Specific identification of fibrin(ogen) degradation products in plasma and serum using blotting and peroxidase labeled antiserum

We describe a method for identifying fibrinogen and fibrin split products using electrophoresis on agarose gel with sodium dodecyl sulfate (SDS) followed by blotting in nitrocellulose paper. Detection of these derivatives after blotting is accomplished with peroxidase-conjugated rather than by isotopically labeled antibodies. This technique can detect diverse fibrinogen derivatives produced in vivo or in vitro by the combined action of thrombin, plasmin, and factor XIII. This methodology is applicable to plasma, serum, and other body fluids including urine and ascitic fluid. This sensitive and specific assay, distinguishing the products of cross-linked fibrin from those of fibrinogen and detecting fibrin polymers in plasma, can be achieved without the use of radioactivity.     

老年不稳定型心绞痛患者纤维蛋白、纤维蛋白原及其降解产物的变化

目的探讨凝血系统在老年人不稳定型心绞痛（ＵＡ）发生和发展中的作用。方法采用一组抗纤维蛋白单克隆抗体（ＳＺ－５８、６４、６５）酶免疫分析法测定２６例老年ＵＡ患者、２４例稳定型心绞痛（ＳＡ）患者和２０例健康老年人（对照组）血浆纤维蛋白原（Ｆｇ）、可溶性纤维蛋白复合物（ＳＦＣ）及血清纤维蛋白降解产物（ＦＤＰ）的浓度。结果ＵＡ患者心绞痛发作时血浆Ｆｇ、ＳＦＣ及血清ＦＤＰ浓度（分别为３．７±０．６ｇ／Ｌ、４９．６±１９．３ｍｇ／Ｌ、３２５．６±７９．４μｇ／Ｌ）显著高于ＳＡ患者心绞痛发作时（分别为３．２±０．６ｇ／Ｌ、２０．９±１０．４ｍｇ／Ｌ、２２４．４±４７．４μｇ／Ｌ）和健康对照组（均为Ｐ＜０．０１），后两者间差异无显著性。ＵＡ发作终止后血浆Ｆｇ和血清ＦＤＰ浓度高于健康对照组，但差异无显著性。结论血栓形成是老年人ＵＡ发生和发展的重要因素之一。     

Plasma D-dimer levels and their relationship to serum fibrinogen/fibrin degradation products in hypercoagulable states

Plasma D-dimer was measured and compared with serum fibrinogen/fibrin degradation product levels (FDPs) in patients with disseminated intravascular coagulation (DIC) and other conditions associated with a hypercoagulable state. D-dimer (N less than 200 ng/ml) was elevated in all 43 patients with DIC, in 48 of 59 patients with liver disease, in 22 of 27 patients with acute leukaemia at presentation, in 17 of 23 patients with malignant disease, in 29 of 39 women in the third trimester of a complicated pregnancy, in 17 of 18 patients with deep venous thrombosis and in only four of 27 patients with acute myocardial infarction. There was a significant correlation between plasma D-dimer and serum FDP levels (P less than 0.01) as follows; DIC: r = 0.58, liver disease: r = 0.57, acute leukaemia: r = 0.84, malignancy: r = 0.87. The frequent elevation of D-dimer observed in liver disease, acute leukaemia, malignancy and complicated pregnancy indicates that a hypercoagulable state is a common occurrence in these conditions although in liver disease elevated levels resulting from a failure of normal clearance mechanisms cannot be excluded. The close relationship between D-dimer and FDP levels suggests that serum FDPs predominantly arise from the interaction of plasmin with crosslinked fibrin rather than with fibrinogen in the conditions in which these were compared.     

Serum crosslinked fibrin (XDP) and fibrinogen/fibrin degradation products (FDP) in disorders associated with activation of the coagulation or fibrinolytic systems

