Rapid detection of cross-linked fibrin degradation products in plasma using monoclonal antibody-coated latex particles

Conformational and structural changes on conversion of fibrinogen to fibrin and its cross-linking by Factor XIIIa lead to the development of new antigenic determinants that permit differentiation between their plasminolytic cleavage products. A monoclonal antibody (DD-3B6/22) that is specific for cross-linked fibrin derivatives containing the D dimer configuration has been used in developing a latex agglutination procedure that can detect fibrin degradation products in either plasma or serum. Fibrinogen or its degradation products do not cross-react with this antibody. Results were calibrated with an enzyme immunoassay, which used a purified D dimer standard. Plasmas from 40 normal subjects, all having D dimer levels below 250 ng/mL measured by enzyme immunoassay, were all negative by latex assay. In contrast, positive latex agglutination titers were obtained with 87 of 88 patients with demonstrated deep venous thrombosis, pulmonary embolism, or disseminated intravascular coagulation. Compared to enzyme immunoassay, latex agglutination assay is less sensitive, but this latex procedure provides a rapid and less elaborate test for elevated levels of cross-linked fibrin degradation products in patients with thrombosis. Plasma assays for fibrin degradation products are preferable to those using serum.     

Evaluation of two new assays for determination of serum fibrinogen/fibrin degradation products

Two new commercial assays for the detection of degradation products of fibrinogen/fibrin (FDP) were evaluated against two standard procedures. The first, a new hemagglutination inhibition (HAI) assay using glutaraldehyde-treated cells, was compared with the tanned red cell hemagglutination inhibition immunoassay (TRCHII). Analysis of 43 samples from patients with a variety of bleeding disorders and thrombotic conditions showed a high degree of correlation between methods (r = 0.934). The second new assay, a rapid slide test using antibody-coated latex particles, was compared with results obtained by electroimmunoassay. There were no significant differences in the results as assessed by two statistical parameters. It was concluded that both new tests are useful for routine use in clinical laboratories.     

Differentiation between fibrin degradation products and fibrinogen degradation products by using newly developed ELISAs

We should distinguish fibrin degradation products (FbDP) from fibrinogen degradation products (FgDP) in order to analyze fibrinolysis in vivo. We analyzed some disorders associated with hyperfibrinolytic states using ELISA for FbDP, FgDP and total fibrin (ogen) degradation products (TDP) (ORGANON TEKNIKA). Each ELISA was useful in terms of reproducibility and dilution linearity of plasma samples. There was no cross-reaction between FbDP and FgDP. The FgDP/FbDP ratio in normal individuals was 1.65. In patients with DIC, it was 0.43, with FgDP level being increased. These results suggest that fibrinolysis is enhanced in patients with DIC, but it is accompanied by fibrinogenolysis. On the other hand, the FgDP/FbDP ratio in patients given urokinase (UK) was 2.88. This suggests that fibrinogenolysis is enhanced in them. In our study, the FgDP/FbDP ratio increased as DIC improved. Thus, we can regard this as an index of therapeutic effects in patients with DIC. We conclude that these three ELISA are useful in analyzing disorders associated with hyperfibrinolytic states.     

Serum fibrin/fibrinogen degradation products as a prognostic index in acute myocardial infarction.

A study of the prognostic value of serum fibrin/fibrinogen degradation products (FDP) in 137 consecutive patients with acute myocardial infarction showed a positive correlation between high FDP levels and poor prognosis. Both the frequency of complications and the mortality were related to increased levels of FDP, the highest of which were found between the fourth and eighth days after infarction.     

Analysis of cross-linked fibrin and fibrinogen degradation products with SDS-PAGE and immunoblot

SDS-PAGE and immunoblot technique with anti-FDP-D, -FDP-E, -fibrinogen antibody or anti-FDP-DD monoclonal antibody were applied to analyze FDP fragments prepared from cross-linked fibrin and fibrinogen with plasmic digestion in vitro. FDP fragments of DY(260K), DD(187K), X(245K), Y(166K), D(77K, 97K), E1(58K), E2(46K) and E3(40K) were identified from data of molecular weight and reactivity to four antibodies referred to reports of other investigators. Serum FDP fragments from five DIC suspected patients were analyzed by the same methods. In two patients' sera, DD fragment was a main component, and in the other three patients' sera, D fragment was a main fraction. Proportions of high molecular weight fragments of FDP were considerably different in patients' sera. Appearance of D fragment in our cases was considered to be derived from unstable fibrin (fibrin monomer and dimer) rather than from fibrinogen. Molecular weight of DD fragments from patients' sera had heterogeneity (160 approximately 180K), and the values were different from that (187K) prepared from cross-linked fibrin. In conclusion, SDS-PAGE and immunoblot analysis of serum fragments of FDP will be an useful technique to investigate the clinical and pathological condition of DIC.     

