Poor correlation between soluble fibrin concentrations measured by two commercially available immunoassays

Soluble fibrin (SF) has attracted considerable interest as a marker for haemostatic activation. Two new enzyme immunoassays for SF, Enzymun-Test^® FM (Boehringer Mannheim, Germany) and Fibrinostika^® Soluble Fibrin (Organon Teknika, Belgium) have recently become commercially available. We measured plasma levels of SF in clinically obtained blood samples in order to compare the two new assays. Blood was drawn from 10 healthy volunteers and from 149 patients on the first day after surgery for fractures of the upper part of the femur. Collection and processing was done according to the manufacturers' recommendations. In the patients, levels found by the two assays were significantly different. The Enzymun-Test^® assay reported a median (range) of 11.87 (2.66 - > 62.20) mg/l, whereas the Fibrinostika^® assay found a median (range) of 3.34 (1.08 - > 10) mg/l. The correlation coefficient was 0.38 (Spearman). A poor correlation was thus found between values obtained by the two assays in the patient category chosen. Further validation of the assays is necessary.     

Cytokines, soluble thrombomodulin and disseminated intravascular coagulation in patients with systemic inflammatory response syndrome

To investigate the relationships between tumor necrosis factor-alpha (TNF), interleukin-1 beta (IL-1β), soluble thrombomodulin (TM), and disseminated intravascular coagulation (DIC) in patients with systemic inflammatory response syndrome (SIRS), twenty-nine SIRS patients were classified into three groups; 4 patients without DIC, 8 DIC patients who recovered, and 17 DIC patients who did not recover. Serum TNF, IL-1β, and soluble TM were measured on the day of the diagnosis of SIRS, and also on the 1st, 3rd, 5th days. All of the DIC patients had multiple organ dysfunction syndrome (MODS) and the number of the dysfunctioning organs showed significant differences between the groups (p = .0017). All of the patients who did not recover from DIC died. The serum soluble TM level was higher in the patients without DIC recovery than in either the DIC recovery patients or the non DIC patients throughout the study period. In DIC patients who did not recover, there were significant correlations between soluble TM and TNF (r² = 0.205, P = .0003) or IL-1β (r² = 0.157, P = .0036). In conclusion, the DIC being associated with endothelial injury is an important pathogenetic factor for MODS and is a main determinant of the outcome of SIRS patients. TNF and IL-1β might be involved in the cause of this endothelial injury. The soluble TM is a good predictor of organ dysfunction and also of a poor prognosis.     

Soluble fibrin in plasma before and after surgery for benign and malignant colorectal disease

In a prospective study, plasma levels of soluble fibrin (SF) were assessed in 97 patients with colorectal cancer immediately before and 1, 2, 7, and 90 days after surgery, 18 patients undergoing surgery for benign colorectal disease serving as controls. Age distribution, routine blood analysis, duration of surgery, perioperative blood loss and anaesthesia was similar in the two groups. SF was quantitated using a commerciel enzyme-linked immunosorbent assay.

The preoperative plasma level of SF was normal in cancer patients as a whole. However, patients with disseminated colorectal cancer had higher levels of SF preoperatively compared to patients with localized colorectal cancer (p<0.01) and controls (p<0.005). On days 1, 2, and 7 days postoperatively, a rather pronounced increase in plasma SF was observed in cancer patients as well as in the controls. Three months after surgery, plasma SF had normalized in controls and in patients undergoing curative cancer treatment. Postoperative deep venous thrombosis (DVT) was detected in 23% of the cancer patients by means of phlebography. The preoperative values of SF in these patients were higher compared to patients not developing DVT (p<0.05).

Patients with colon cancer displayed higher SF in plasma than patients with rectal cancer (p<0.05).     

Effect of dipyridamole on experimental disseminated intravascular coagulation in rats

Experimental disseminated intravascular coagulation (DIC) can be induced by 4-h sustained infusion of endotoxin in a dose of 100 mg/kg in rats. The experimental model of DIC in rats was used to study the preventive effect of dipyridamole against DIC. Before the infusion of endotoxin, 0.5, 5.0 or 50.0 mg/kg of dipyridamole was injected intraperitoneally. The preventive effect against DIC was noted in all the parameters, such as fibrinogen and fibrin degradation products, fibrinogen level, prothrombin time, partial thromboplastin time, platelet count, and the number of renal glomeruli with fibrin thrombi, in rats treated with 5.0 or 50.0 mg/kg of dipyridamole. From these results, it was shown that dipyridamole inhibited the aggravation of endotoxin-induced experimental DIC in rats.     