Soluble crosslinked fibrin derivatives (XDP) in serum were determined by enzyme immunoassay utilizing monoclonal antibodies and compared with serum fibrinogen/fibrin degradation products (FDP) assayed by conventional techniques. In healthy subjects and patients with miscellaneous disorders not usually associated with activation of the haemostasis mechanism, mean XDP levels were 45 and 70 ng/ml respectively. However, elevated levels of XDP occurred in conditions commonly associated with intravascular and possibly extravascular activation of the coagulation system. Markedly raised mean XDP values (677-6900 ng/ml) occurred in treated pulmonary embolism, disseminated neoplasia, severe inflammatory disorders and complicated postoperative states, and lesser but significant elevation (mean 150-400 ng/ml) in treated venous thrombosis, uneventful postsurgical states, localized neoplasia, liver disease and symptomatic arterial disease. Levels during initial streptokinase therapy (mean 24 000 ng/ml) fell tenfold as treatment was continued. The degree of XDP elevation over normal values was significantly higher than that of FDP in conditions with a propensity for venous thrombosis (post-operative states, disseminated neoplasia and inflammatory diseases) than in liver disease, localized neoplasia or patients receiving heparin therapy for venous thromboembolism.     

Fibrin and fibrinogen degradation products in plasma of patients with colorectal adenocarcinoma

PURPOSE: The aim of the present study was to correlate the preoperative plasma levels of TDP in patients with colorectal cancer to tumor stage, metastasis, and postoperative thromboembolic complications. METHODS: Ninety-one patients with colorectal cancer, 20 patients with colorectal adenoma, and 71 patients without neoplastic lesions in the colon or rectum were included in this prospective study. Before surgery, total fibrin and fibrinogen degradation products (TDP) were measured in plasma of all patients with a specific enzyme-linked immunosorbent assay test. Phlebography was performed postoperatively in 82 of 91 patients with colorectal cancer. RESULTS: Median TDP in plasma of patients with colorectal cancer (805 (range, 339–5,024) ng fibrinogen equivalents (ngFE)) was significantly higher than TDP in patients with colorectal adenoma (591 (range, 417–1386) ngFE/ml) and TDP in patients without neoplastic lesions in the colon (632.8 (range, 180–2622) ngFE/ml;P <-0.003). In patients with colorectal cancer and liver metastasis, TDP in plasma (1085.5 (range, 468–5024) ngFE/ml) was significantly higher than in patients with localized tumor growth (753 (range, (339–2,780) ngFE/ml;P <-0.02). Twenty of 82 patients (24 percent) with cancer developed thromboembolic complications. TDP was preoperatively significantly higher in this group of patients (1,101 (range, 468–2,167) ngFE/ml) compared with patients without thromboembolic complications (753 (range, 339–5024) ngFE/ml;P <-0.04). CONCLUSION: Preoperative plasma levels of TDP were elevated in patients with colorectal cancer, especially in patients with liver metastasis and in patients developing postoperative deep venous thrombosis.     

Specific determination and identification of cross-linked fibrin degradation products in patients under thrombolytic therapy for myocardial infarction

We have demonstrated that the specific determination and identification of plasma FbDP alone is not sufficient to follow the effectiveness of thrombolytic therapy. Some patients who received an intravenous infusion of rt-PA for myocardial infarction had very high plasma FbDP levels, although no recanalization was observed angiographically. Since in many cases the FbDP levels increased after recanalization, it is assumed that rethrombosis may occur during and after t-PA treatment.     

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.     

Mechanisms for elevated fibrin/fibrinogen degradation products in acute experimental pulmonary embolism

The mechanism and significance of elevated levels of serum fibrin degradation products (FDP) in pulmonary embolism were investigated experimentally. Dogs were embolized with autologous blood clot- incorporating canine 125I-fibrin and were infused with either saline, heparin, or streptokinase. Serial measurements were made of total FDP by hemagglutination inhibition assay and of radioactive FDP. After saline, the peak level of total FDP was 323 mug/ml, but radioactive FDP was only 8 mug/ml. After heparin, these values were 44 and 11 mug/ml, respectively, and after streptokinase, 415 and 20 mug/ml. The results suggest that under these experimental conditions the elevated levels of FDP in pulmonary embolism are derived mainly from lysis of fibrin deposited after embolization rather than from lysis of the original embolus. Heparin inhibits both fibrin deposition and elevation of FDP levels after embolism.     

