Effect Of Fibrinogen Degradation Products Research Articles

Evaluation of an automated photometric fibrinogen assay.

A recently-introduced automated method for the determination of plasma fibrinogen is based on the principle of von Clauss, combined with photometric detection: after addition of thrombin, the coagulation time is determined by measuring the change in absorption at 405 nm. This method was evaluated and compared with the original coagulometric Clauss assay and with the prothrombin time (PT)-derived automated method. The inter-assay coefficient of variation of the Clauss-derived assay was lower (14.1, 3.8 and 4.6%) than the PT-derived assay (16.1, 7.5 and 10.5%, respectively) at all three fibrinogen levels tested (1.2, 4.0 and 7.5 g/l). The correlation between the assays was investigated according to the method of Passing and Bablok and could be described as follows: Clauss-derived = 0.79 (PT-derived) + 0.66; Clauss-derived = 1.12 (Clauss) + 0.143. The interference of heparin (< 1.5 U/ml), haemoglobin (< 30 micromol/l), bilirubin (< 200 micromol/l) and triglycerides (< 5.5 mmol/l) in the Clauss-derived assay was negligible. The effects of fibrinogen degradation products on the Clauss-derived assay were comparable with the effects on the Clauss assay, in contrast to the effects on the PT-derived assay. In conclusion, the Clauss-derived assay is a specific and precise automated method to determine fibrinogen concentrations in plasma, which is not liable to interference from different pathophysiological substances.

Enhancement of interleukin-6 production by fibrinogen degradation product D in human peripheral monocytes and perfused murine liver.

The effect of fibrinogen degradation products D and E (FDP-D, FDP-E) on IL-6 production in perfused mouse livers and peripheral monocytes is studied. Similarly to bacterial endotoxin FDP-D is highly potent to augment the IL-6 production measured in perfused mouse livers, while FDP-E is not stimulatory. FDP-D but not FDP-E is able to stimulate the in vitro IL-6 production of human peripheral monocytes, as well. Plasmin alone is almost ineffective on IL-6 production both in perfused livers and monocytes. Our findings suggest a direct positive feedback circuit, among fibrinogen, FDP and IL-6.

Streptokinase and APSAC inhibit platelet aggregation in vitro by fibrinogenolysis: Effect of plasma fibrinogen degradation products X and E

Summary Streptokinase (SK) in vitro can both activate and inhibit platelets. Inhibition has been ascribed either to proteolysis of membrane glycoproteins (GPs) or to the inhibitory effect of fibrinogen degradation products (FgDPs). Addition of a low concentration of SK (300 U/ml) to human citrated platelet rich plasma (cPRP) has been shown to lead to an aggregation defect, related to fibrinogenolysis, without discernible changes in the glycoprotein IIb/IIIa complex. In the present study, similar results were obtained with SK (300–5000 U/ml), and the acylated plasminogen streptokinase activator complex (APSAC) (0.01 U/ml). The aggregation defects appeared to be related to the extent of fibrinogenolysis. The knowledge of the FgDPs profile might help in the understanding of this aggregation defect. We have chosen to generate the FgDPs in plasma by activating plasminogen with SK 300 U/ml, 15 min at 37°C. Fragments X and E were separated by gel filtration and identified by electrophoresis and immunoblot. The effects of the fractions containing fragments X and E on aggregation were studied using human washed platelets resuspended in Tyrode buffer, 2 mM Ca++, 1 mM Mg++, after stimulation with ADP (10−5 mol/l). The results (expressed as percentage of the response obtained with intact fibrinogen) were: 72%±18 (p In conclusion, our data confirm and extend previous observations on the mechanisms of platelet aggregation inhibition by SK or APSAC. The main factor of platelet inhibition in plasma appears to be the FgDP E.