Alterations of haemostasis parameters with special reference to fibrin stabilization, factor XIII and fibronectin in patients with obliterative atherosclerosis

Intravascular and endoparietal fibrin deposition are thought to be involved in atherosclerotic process, especially when fibrin is stabilized by factor XIII of coagulation system. The study carried out on a group of 50 patients with obliterative atherosclerosis of the lower limbs revealed an increased plasma fibrin stabilizing activity apparently due to an increased F.XIII level. Furthermore, alterations in coagulation and fibrinolysis indicating hypercoagulable tendency were found. It is concluded, that the observed changes may contribute to the development of atherosclerotic process.     

Release of N-terminal peptides from glu-plasminogen by plasmin in the presence of fibrin

The rate of plasmin-catalyzed Glu-plasminogen to Lys-plasminogen conversion was faster in the presence of fibrin than in its absence when assayed by SDS-polyacrylamide gel electrophoresis followed by quantification of stained bands by densitometer. Experiments with the isozymes Glu-plasminogen I and Glu-plasminogen II showed that plasmin catalyzed formation of Lys-plasminogen II was especially facilitated in the presence of fibrin.     

Temperature dependent dissociation of soluble fibrin monomer complexes demonstrated by agarose gel filtration

The temperature dependent dissociation behaviour of soluble fibrin monomer complexes (SFMC) in plasma of post surgical patients was studied using gel filtration chromatography of β-Ala precipitated plasma samples. A temperature dependency in the investigated range between 12° and 37°C was demonstrated with a linear regression y = 5.56?0.07 x and a correlation coefficient r = 0.83. Additional experiments using 125 I-des- A fibrin injected into heparinized rabbits revealed that after gel filtration of whole plasma samples at 20°C 68% of the activity was eluted in front of the fibrinogen elution volume and 32 % with the fibrinogen volume. At 37°C 24 % of the activity was eluted in front of fibrinogen, while 76 % eluted with the fibrinogen peak. The experiments indicate that SFMC are subject to a temperature dependent dissociation behaviour which is also influenced by the type of the complexes.     

Interaction of fibrinogen with mercury

The interaction of bovine fibrinogen with mercuric chloride was studied. Gel filtration on Sephadex G-25 revealed that fibrinogen bound twice the amount of mercury such as fibrin or fibrin monomers (8.8, 4.5, and 3.4 μg Hg²⁺ ions/ mg protein, respectively). Fibrinogen complexed with mercury or in the presence of Hg²⁺ ions at concentration above 10⁻⁶ M was clotted by thrombin more effectively than in the control system which was devoid of this metal. Reaggregation of the purified fibrin monomers was not affected by mercury.     

Acceleration of fibrin gel formation by unrelated proteins

Addition of γ-globulin, serum albumin, hemoglobin, or ovalbumin in concentrations of 1–10 g/dl to solutions of purified fibrinogen results in a substantial (up to six-fold) decrease in the lag time preceding appearance of a firm fibrin gel following addition of thrombin at 24°C. The effect does not appear to be due to a protein-induced enhancement in the enzymatic activity of thrombin, nor does it appear to be due to the co-condensation of the added protein with fibrin/fibrinogen. It is suggested that the observed effect is primarily due to nonspecific volume exclusion arising from increased fractional occupancy of solution volume by macromolecules.     

Altered influence of polymorphonuclear leukocytes on coagulation in acute ischemic stroke

Polymorphonuclear (PMN) and mononuclear (MN) leukocytes possess procoagulant and anticoagulant activity which could be involved in both formation and dissolution of thrombi. We investigated if coagulant properties of circulating leukocytes are altered in patients within three days after acute ischemic stroke (n=22) as compared to a control group (n=22) matched for sex and age. The recalcification time with autologous plasma did not differ between patients and control subjects. Circulating PMNs were procoagulant in all subjects, however, they were less procoagulant in patients (−18.1 [−13.4 − −22.8] % of control experiments; mean [95% confidence interval]) than in controls subjects (−31.9 [−27.4 − −36.4] %; P=0.0002). In contrast, MNs were similarly procoagulant in both groups. In the activated partial thromboplastin time (aPTT), there was a non-significant trend to less procoagulant PMNs in patients (−6.7 [−5.1 − −8.2] %) than in control subjects (−8.4 [−6.4 − −10.5] %). The recalcification time with pooled human plasma showed similar results as with autologous plasma. The procoagulant activity of PMNs increased in follow-up measurements in patients. Upon stimulation with FMLP, the procoagulant activity of PMNs decreased in control subjects but did not change significantly in patients. In the acute stage after ischemic stroke, circulating PMNs exhibit a decreased capability to stimulate coagulation, a feature which reflects cell activation and which may be a reaction on thrombus formation and ischemic tissue damage.     