The role of plasma fibrinogen,D-dimer and fibrinogen degradation products in diagnosis and disease activity in patients with ankylosing spondylitis

Aim of the workTo evaluate the role of coagulation-related markers such as plasma fibrinogen, D-dimer and fibrinogen degradation product (FDP) in ankylosing spondylitis (AS) patients and their relationship with disease activity.Patients and methodsData were collected from 210 AS patients and 204 age and gender matched healthy controls. The Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) was used to divide AS patients into active (≥4) and inactive (score < 4) groups.ResultsThe mean age of the patients was 35.3 ± 16.3 years. They were 156 males and 54 females (M:F 2.9:1) and had a disease duration of 9.4 ± 7.2 years. The mean fibrinogen, D-dimer and FDP were significantly increased in the patients (375.4 ± 125.01 mg/dl, 2874.8 ± 1884.6 ug/l and 18.3 ± 11.3 mg/l) compared to the control (276.7 ± 71.9 mg/dl, 913.3 ± 540.6 ug/l and 3.01 ± 1.2 mg/l respectively; p < 0.001 each). Plasma fibrinogen, D-dimer and FDP increased in active compared to inactive patients (p < 0.001) and were significantly associated with BASDAI (p < 0.001). The optimal cut-off value of plasma fibrinogen, D-dimer and FDP for the diagnosis of AS were >288 mg/dl, >472 ug/l and >1.44 mg/l while to discriminate active from inactive the values were 393 mg/dl, 1228 μg/L and 1.82 mg/L, respectively. Logistic regression analysis showed that D-dimer is an independent predictor for AS disease activity (OR = 2.85, 95%CI: (1.85--4.43), p < 0.001).ConclusionFibrinogen, D-dimer and FDP increased in AS patients and significantly correlated with disease activity. D-dimer may play a role as a novel inflammatory parameter to predict disease activity in AS patients.     

The fibrinolytic response to ancrod therapy: characterization of fibrinogen and fibrin degradation products

Ancrod is a purified coagulant venom which renders blood incoagulable by cleaving fibrinopeptide A (FPA) from fibrinogen, but the mechanism involved in the clearance of fibrin from the circulation is unknown. To investigate the fibrinolytic response to ancrod, and to increase understanding of clearance mechanisms, six patients with peripheral vascular disease causing claudication were infused with ancrod at 2 u/kg over 6 h followed by 2 u/kg at 12 h intervals for 38 h. Venous blood samples were taken at time 0, 3, 6, 25 and 49 h for assay of fibrinogen (Fbg), fibrinopeptide A (FPA), total fibrin(ogen) degradation products (TDP), fibrin degradation products (FbDP), fibrinogen degradation products (FgDP), cross-linked fibrin degradation products (XL-FDP), tissue plasminogen activator (tPA), urinary type plasminogen activator (u-PA), plasminogen, α₂ antiplasmin (α₂AP) and plasminogen activator inhibitor-1 (PAI-1). Fibrinogen (median and range) was 2.3 (1.4–3.90) g/l at time 0 and thereafter was undetectable. FPA rose from 2.5 (1.8–3.6) to 600 and 188 pmol/l at 3 h and 6 h and remained elevated. TDP, FbDP and FgDP increased greatly following ancrod while there was no evidence of XL-FDP. The surprising increase in FgDP during defibrination suggests either that fibrinogen is digested following its incorporation into circulating fibrin protofibrils or that some of the fibrin subunits in the photofibril retain one of the two fibrinopeptide A's. tPA and uPA remained unchanged. Plasminogen fell from 125 (100–155)% to 79 (40–118)% at 49 h and α₂AP fell from 91 (75–107)% to 24 (10–35)% at 49 h. The level of PAI-1 was depressed during defibrination, with the exception of the 6 h data. The results demonstrate that ancrod removes FPA from fibrinogen to produce non-cross-linked (soluble) fibrin. This is cleared from the circulation without evidence of an increase in the circulating activities of the plasminogen activators, tPA or UK, but with evidence of plasminogen activation and consumption.     

