Reactivity of D-dimer assays with the fibrinogen--fibrin split products generated by thrombolytic agents.

The reactivity of two D-dimer assays (a latex agglutination method, D-Di test and an ELISA procedure, Asserachrom D-Di) to the various fibrin or fibrinogen degradation products generated in plasma by three different thrombolytic agents was analysed, in the presence or absence of a fibrin clot. Other assays performed in parallel were an ELISA assay for (DD)E complexes and the conventional fibrinogen degradation products (FDP) latex test on serum. The thrombolytic agents urokinase, streptokinase or tPA were added at various concentrations and incubated for different times ranging from 10 min to 24 h. The data showed that the D-dimer latex assay was always negative provided there was no fibrin in plasma and despite the presence of high FDP levels (greater than 600 micrograms/ml) in serum. In contrast, D-dimer or (DD)E complexes were measured by ELISA, but up to a given concentration (15-20 micrograms/ml) which reached a plateau and remained stable irrespective of the thrombolytic concentrations or the degradation times. In the presence of fibrin clot, fibrinolysis was extremely fast with tPA and the FDP were generated at a much higher concentration that that expected from the size of the fibrin clot. This suggests the existence of fibrinogenolysis targeted by the presence of fibrin but negative in its absence. Urokinase and streptokinase generated FDP very quickly but a much slower degradation rate of fibrin was observed. The immunoblotting confirmed these data and showed that no late FDP were formed in plasma even at high thrombolytic concentrations except when fibrin was present.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibodies to different neo-epitopes on fibrinogen and fibrin degradation products.

The measurement of fibrin or fibrinogen degradation products is widely used in clinical practice for the diagnosis and follow up of coagulolytic disturbances. Recently D-dimer assays have become very popular owing to their direct application to plasma. However, in some clinical situations there is a need to differentiate fibrin from fibrinogen degradation products. These are still routinely measured by conventional assays on serum. We tried to develop various monoclonal antibodies specific for the neo-epitopes unmasked during the degradation of fibrin or fibrinogen. Fifteen mice hybridomas producing the expected antibodies were obtained and ten were extensively characterized. They could be classified in three reactivity classes: D and D-dimer, D-dimer and early fibrinogen or fibrin degradation products. These monoclonal antibodies were used to develop latex slide assays and ELISA techniques. Two types of assays were obtained; those which were specific for fibrin-related products and those evaluating the totality of fibrin or fibrinogen degradation products. Assays discriminating the fibrinogen split products from those derived from fibrin, and performed directly on citrated plasma can be proposed. They provide complementary information in clinical states such as DIC, pulmonary embolism, leukaemias, thrombolysis, etc.

Measurement of tPA and tPA-PAI-1 complexes by ELISA, using monoclonal antibodies: clinical relevance.

Two ELISA methods using monoclonal antibodies, are described for the measurement of tPA:Ag and tPA-PAI-1 complexes. On normal population tPA:Ag was found with a mean value of 5 ng/ml. Furthermore, despite that tPA activity was very low, only 50% (mean value) was measured as stable complexes with PAI-1. Important increase of tPA:Ag was observed in various pathologies (cardiac infarction, septicemia, respiratory distress syndrome). In liver disease, tPA:Ag reached high levels up to 100 ng/ml. Impaired liver clearance can potentiate the increased concentration which results from endothelial release. In all patients with elevated tPA:Ag level, 70 to 100% of tPA was complexed to PAI-1. Excess release of PAI-1 accompanys the increased release of tPA as it is proved by presence of high residual PAI-1 activity. Addition of exogeneous tPA to these pathological plasmas induced a high increase in tPA-PAI-1 complexes. Venous stasis in normal population resulted in a parallel increase of tPA:Ag and tPA-PAI-1 complexes. Although about a two fold increase was obtained for both parameters, post venous stasis plasma presented a much higher fibrinolytic activity while PAI-1 activity was moderately elevated. tPA:Ag and tPA-PAI-1 complexes have diagnosis and prognosis value in various pathologies as indicators of stimulated release of fibrinolysis activator and inhibitor.