Effects of fibrinogen and fibrin on the activation of glu- and lys-plasminogen by urokinase

Glu- and Lys-plasminogen (plg) were mixed with fibrinogen or fibrinogen plus thrombin in the presence of various units of urokinase (UK) and S-2251 (H-D-Val-Leu-Lys-pNA). Time course of the hydrolysis of S-2251 and the increment of OD₄₀₅/min were monitored by using a spectrophotometer. Glu-plg was activated better by UK in the presence of fibrinogen and fibrin than in their absence. The effects of fibrin were larger than those of fibrinogen in the activation of Glu-plg. When Lys-plg was used for the similar experiments, the activation rate of Lys-plg was slightly increased in the presence of fibrinogen and fibrin (fibrin > fibrinogen), but their effects were smaller than in the case of the activation of Glu-plg.     

Local activation of the coagulation and fibrinolysis systems in lung disease

Extravascular coagulation and fibrinolysis is an integral part of inflammatory reactions. Disordered expression of procoagulant and profibrinolytic factors by mononuclear phagocytes of the lung (i.e. lung alveolar macrophages (LAM) and interstitial macrophages) may have important bearings on inflammatory lung tissue destruction and repair. Based on this hypothesis we have measured the presence of trigger molecules and activation products of the coagulation and fibrinolytic system in cell-free bronchoalveolar lavage fluid and in bronchoalveolar cells. Patient groups with chronic obstructive disease (COLD)(n=76), idiopathic pulmonary fibrosis (IPF)(n=29), sarcoidosis (n=22), lung cancer (n=36), pneumonia (n=39), aquired immunodeficiency syndrome (AIDS)(n=17) and a control group (n=60) were studied by bronchoalveolar lavage (BAL). In all patient groups tissue thromboplastin (TPL) and fibrinopeptide A (FPA) were significantly increased compared to controls. Plasminogen activator (PA) activity was significantly lower in patients than in normals, and usually associated with high levels of antifibrinolytic activity. The level of PA inhibitor (PAI-2) was not significantly higher in any patient group compared to controls. The sensitivity of the method for fibrin degradation products (FDP) analysis was not high enough to detect FDP in BAL fluid of control individuals, whereas such products could be demonstrated in 25–53% of patients in various categories. We conclude that disordered expression of procoagulant and plasminogen activator activities in bronchoalveolar lavage fluid may reflect a milieu that favours accumulation of fibrin in inflammatory lung tissue and form the basis for the development of pulmonary fibrosis.     

Effects of subcutaneously administered dermatan sulfate (MF 701) on the coagulation and fibrinolytic parameters of healthy volunteers

Eight healthy volunteers were given single subcutaneous doses of dermatan sulfate (DS, 100, 200 and 400 mg), heparin (5,000 IU) and placebo in random order. Wash-out between treatments was > 10 days. Serial blood samples were taken before and up to 24 hours after treatment to measure coagulation and fibrinolytic parameters. Thrombin generation was significantly inhibited by DS and heparin as compared to placebo. The effect of DS was dose-dependent. Peak inhibition after 200 mg DS was comparable to that of 5,000 IU heparin, but lasted longer. A small, bordeline significant prolongation of APTT was observed after 400 mg DS and heparin. The changes in PAI and fibrinolytic activities were those of the circadian variation. No changes were seen in the other parameters tested. In conclusion, single s.c. doses of DS (200, or 400 mg) inhibit thrombin generation equally or more than 5,000 IU heparin and for a longer time. The effect of both treatments on fibrinolysis is negligible.     

Estimation and characterization of soluble fibrin monomer complexes during endotoxin-induced intravascular coagulation.

This study is concerned with thrombin-mediated intravascularly occurring changes in the fibrinogen molecule in rabbits that developed generalized intravascular coagulation after two endotoxin injections spaced 24 hours apart. Intravascular coagulation was evidenced by glomerular fibrin deposition. 24 hours after the first endotoxin injection an increase in the concentration of soluble fibrin monomer complexes indicated intravascular activation of the coagulation system. The second endotoxin injection was followed by a decrease in fibrinogen concentration and an increase in soluble fibrin monomer complexes in relation to the total fibrinogen content. Characterization of the soluble fibrin monomer complexes revealed that they were to a larger extent not dissociable in SDS and urea, and that they contained α-dimers. This indicates that circulating crosslinked fibrin oligomers were present.     

Determination of soluble fibrin: A comparison of four different methods

The determination of soluble fibrin (SF) in plasma was compared using four different methods. The SF-ELISA immunologically measures the concentration of desAA- and desAABB-fibrin while the SF-tPA-test is based on activation of plasminogen by tissue plasminogen activator (tPA) in the presence of fibrin; the SF-PS-turbidimetry assay relies on the protamine sulphate (PS) -induced aggregation of fibrin in plasma whereas the SF-erythrocyte-agglutination-test (SF-EAT) detects soluble fibrin by its aggregation with fibrin monomers attached to test erythrocytes.