Effects of fibrinogen, fibrin and their degradation products on the behaviour of vascular smooth muscle cells

The transition of fibrinogen to fibrin and to their degradation products within the arterial wall has been reported to be accompanied by atherosclerotic progression. A major step in the pathogenesis of atherosclerosis is the vectorial migration of vascular smooth muscle cells (SMCs) from the arterial media through the internal elastic lamina into the intima and their subsequent proliferation in the intima. I have been studying the effects of fibrinogen, fibrin and their degradation products on the behaviour, particularly migration, of SMCs. Fibrinogen/fibrin stimulates the adhesion and migration of SMCs and their effects are mediated by both the RGD-containing region of the alpha chain of fibrinogen/fibrin and integrin alpha v beta 3 on the cell surface. SMCs migrate into fibrin gel even with no other chemotactic stimuli. SMCs displayed two-fold increase in migration into crosslinked fibrin gels compared to non-crosslinked gels, suggesting the importance of fibrin crosslinking by factor XIIIa on its three-dimensional structure for the migration of SMCs. Fibrin gels prepared with batroxobin, which cleaves only fibrinopeptide A, with ACTE, which cleaves only fibrinopeptide B, and with protamine sulfate, which cleaves nothing, but forms a fibrin-like gel, induce migration of SMCs in a manner similar to the gel prepared with thrombin, suggesting that the cleavage of fibrinopeptides is not involved in the migration of SMCs. Both anti-fibrinogen fragment D and E antibodies inhibit the migration of SMCs into fibrin gel, suggesting that both D and E regions of fibrin are involved in the migration of SMCs into fibrin gel. The migration of SMCs into fibrin gel also depends on the RGD-containing region and integrin alpha v beta 3. Both fibrinogen fragments D and E inhibit the migration of SMCs into fibrin gels, suggesting that these fragments may be involved in the regulation of SMC migration into fibrin gel as the result of fibrinolysis. Although subcultured SMCs usually show a synthetic phenotype, the behaviour of contractile SMCs may be crucial for the subsequent migration of the cells. We employed an in vitro assay system to evaluate the effects of fibrin gels on the migration of SMCs from explants taken from rabbit aorta. alpha v beta 3 integrin and the RGD-containing region are involved in the migration of SMCs into the fibrin gels. SMCs which migrated from the explants showed positive staining with monoclonal antibodies against SMC myosin heavy chain isoforms, SMemb, SM1 and SM2, suggesting that they are in an intermediate state changing from a contractile to synthetic state. These findings show that fibrin (ogen) itself induces adhesion and migration of SMCs without other chemotactic or chemokinetic substances, suggesting a crucial role for fibrin (ogen) in the development and progression of such vascular diseases as atherosclerosis, thrombosis and restenosis following balloon angioplasty.     

Association between plasma levels of D-dimer and fibrinogen/fibrin degradation products (FDP) for exclusion of thromboembolic disorders

Background: D-dimer and fibrinogen/fibrin degradation products (FDP) levels are elevated in subjects with thromboembolic disorders, and the assays for detection of D-dimer and FDP are used in many laboratories for the investigation of these disorders. The aim of this study was to evaluate the association between the plasma levels of D-dimer and FDP in the investigation of thromboembolic disorders. Methods: D-dimer and FDP immunoassays were performed in 217 consecutive blood samples from subjects with suspected of thromboembolic disorders by use of Liatest D-dimer and Plasma FDP. Results: FDP results were classified in: <5, 5–20, >20 μg/mL, and D-dimer levels obtained in these groups ranged to 350–1210 ng/mL, 420–1960 ng/mL, and 1190–51170 ng/mL, respectively. A significant association between D-dimer levels and the reaction times necessary to occur agglutination in latex agglutination test for FDP was observed. Conclusions: There was an association between plasma levels of D-dimer and FDP. The preliminary determination of FDP levels could be useful because it allows estimating the D-dimer levels before of the automated systems analysis, reducing costs associated to dilutions of plasma samples.     

Content of fibrinogen degradation products, soluble fibrin and the fibrinolytic activity of blood in myocardial infarct]

The blood content of soluble fibrin and D and E fibrinogen fragments, the protamine sulfate test and the activity of plasmin and plasminogen activator were studied in patients with macrofocal myocardial infarction. It was established that the content of soluble fibrin in blood considerably increases in the first days of the disease, and the level of products of fibrinogen D and E disintegrationand the value of the protamine sulfate test increase in the 2nd--3rd week of the disease. The appearance in the blood of fibrinogen derivates was attended by low activity of plasmin and plasminogen activator in blood. The highest content of fibrinogen derivates and low activity of plasmin and plasminogen activator were noted in patients with arrhythmias and congestive circulatory insufficiency. It is suggested that the appearance in circulation of high-molecular fibrinogen compounds is important in the development of rheological disorders.