Streptokinase and APSAC inhibit the platelet aggregation in vitro by fibrinogenolysis: Effect of fibrinogen degradation products X and E generated in plasma

Streptokinase and APSAC inhibit the platelet aggregation in vitro by fibrinogenolysis: Effect of fibrinogen degradation products X and E generated in plasma

Fibrinogen degradation products generation is the major determinant of platelet inhibition induced by plasminogen activators in platelet-rich plasma

The platelet function defect induced by thrombolytic agents has been referred either to the degradation of platelet surface receptors or to the anti-aggregatory effect of fibrinogen degradation products (FgDPs). In the present study we have evaluated platelet aggregation induced by ADP, collagen and ristocetin after incubation of washed platelets or platelet-rich plasma (PRP) with plasmin (1.1–3.4IU/ml), plasminogen activators (PAs) (streptokinase 250–1000 IU/ml; urokinase, 10–1000 IU/ml; t-PA 0.5–10 μg/ml) or FgDPs (0.062–2 mg/ml). In parallel the surface levels of platelet GP lb and IIb/IIIa complex were determined by fluorescence flow cytometry using specific monoclonal antibody. Washed platelets treated with plasmin (1.1IU/ml) for 10 to 90 min showed a progressive reduction of ristocetin-induced platelet agglutination and a progressive reduction of surface GP Ib. Surface expression of GP IIb/IIIa complex was significantly increased after plasmin exposure. The addition of PAs to PRP resulted in a marked reduction of ADP-induced platelet aggregation. Collagen-induced platelet aggregation was only slightly affected. Similar changes were observed when PRP was preincubated with high concentrations of FgDPs. In PRP treated with PAs platelet surface levels of GP Ib and GP IIb/IIIa complex did not show any significant changes. In conclusion our results show that in plasma no proteolysis of platelet adhesive receptors occurs after plasminogen activation. The platelet inhibition observed after incubation of PRP with PAs is likely to be caused by FgDPs generation.

In vitro effect of plasmin on human platelet function in plasma. Inhibition of aggregation caused by fibrinogenolysis.

Plasmin has been reported both to activate platelets and to inhibit platelet functions. The latter effect was thought to be caused by proteolysis of the main membrane glycoproteins. We found that incubation of citrated human platelet-rich plasma with streptokinase (SK) (300 IU/ml) does not produce any detectable activation but leads to a time-dependent inhibition of ADP-induced aggregation accompanied by substantial fibrinogenolysis. These effects were abrogated by previous addition of a plasmin inhibitor, aprotinin. Crossover experiments (SK-treated or control platelets mixed with SK-treated or control plasma) demonstrated that the platelets remained functional and that the aggregation defect was caused by fibrinogenolysis. Further experiments (addition of purified fibrinogen to fibrinogen-depleted plasma with either SK or thrombin) suggested that in addition to the low residual level of fibrinogen, fibrinogen degradation products had an inhibitory effect. Under the same conditions, tissue-type plasminogen activator (t-PA) (3,000 ng/ml) had no effect on platelet aggregation, and plasma fibrinogen was not significantly lowered. The effects on glycoproteins IIb-IIIa of incubation with SK, t-PA, or plasmin were assessed with immunoblots with murine monoclonal antibodies directed against either part of the complex, which is the receptor for fibrinogen. Proteolysis was detected only in the presence of EDTA, a potent chelator of divalent cations. The incubation of human platelets in citrated plasma with SK concentrations obtained during therapy leads to an aggregation defect that is related to the decrease in fibrinogen, the adhesive protein involved in this function, and to the impeding effect of fibrinogen degradation products on its binding onto platelets but not to an alteration of the corresponding platelet receptor, the heterodimer glycoproteins IIb-IIIa.