Soluble fibrin was generated in vitro by addition of thrombin or ancrod to plasma. In these experiments the soluble fibrin values of the four methods correlated well with each other and with the fibrinopeptide A release, especially in ancrod-induced fibrinogen turnover (r > 0.93). This high correlation is remarkable, considering the fact that the methods are based on different principles. Detection of thrombin-induced soluble fibrin was more sensitive; differences between ancrod and thrombin action were observed as well, probably due to different forms of soluble fibrin. A delayed increase of SF-PS-turbidimetry values in particular during the thrombin action can be attributed to a lack of detectable aggregation of soluble fibrin at low concentrations due to its solubility in plasma.

Subsequently, soluble fibrin was measured in samples from patients. The SF-ELISA and SF-tPA-test were highly sensitive and correlated better than the other methods with each other, but all correlations were less satisfactory compared with the in vitro studies. These weaker correlations might be explained by the heterogeneity of soluble fibrin determined by inter- and intraindividually varying concentrations of fibrinogen and its different derivatives in plasma samples from patients.

All methods provided reliable results with differences in sensitivity, specificity and practicality. The SF-tPA-test, SF-PS-turbidimetry, and SF-EAT are practical methods for routine use whereas the SF-ELISA is a highly reliable and by far the most sensitive and specific method thus offering new insights into pathogenesis of fibrinaemia and related diseases.     

Isolation and characterization of a monoclonal antibody specific for fibrinogen and fibrin of human origin

A hybridoma cell line secreting a monoclonal antibody directed against human fibrinogen has been isolated. The antigenic determinant recognized is present on both the fibrinogen and fibrin molecules but is apparently absent from the D and E fragments and from fibrinopeptides A and B. The antibody seems to recognize a conformational structure present in native fibrinogen and in which several or possibly all the fibrinogen polypeptide chains may participate.     

The monoclonal antibody that recognizes an epitope in the C-terminal region of the fibrinogen alpha-chain reacts with soluble fibrin and fibrin monomer generated by thrombin but not with those formed as plasmin degradation products

INTRODUCTION: The presence of soluble fibrin (SF) provides evidence of thrombin activation in the blood; therefore, SF is a useful marker for diagnosing blood coagulation diseases such as disseminated intravascular coagulation (DIC). The antibody that specifically detects SF could be a useful tool for diagnosing thrombotic diseases. MATERIALS AND METHODS: By using an acid-solubilized desAA-FM (fibrin monomer) as an immunogen, we developed a monoclonal antibody, namely J2-23, which specifically reacts with SF and FM. We examined the specificity of J2-23 by ELISA and immunoblotting and confirmed the reactivity of J2-23 with SF and FM by gel filtration. RESULTS AND CONCLUSIONS: J2-23 specifically reacted with SF, but not with fibrinogen or plasmic fibrinogen-derived Fbg-X, Fbg-Y, Fbg-E, and D; thrombin-treated Fbn-X, Fbn-Y, and Fbn-E; and plasmic cross-linked fibrin (DD, XDP). The epitope recognized by J2-23 was located within the Aalpha 502-521 region on the C-terminal of the fibrinogen alpha-chain. The reactivity of J2-23 disappeared following the action of the fibrinolytic enzyme plasmin. Furthermore, J2-23 reacted not only with SF but also with FM in plasma from DIC patients. This indicated that J2-23 specifically detected coagulation without reflecting the plasmin action. We demonstrated the potential of J2-23 as a useful antibody for detecting SF for diagnosing blood coagulation.     

A latex immunoassay of fibrin/fibrinogen degradation products in plasma using a monoclonal antibody

We have developed a latex immunoassay using an anti D neo monoclonal antibody (F2C5) which recognizes an epitope present in fragment D but which is hidden in intact fibrinogen and in early fibrinogen degradation products. This technique was applied directly to plasma of both healthy donors and patients, and was shown to be very convenient for clinical investigation, especially in emergency cases for diagnosis of intravascular coagulation. The use of plasma samples instead of serum offers several advantages: it is not time consuming since blood clotting is not required; it avoids overestimation when fibrinogen cannot be totally clotted, and underestimation due to the binding of nonclottable fibrin degradation products to the clot during clotting in vitro. This monoclonal antibody, which reacts more with FbDP (expressed infragment D) than with fragment D, does not allow fibrin and fibrinogen degradation products to be differentiated. However, this discrimination does not seem critical for its clinical use since the level of fragment D neo antigen remained within the normal range in 12 cases of spontaneous or venous occlusion induced hyperfibrinolysis.