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In 12 patients treated with 100 mg rt-PA/3 h for acute myocardial infarction (AMI), serial fibrinogen levels were measured with the Clauss clotting rate assay ("functional fibrinogen") and with a new enzyme immunoassay for immunologically intact fibrinogen ("intact fibrinogen"). Levels of functional and "intact fibrinogen" were strikingly different: functional levels were higher at baseline; showed a more pronounced breakdown during rt-PA therapy; and a rebound phenomenon which was not seen for "intact fibrinogen". The ratio of functional to "intact fibrinogen" was calculated for each individual patient and each time point. The mean ratio (n = 12) was 1.6 at baseline, 1.0 at 90 min, and increased markedly between 8 and 24 h to a maximum of 2.1 (p < 0.01), indicating that functionality of circulating fibrinogen changes during AMI and subsequent thrombolytic therapy. The increased ratio of functional to "intact fibrinogen" seems to reflect a more functional fibrinogen at baseline and following rt-PA infusion. This is in keeping with data that the relative amount of fast clotting "intact HMW fibrinogen" of total fibrinogen is increased in initial phase of AMI. The data suggest that about 20% of HMW fibrinogen are converted to partly degraded fibrinogen during rt-PA infusion. The rebound phenomenon exhibited by functional fibrinogen may result from newly synthesized fibrinogen with a high proportion of HMW fibrinogen with its known higher degree of phosphorylation. Fibrinogen- and fibrin degradation products were within normal range at baseline. Upon infusion of the thrombolytic agent, maximum median levels of 5.88 micrograms/ml and 5.28 micrograms/ml, respectively, were measured at 90 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Soluble fibrin is considered as a molecular marker for intravascular fibrin formation, and impending thrombotic events. Most of the existing assays are less suitable for routine clinical applications and their specificity may be limited. We have developed a sandwich-type EIA with a fibrin-specific MoAb described by us before (Proc Natl Acad Sci 1989; 86: 8951) as the capture antibody. An other MoAb (G8; Thromb Haemostas 1988; 60: 145) with an epitope in the carboxyl-terminal sections of the fibrin alpha-chains, was labeled with peroxidase and used as the tagging antibody. The EIA is calibrated against plasma spiked with known concentrations of soluble fibrin. The time-to-result of the EIA is only 1.5 h. Concentrations as low as 0.5 micrograms soluble fibrin/ml plasma are readily measureable. Heparin has no effect on the results. Fibrinogen and fibrin(ogen) degradation products are not detected. The values of fibrinopeptide A and soluble fibrin values found with the "COA-SET soluble fibrin" assay correlated well with the soluble fibrin values found with our EIA i.e. r = 0.998 and 0.984, respectively. The run-to-run variabilities were 7.9% and 6.6% for samples with low and high soluble fibrin concentrations, respectively. The within-run variabilities were 2.5, 1.8, 4.0 and 4.6% for samples with 1, 0.5, 0.25 and 0.125 micrograms soluble fibrin/ml, respectively. The sensitivity, specificity, accuracy and short time-to-result make our EIA suitable for routine clinical applications and the monitoring of the effectivity of heparinization.

We have developed a sandwich-type enzyme immunoassay (EIA) for the quantitation of fibrin degradation products (FbDP) in plasma with a time-to-result of only 45 minutes. The assay is based on the combination of the specificities of two monoclonal antibodies (FDP-14 and DD-13), developed in our institute. FDP-14, the capture antibody, binds both fibrinogen degradation products (FbgDP) and FbDP, but does not react with the parent fibrin(ogen) molecules. It has its epitope in the E-domain of the fibrinogen molecule on the B beta-chain between amino acids 54-118. Antibody DD-13 was raised using D-dimer as antigen and is used as a tagging antibody, conjugated with horse-radish peroxidase. A strong positive reaction is obtained with a whole blood clot lysate (lysis induced by tissue-type plasminogen activator) which is used as a standard. The EIA does virtually not detect FbgDP i.e. purified fragments X, Y, or FbgDP generated in vitro in plasma by streptokinase treatment. This indicates that the assay is specific for fibrin degradation products. We have successfully applied this assay to the plasma of patients with a variety of diseased states. In combination with the assay previously developed by us for FbgDP and for the total amount of FbgDP + FbDP (TDP) in plasma, we are now able to study the composition of TDP in patients plasma in terms of FbgDP and FbDP.

Various plasmin preparations were tested for their suitability for use in the assay of alpha 2-antiplasmin in blood plasma by the immediate plasmin inhibition test. Activation of plasminogen, viz., 1-Glu-plasminogen and/or 77-Lys-plasminogen, by immobilized urokinase results in plasmin preparations suitable for this alpha 2-antiplasmin test. Plasmins obtained by "spontaneous" activation procedures in glycerol containing solutions, however, appeared not to be suitable. In this second group, the behaviour of the plasmins resembles that of 442-Val-plasmin (miniplasmin), which is known to show a low inactivation rate with alpha 2-antiplasmin due to the absence of lysine-binding sites in the plasmin molecule. Evidence is presented that, in the nonsuitable plasmins, the lysine-binding sites, although not completely absent, have at least partly lost their functional integrity.

The most commonly used fibrinogen assays in the clinic are clotting rate assays, e.g. the Clauss method. Such functional assays may be disturbed by e.g. heparin, anticoagulant fibrinogen degradation products (FgDP) and in the case of a dysfibrinogenemia. Immunological methods would not suffer from these interferences. However, immunological assays for fibrinogen, which do not measure FgDPs, do not exist. To set up such an enzyme immunoassay (EIA) we developed two monoclonal antibodies. The first monoclonal antibody (G8) has its epitope in the carboxyl-terminal 150 amino acid stretches of the fibrinogen A alpha-chains. G8 is used to coat the wells of microtitration plates, and is the capture antibody in this EIA. The second antibody (Y18) has been described by us previously (Blood 1985; 66: 503). It is directed against fibrinopeptide A, covalently bound to the alpha-chains i.e. against the amino-terminal stretches of the A alpha-chains. Y18 is conjugated with horse-radish peroxidase, and used as tagging antibody. The EIA does not react with, and is not interfered by FgDP such as purified fragments X and Y, up to a concentration of 800 micrograms/ml. An FgDP mixture such as generated by Streptokinase treatment of plasma does not respond. Fibrin degradation products (whole blood lysate) up to 800 micrograms/ml do not interfere nor do heparin, EDTA or oxalate. The time-to-result of the EIA is only 45 minutes. Some patient plasmas yielded dose-response curves which are not parallel with the calibration curve of the EIA. An explanation for this phenomenon could not be given.(ABSTRACT TRUNCATED AT 250 WORDS)