FDP D-Dimer Induces the Secretion of Interleukin-1, Urokinase-Type Plasminogen Activator, and Plasminogen Activator Inhibitor-2 in a Human Promonocytic Leukemia Cell Line

We studied the effect of fibrinogen degradation products D, E, and D-dimer on a human promonocytic leukemia cell line, NOMO-1. After exposure to a 10(-5)-mol/L fragment D or D-dimer, the cells displayed macrophage-like characteristics, such as adherence to plastic surfaces, and showed approximately a twofold increase in response to the nitroblue tetrazolium reduction test. The secretion of interleukin-1 alpha (IL-1 alpha) into the medium was markedly stimulated by a 10(-5)-mol/L fragment D, E, and D-dimer, whereas a significant increase in IL-1 beta secretion was observed only in D-dimer-stimulated cells. In addition, D-dimer induced a rapid increase in urokinase-type plasminogen activator on day 1 (0.52 +/- 0.02 ng/mL v 0.07 +/- 0.01 ng/mL in the control culture) and a slow increase in plasminogen activator inhibitor-2 on day 5 (3.9 +/- 1.6 ng/mL v 1.2 +/- 0.2 ng/mL in the control culture). An increase in tissue factor (TF) was also demonstrated on the cell surface of NOMO-1 cells exposed to fragment D or D-dimer by indirect immunofluorescence using an anti-TF monoclonal antibody. Scatchard plot analysis showed that fragment D and D-dimer bound to the NOMO-1 cells with a kd of 3.3 nmol/L and 2.7 nmol/L, respectively. These results suggest that fragment D-dimer specifically stimulates cells of monocyte-macrophage lineage to secrete key substances that regulate blood coagulation, fibrinolysis, and inflammation.

FDP D-dimer induces the secretion of interleukin-1, urokinase-type plasminogen activator, and plasminogen activator inhibitor-2 in a human promonocytic leukemia cell line

We studied the effect of fibrinogen degradation products D, E, and D- dimer on a human promonocytic leukemia cell line, NOMO-1. After exposure to a 10(-5)-mol/L fragment D or D-dimer, the cells displayed macrophage-like characteristics, such as adherence to plastic surfaces, and showed approximately a twofold increase in response to the nitroblue tetrazolium reduction test. The secretion of interleukin-1 alpha (IL-1 alpha) into the medium was markedly stimulated by a 10(-5)- mol/L fragment D, E, and D-dimer, whereas a significant increase in IL- 1 beta secretion was observed only in D-dimer-stimulated cells. In addition, D-dimer induced a rapid increase in urokinase-type plasminogen activator on day 1 (0.52 +/- 0.02 ng/mL v 0.07 +/- 0.01 ng/mL in the control culture) and a slow increase in plasminogen activator inhibitor-2 on day 5 (3.9 +/- 1.6 ng/mL v 1.2 +/- 0.2 ng/mL in the control culture). An increase in tissue factor (TF) was also demonstrated on the cell surface of NOMO-1 cells exposed to fragment D or D-dimer by indirect immunofluorescence using an anti-TF monoclonal antibody. Scatchard plot analysis showed that fragment D and D-dimer bound to the NOMO-1 cells with a kd of 3.3 nmol/L and 2.7 nmol/L, respectively. These results suggest that fragment D-dimer specifically stimulates cells of monocyte-macrophage lineage to secrete key substances that regulate blood coagulation, fibrinolysis, and inflammation.

Interaction of plasminogen and fibrin in plasminogen activation.