Clinical significance of a new fibrinogen/fibrin degradation products(FDP) test using plasma samples for the diagnosis of fibrinogenolysis and fibrinolysis

Recently, a new fibrinogen/fibrin degradation products(FDP) test using monoclonal antibodies against FDP(LPIA FDP-P: FDP-P) has been developed, which is able to measure FDP directly in plasma. The objective of this study is to clarify clinical significance of the test in the diagnosis of fibrinogenolysis and fibrinolysis in comparison with a conventional FDP test using polyclonal antibodies against fibrinogen(FDP-S) and D-dimer test using monoclonal antibodies against D-dimer(D-D). The monoclonal antibodies used in FDP-P test was shown to recognize fragment X, Y and D1 derived from fibrinogen digested by urokinase, and was also to recognize XDP fragments, D-dimer and D derived from cross-linked fibrin digested by tissue plasminogen activator using SDS-PAGE and immunoblotting analysis. There was a good correlation of FDP levels between FDP-P test and FDP-S test. However, levels of FDP in both tests were discrepant in several samples. There was a tendency that the levels of FDP were higher in FDP-S test than in FDP-P test. Such discrepancy was suggesting that soluble fibrin monomer complex(FM) was recognized by the antibodies used in FDP-S test, but not recognized by the antibodies used in FDP-P test. There was also a good correlation of FDP levels between FDP-P test and D-D test. However, the levels of FDP in both tests were discrepant in several samples. The levels of FDP were higher in FDP-P test than in D-D test. These discrepant samples had lower levels of antiplasmin and higher levels of plasmin antiplasmin complex(PIC), and also showed XDP fragments, D-dimer, X, Y, and D1 by using SDS-PAGE. These observations suggest that D-D test measures only fibrinolytic fragments, while FDP-P test measures fibrinogenolytic fragments as well as fibrinolysis. In results, the FDP-P test was confirmed to be a useful tool to examine fibrinogenolysis as well as fibrinolysis more specifically than the conventional FDP test.     

Pharmacokinetics of degradation products of fibrin and fibrinogen during alteplase therapy of acute myocardial infarction

Pharmacokinetics of fibrin degradation products (FbDP), D-dimers, fibrinogen degradation products (FgDP) and total degradation products (TDP) in plasma were investigated using ELISA methods in 12 patients undergoing therapy with alteplase (100 mg/3 h) for acute myocardial infarction. Peak concentrations of all degradation products occurred significantly later (1.6–2.5 h) than peak alteplase levels (2 min). Peak D-dimer concentrations (mean 2.6 μg/ml) were significantly lower than those of FbDP (7.1 μg/ml) or FgDP (7.8 μg/ml). However, the half-life of D-dimers (mean 13.3 h) was more than 4.5-fold longer than that of FbDP (2.9 h) or FgDP (2.8 h) (p<0.001). Consequently, areas under the plasma concentration-time curve (AUC) of D-dimers were comparable with those of FbDP or FgDP The ratios AUC(FbDP)/AUC(FgDP) and AUC(D-dimers)/AUC(FgDP) are proposed as new indices of fibrin specificity. Their values in this study (geometric mean, 95% CI) were 1.15 (0.60–2.20) and 1.30 (0.52–3.24) respectively. It is concluded that cross-linking may prolong the half-life of fibrin degradation products, and that complete time profiles of fibrin- and fibrinogen degradation products in plasma, rather than single point measurements, are essential for reliable quantification of fibrin specificity.     

Fibrinogen and fibrinogen/fibrin degradation products in the urine of rabbits infected with Trypanosoma (Trypanozoon) brucei

Summary Studies have been carried out on the urine of rabbits infected with Trypanosoma (Trypanozoon) brucei to determine whether fibrinogen or fibrinogen/fibrin degradation products (FDP) could be detected. No fibrinogen was found but during the last two weeks of this 7-week infection low levels of FDP were present in the urine which did not exceed 5 g/ml. Rabbit urine was shown to contain a potent proteolytic enzyme capable of breaking down rabbit fibrinogen and both early and late FDP were present in the cleavage products. No deposits of fibrin were detected in the kidney, but casts were present in the urine suggesting renal damage. The most likely explanation of the urinary FDP is that either an increase in the glomerular permeability occurs allowing filtration of plasma FDP or a local fibrinogenolysis in the kidney tubules.     

纤维蛋白原对人血管平滑肌细胞株增殖和移行的影响

目的:探讨纤维蛋白原(Fg)、纤维蛋白(Fb)及其降解产物(FDPs)对体外人血管平滑肌细胞(VSMC)增殖与移行的影响。方法:采用细胞计数法和四唑盐(MTT)比色法测定VSMC增殖,通过刮伤实验(woundingmodel)和Transwell嵌套装置观察VSMC的移行。结果:Fg本身对VSMC增殖无促进作用,但可促进VSMC的移行,且呈浓度依赖关系;Fb及FDPs均可明显促进VSMC增殖和移行,Fb的作用还呈现浓度依赖关系,而FDPs作用呈现先增强后减弱的趋势,即在一定浓度范围内作用明显,高于或低于此范围作用较弱。结论:Fb及FDPs通过促VSMC的增殖和移行作用,可能在再狭窄和动脉粥样硬化中起重要作用。     

Assay of serum fibrin degradation products by agglutination-inhibition of coated erythrocytes

An immunological method for detecting fibrin degradation products by tanned cells agglutination has been standardized. The effects of different variables, such as cell concentration, pH, and length of incubation, and the specificity of the reaction, are described. The method is compared with other immunological techniques.