA, these markers were of little value for the diagnosis of DVT in this specific population.

Summary.  Objective: The objective of the present study was to evaluate the pharmacokinetics and the clearance pathways of rFVIIa after intravenous administration to hemophilia patients. Methods: Ten severe hemophilia patients were included in the study; all patients were intravenously administered a clinically relevant dose of 90 μg kg−1 (1.8 nmol kg−1) rFVIIa. Blood samples were collected consecutively to describe the pharmacokinetics of rFVIIa. All samples were analyzed using three different assays: a clot assay to measure the activity (FVIIa:C), an enzyme immunoassay (EIA) to measure the antigen levels (FVII:Ag), and an EIA (FVIIa‐AT) to measure the FVIIa antithrombin III (AT) complex. Pharmacokinetic parameters were evaluated both by use of standard non‐compartmental methods and by use of mixed effects methods. A population pharmacokinetic model was used to simultaneously model all three datasets. The total body clearance of rFVIIa:C was estimated to be 38 mL h−1 kg−1. The rFVII‐AT complex formation was responsible for 65% of the total rFVIIa:C clearance. The initial and the terminal half‐life of rFVIIa:C was estimated to be 0.6 and 2.6 h, respectively. The formation of rFVII‐AT complex was able to explain the difference observed between the rFVIIa:C and the rFVII:Ag concentration. The non‐compartmental analysis resulted in almost identical parameters.