Glu1-, Lys77-, miniplasminogens, kringle 1-3, kringle 1-5A, and kringle 1-5R were able to bind with fibrin, while microplasminogen and kringle 4 did not bind significantly. Kringle 1-5A, but not kringle 1-3, effectively inhibited the binding of Glu1-, Lys77-, and miniplasminogens with fibrin. Miniplasminogen also inhibited the binding of Glu1-plasminogen with fibrin. The binding of kringle 1-3 with fibrin was blocked by mini- or Glu1-plasminogen. It is therefore evident that there are two fibrin-binding domains in plasminogen and that the one in kringle 5 is of higher affinity than that in kringle 1-3. CNBr cleavage products of fibrinogen effectively enhanced the activation of Glu1-, Lys77-, or miniplasminogens, but not microplasminogen, by tissue-type plasminogen activator. Kringle 1-5, but not kringle 1-3, dose-dependently inhibited the enhancement by fibrinogen degradation products of Glu1-plasminogen activation by the activator. Lysine and epsilon-aminocaproic acid could inhibit the binding of plasminogens and plasminogen derivatives with fibrin and block the enhancement effect of fibrinogen degradation products on plasminogen activation. The data clearly illustrate that the binding of plasminogen with fibrin, mainly determined by kringle 5, is essential for effective activation by tissue-type plasminogen activator. However, the presence of kringle 1-4 in the plasminogen molecule is required for the full enhancing effect since the kcat/Km of miniplasminogen activation in the presence of fibrinogen degradation products was 8.2 microM-1 min-1 which is significantly less than 52.0 microM-1 min-1 of Glu1-plasminogen.

Effect of Fibrinogen Degradation Products and Lung Ground Substance on Surfactant Function

Acute lung injury syndromes have many characteristics including protein-rich alveolar edema, hyaline membranes, and abnormal surface tension at the alveolar air-liquid interface. Increased surface tension can occur because of a relative surfactant deficiency and/or dysfunction. It has been previously demonstrated that surfactant dysfunction occurs when plasma protein inhibitors leak into the alveolar space during the induction of the lung injury and edema formation. The present study investigated whether inhibitors that would be generated during the stage of repair from lung injury could impair surfactant function. We determined whether fibrinogen degradation products (FDP) which would be released during lysis of the fibrin(ogen)-containing alveolar exudate and hyaline membranes, and components of the lungs' ground substance could inhibit the in vitro function of a lipid extract surfactant preparation. FDP were prepared by incubating human fibrinogen with plasmin or neutrophil elastase for 4 min to 60 h and were characterized by SDS-PAGE. Early (fragment X and Y) and late (fragment D and E) plasmin-derived FDP (MW greater than 40,000) inhibited surfactant function as assessed by a bubble surfactometer. The early elastase-derived FDP also inhibited surfactant, but the later and much smaller fragments (MW less than 15,000) did not affect surfactant function. Laminin also inhibited surfactant in a dose-dependent manner. Neither hyaluronic acid nor heparan sulfate affected surfactant performance in vitro. We conclude that plasmin-induced lysis of intraalveolar fibrinogen and hyaline membranes will result in prolonged generation (i.e. days) of surfactant inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of fibrinogen degradation products on production of hepatocyte stimulating factor by a macrophage cell line (P388D1)

Hepatocyte Stimulating Factor (HSF) is a monocyte/macrophage-derived regulatory protein which stimulates the hepatic synthesis of several plasma proteins. Plasminolytic peptides of fibrinogen or fibrin were tested for their ability to stimulate the secretion of HSF in an established macrophage cell line (P388D 1). One of the peptides, fragment D, induced the production of HSF in the cells in a dose-dependent manner, while intact fibrinogen did not. A hierarchy of potency was established for the major plasminolytic fragments which is as follows: D ≥ DD > X ≥ E. Fragment D had no effect on production of interleukin-1, a potent monokine released by P388D 1 cells. Taken together these results demonstrate that fragment D is a specific and potent inducer of HSF in P388D 1 cells.

Hemorrhage vs Rethrombosis After Thrombolysis for Acute Myocardial Infarction

To determine whether or not the timing of heparin infusion affects either bleeding or reocclusion following intracoronary streptokinase for acute myocardial infarction, heparin was infused immediately after streptokinase in 89 patients and was delayed for 12 hours in the subsequent 93. Bleeding occurred in 22 immediate-heparin patients and was major in five (one fatal); there were 14 hemorrhages in the delayed-heparin group, all minor. At discharge, reocclusions were observed in 18% (12/68) of immediate-heparin patients, and 11% (3/27) of the latter. Bioassayed fibrinogen levels displayed sustained depression regardless of bleeding for 20 hours after streptokinase; however, defibrinogenation measured by immunoassay was much less striking. This suggests that high levels of fibrinogen degradation products spuriously affect the bioassay of fibrinogen. We conclude that bleeding is related to the anticoagulant effects of fibrinogen degradation products interacting with heparin, and may be largely independent of hypofibrinogenemia. Delaying heparin for 12 hours may decrease the risk of bleeding while little affecting the risk of reocclusion.

