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SummaryPrevious in vitro studies using spontaneously clotting whole blood revealed thrombin formation and high fibrinopeptide A (FPA) concentrations measured during incubation time. This occurred in spite of normal concentrations of thrombin antagonists present in the blood of the healthy subjects examined. However, there are several reports showing that in vivo increased thrombin- anti thrombin III-complex (TAT) concentrations and relatively low FPA concentrations may occur e. g. in patients with (pre)thrombotic disorders. These in vivo findings indicate more effective thrombin inhibition by antithrombin III, with almost no fibrin formation. To find an explanation for the differences observed in vitro and in vivo, we extended the in vitro studies by measuring concentrations of prothrombin fragment 1 + 2 (F1 + 2), TAT and FPA at several time points until 30 min. Our goal was to test whether thrombin at least initially is neutralized by antithrombin III, resulting in a lack of fibrin formation, either in the absence or in the presence of heparin (0.2 and 0.5 U/ml whole blood, respectively).In the absence of heparin a simultaneous increase in the concentrations of F1+2, TAT and FPA was observed. Thrombin was only partially neutralized by antithrombin III and large amounts of fibrin were formed. The addition of heparin virtually suppressed thrombin formation since the F1 + 2 concentration remained low. Moreover, the small amounts of thrombin formed were neutralized by antithrombin III to a greater extent than in the absence of heparin. Thus, in the presence of heparin less fibrin was produced as evidenced by significantly lower FPA concentrations.Experiments using blood of two patients with antithrombin III deficiency showed that fibrin formation was not different from the healthy controls in spite of the significantly higher initial F1 + 2 concentration measured. During incubation, the patients tended to form more thrombin, of which proportionally less was neutralized by antithrombin III, and more fibrin as compared to the healthy controls. Heparin addition suppressed thrombin formation less efficiently.

SummaryWe compared F 1 + 2 results obtained with two commercial ELISA methods (Behring and Baxter) assaying the same plasma samples. There was little correlation between the results of the two methods, as shown by the low correlation coefficient (r = 0.50) and by low percentage of concordant classification (normal or abnormal) of the samples (24%). Such poor correlation is probably due to the different anticoagulants suggested, because correlation improved when both methods were carried out in plasmas collected with the same anticoagulant. However, the Baxter method still gave significantly lower F 1 + 2 values than the Behring method. Assuming that this difference is due to the use of standards with different F 1 +2 concentrations, the standards from Behring and Baxter were evaluated by both methods. Parallel dose-response curves were obtained when the standards were run by the Behring method but not by the Baxter method, indicating that the two standards are qualitatively different. This study demonstrates that the two F 1 + 2 methods give different values for the same samples and that these values are poorly correlated. Standardization of the F 1 + 2 assays cannot be achieved easily simply by using a common standard and the use of different anticoagulants appears to be the main reason for poor standardization.

SummaryWe have investigated the influence of long term oral anticoagulants (OAC) upon the plasma levels of prothrombin fragment 1 + 2 (F1 + 2), of thrombin-antithrombin III complexes (TAT) and of D-Dimer in 20 patients affected by a proximal deep vein thrombosis (DVT) diagnosed by ultrasonic duplex scanning. Patients (63 ± 17 years, mean ± SD) were sampled at the beginning of the OAC treatment (day 1), which was started 1 to 6 days after diagnosis confirmation and full heparinization, and then 8, 35 and 92 days after. The results were compared to those obtained in a blood donor population (39 ± 10 years) and to an age-matched healthy population (63 ± 19 years). The mean INR determined on days 8, 35 and 92 were almost identical (2.8 ± 0.7, 2.9 ± 0.9 and 2.8 ± 0.6 respectively). In contrast, highly significant variations of the three markers were recorded during the observation period. Eight days after the beginning of OAC, increased levels of TAT complexes were associated with subnormal levels of F1 + 2 suggesting persistence of a hypercoagulable state. On the further sampling times, TAT complexes were in the normal range while F1 + 2 were far below the normal range. Between day 1 and day 92, the levels of D-Dimer continuously decreased reflecting a long-term fibrinolytic process.This study clearly indicates that high INR are not systematically associated with very low F1 + 2 levels, particularly in the acute phase of thrombosis. Whether or not it is possible to reduce the intensity of the anticoagulant treatment 1 month after its initiation on the basis of very low levels of F1 + 2 can only be determined by prospective clinical trials.

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.

SummaryWe have investigated the antithrombotic properties of prothrombin fragment 1.2 (F1.2) in this study. To do this, we established the minimum concentration of human placental tissue factor or human a-thrombin that was lethal in mice within 5 min after intravenous injection. Prothrombin F1.2 protected the mice from the lethal effect of tissue factor or α-thrombin in a dose dependent manner, with 500 μg (14 nmoles) of prothrombin F1.2 per mouse being the minimum amount required to protect all mice from the lethal effect of either thrombogenic stimulus. The minimum dose of heparin which protected mice from the lethal effect of thrombin or tissue factor was 6 units or ∼3.3 nmoles. The observation that prothrombin F1.2 has antithrombotic properties suggests prothrombin F1.2 can modulate coagulation in vivo, as it has previously been shown to do in vitro.

An 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 microgram/l. Mean coefficients of variation of 4.2% (intraassay) and 3.5% (interassay) were found for TAT concentrations between 2 and 60 micrograms/l. A reference range from 0.85 to 3.2 micrograms/l was calculated from TAT concentration in plasma samples from 88 healthy donors (mean value +/- SD: 1.45 +/- 0.4 micrograms/l). In plasma samples from patients with pulmonary embolism (n = 17), TAT concentrations between 3 and 25 micrograms/l were measured. In 15 patients with deep vein thrombosis, TAT was found up to 3 to 25 micrograms/l. 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.