Stimulation of fibrinogen synthesis in cultured rat hepatocytes by fibrinogen fragment E

Because of the inherent difficulties of experimentation in intact animals, we used primary monolayer cultures of non-proliferating adult rat hepatocytes to study the effects of fibrinogen degradation products on fibrinogen biosynthesis. The freshly isolated hepatocytes obtained by collagenase perfusion of the liver in situ were cultured in a chemically defined serum-free medium. The rate of fibrinogen synthesis in control cultures was 40–50 pmol 2.5·10 6 cells per 24 h. Additions of 20, 60 or 100 μg of homologous stage I fibrinogen degradation products had no effect on fibrinogen synthesis. In contrast, addition of the same amounts of homologous or heterologous (human) stage III fibrinogen degradation products resulted in a concentration-dependent increase in fibrinogen biosynthesis without affecting the rate of synthesis of albumin. When purified stage III fibrinogen degradation products D and E (human) were tested in 10, 30 or 50 μg/3 ml medium only fragment E showed a significant increase in fibrinogen biosynthesis (1.9-, 2.8- and 5.6-fold, respectively, over the control cultures). The presence of excess fibrinogen had no effect. These results suggest that fibrinogen fragment E may be a specific stimulator of fibrinogen biosynthesis which may play an important role in maintaining normal levels of plasma fibrinogen.

Effects of fibrinogen degradation products on glomerular mesangial cells in culture

We investigated effects of fibrin, fibrinogen, and fibrinogen degradation products (FDPs) on human glomerular mesangial cells in culture, using the methods of cell count, 3[H] thymidine uptake and 51Cr release. Incorporation of 3[H] thymidine by the cells was much the same with various concentrations of fibrin and similar to findings in the control without fibrin. Fibrinogen, FDP-D, -E, low molecular weight FDP (LMWFDP) fractions 1 and 3 had no promoting effects on mesangial cell proliferation. The LMWFDP fraction 2 showed a suppressive effect on the proliferation of cultured cells and increase of 51Cr release from the cells. The cytotoxic effect of this fraction was dose-dependent. These results suggest that while fibrin deposition in the renal glomeruli does not relate with mesangial hypercellularity, this deposition plays a role in mesangiolysis, through the production of FDPs.

Hemorrhage and the products of fibrinogen digestion after intracoronary administration of streptokinase.

Hemorrhage was prospectively identified in 26 of 116 consecutive patients (23%) who were receiving intracoronary streptokinase for occlusive coronary thrombi producing infarction. Bleeding was not influenced by the dose of streptokinase or the method of cardiac catheterization. Before treatment, prothrombin time and partial thromboplastin time were normal in both bleeders and nonbleeders. Fibrinogen levels measured by bioassay after streptokinase (mean +/- SEM) were 62 +/- 29 mg/dl in patients with major bleeding, 111 +/- 26 mg/dl in patients with minor bleeding, and 109 +/- 13 mg/dl in nonbleeders (p = NS). The regression slope b calculated from poststreptokinase fibrinogen time-concentration data in 71 patients was 4.7 mg/dl/hr. However, mean fibrinogen concentrations calculated at sequential 5 hr intervals revealed no net regeneration for the first 20 hr after thrombolysis. The apparent fibrinogen regeneration rate was less than normal (31 mg/kg/day) for more than 10 hr but subsequently increased to 94 +/- 10 mg/kg/day by the second day. The initial apparent latency of fibrinogen regeneration paralleled the sharp rise in fibrinogen degradation products, which began to decline after 20 hr of treatment but remained elevated well into the second day. Because of their anticoagulant effects, these products may interfere with the fibrinogen assay, causing spuriously low results. Thus, whether the early delay in fibrinogen regeneration is real or simply a reflection of the effects of fibrinogen degradation products on the bioassay, it signals the time for caution in initiating systemic heparin therapy.

The effect of fibrinogen degradation products and some lysine analogues on the dissociation of plasmin(ogen) - fibrin complexes

Fibrin carries sites which bind plasmin and preactivated plasminogen (mol.wt. 84,000) allowing the formation of stable complexes. Fibrinogen degradation product E interferes with this complex formation suggesting that the plasmin(ogen) binding site is present in this fragment. The complexes are also dissociated by the anti-fibrinolytic agents 6-aminohexanoic acid and aminomethyl cyclohexanoic acid which both compete for the fibrin binding site of plasmin and plasminogen.

Methods for correcting inhibitory effects of fibrinogen degradation products in fibrinogen determinations

To assess the influence of fibrinogen degradation products (FDP) on fibrinogen determinations, Clauss and Ratnoff-Menzie assays were performed with different concentrations of human plasma and FDP. When varied concentrations of plasma were mixed with an equal volume of FDP at a final titer of 1:512 (tanned red cell assay), fibrinogen levels were decreased by approximately 30% in the Ratnoff assay. Inhibition was overcome by clotting the fibrinogen in a smaller volume of saline. Inhibition was also corrected by clotting more concentrated mixtures of plasma and FDP with thrombin. The influence of FDP in fibrinogen assays is significant when low levels of fibrinogen are measured.

The sulphite precipitation method for fibrinogen measurement; Its use on small samples in the presence of fibrinogen degradation products

A technique based on precipitation by sodium sulphite (Na 2SO 3) has been developed for measuring the fibrinogen concentration of small (100 μ1) samples. This technique offers technical advantages over the throm bin-clotting method, especially when measuring dilute solutions. Measurements made with the sulphite technique on fibrinogen solutions and on plasma samples were not significantly different from those obtained with the thrombin-clotting method. The effect of fibrinogen degradation products (FDP) on the two methods was investigated. Sulphite was found to precipitate fibrinogen fragments X and Y in conjunction with fibrinogen, but not D or E; thrombin clotted some forms of fragment X and fibrinogen but its action was inhibited in the presence of substantial amounts of Y, D or E. Neither technique was very sensitive to the presence of FDP in plasma, concentrations of at least 15% of that of the fibrinogen being required in order to affect the measurement of fibrinogen. The most prevalent FDP in plasma, namely fragments X and Y, are measured together with fibrinogen by the sulphite method, their effect on the thrombin-clottable fibrinogen is difficult to predict. Thus, the sulphite method could prove useful for measuring fibrinogen in plasma samples containing high levels of FDP.

Effect of fibrinogen degradation products /FDP/ on the action of certain centrally acting drugs

The effect of FDP upon the action of pethidine, amphetamine, caffeine, cardiazol and chlorpromazine in rats was investigated. FDP were derived from in vitro trypsin-digested fibrinogen for 3 min and 120 min. The products of degradation given together with the drugs increased their potency by enhancing their level in the brain tissue. Noteworthy is the observation that FDP were more effective after 120 min digestion, thus peptides of small molecular weight. A possible mechanism of these changes is discussed.

The Effect of Fibrinogen Degradation Products on Plasmin Activity

SummaryPurified fibrinogen degradation products Y, D or E were not found to exert antiplasmin activity as measured by the clot lysis time.Spontaneous inactivation of plasmin was observed during incubation at 37° C. This inactivation was partly prevented by the presence of fibrinogen degradation products, as well as by other proteins.