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PubMed1969

Congenital dysfibrinogenemia: fibrinogen Detroit.

A 17 yr old female with a congenital bleeding disorder was found to suffer from dysfibrinogenemia. Whole blood and plasma coagulation times were delayed and thrombelastograms were grossly abnormal. Clottability of plasma fibrinogen by addition of thrombin was not demonstrated during the 30 min test period. Fibrinogen was revealed by turbidometric and immunologic techniques. Other coagulation factors were present in normal amounts and prothrombin activation was normal. Patient's plasma inhibited thrombin clotting times of normal plasma and purified normal fibrinogen. Fibrinolysis was not detected. The plasma fibrinogen migrated normally on paper and cellulose acetate electrophoresis, but on immunoelectrophoresis it displayed a faster mobility than normal fibrinogen. On immunodiffusion the antigenic determinants were similar to those of normal fibrinogen. The patient's fibrinogen-antifibrinogen precipitins required longer to appear and the resultant precipitin was broader and hazier than those elicited with normal fibrinogen. These findings suggest the presence of two discrete populations of fibrinogen molecules. Investigation of the family of the patient suggested that the defect has an autosomal dominant pattern of heredity. Immunologic comparisons of our patient's plasma and of her relatives with plasma of patients with "Fibrinogen Baltimore" and "Fibrinogen Cleveland" revealed certain differences in immunoelectrophoretic mobility as well as in immunodiffusion. In keeping with the nomenclatures of abnormal fibrinogens in the literature, we propose the term "Fibrinogen Detroit" for this fibrinogen.Physicochemical properties of "Fibrinogen Detroit" were investigated also and compared with those of normal fibrinogen. Purified normal fibrinogen (clottability 96.7%) and "Fibrinogen Detroit" revealed homogeneity when studied by ultracentrifugation and immunoelectrophoresis. Native and cleaved "Fibrinogen Detroit" had the same sedimentation constants and molecular weights as the normal. In fresh samples. 3 moles of free SH groups/mole of fibrinogen were titrated in both. Determination of the amino acid composition revealed a decreased content of lysine, glucosamine, and galactosamine in abnormal fibrinogen. Total carbohydrates, protein-bound hexoses, sialic acid, and hexosamine were decreased in the abnormal fibrinogen. In an investigation with Doctors Blombäck a specific molecular defect was revealed in the N-terminal disulfide knot of the alpha (A) chain in which the arginine at the 19th position was replaced by serine. It is believed that the substitution of a strongly basic amino acid with a neutral hydroxy acid may result in considerable conformational changes in the N-terminal disulfide knot of fibrinogen which might affect the "active site" for polymerization. The lower carbohydrate content observed in "Fibrinogen Detroit" may have been the result of a change in primary and tertiary structure of the protein.

Adolescent↗
PubMed2003

High plasma fibrinogen level is associated with poor clinical outcome in DIC patients.

We measured the plasma level of fibrinogen in 560 patients with disseminated intravascular coagulation (DIC) and evaluated its relationship with outcome and with other hemostatic markers. Forty-seven percent of patients had >200 mg/dL of plasma fibrinogen and 24% had <100 mg/dl of plasma fibrinogen, suggesting that plasma fibrinogen level is not a sensitive marker for DIC. In our analysis of outcome and plasma fibrinogen levels, the rate of death was high in leukemia/lymphoma patients with high fibrinogen concentration, but no significant difference in outcome was observed in relation to plasma fibrinogen concentration in non-leukemia/lymphoma patients with DIC. Among patients with leukemia/lymphoma, the frequency of organ failure was markedly high in patients with high plasma levels of fibrinogen. Among patients without leukemia/lymphoma, the frequency of organ failure increased concomitantly with the increase in plasma fibrinogen levels. The international normalized ratio was significantly increased in leukemia/lymphoma patients with low fibrinogen. FDP levels were slightly increased in patients with low fibrinogen. Platelet count was significantly low in patients without leukemia/lymphoma with high fibrinogen. DIC score increased concomitantly with the reduction in plasma fibrinogen levels. Plasma levels of thrombomodulin and tissue factor were significantly high in patients with high fibrinogen levels. Plasma levels of antiplasmin and plasminogen were significantly decreased in patients with low fibrinogen. Plasma levels of plasmin plasmin-inhibitor complex and tissue type plasminogen activator/plasminogen activator inhibitor-1 complex (PAI-I) were significantly higher in patients with low fibrinogen than in those with high fibrinogen. Plasma levels of PAI-I and IL-6 were significantly higher in patients with high fibrinogen than in those with low fibrinogen. Patients with high fibrinogen levels showed less activation of secondary fibrinolysis, which might explain the occurrence of organ failure and poor outcome.

Adult↗
PubMed1967

Studies of the metabolism and distribution of fibrinogen in patients with hemophilia A.

Using autologous (131)I-fibrinogen, we made studies of the metabolism and distribution of fibrinogen in 10 patients with hemophilia A. In two patients simultaneous studies of autologous (131)I-fibrinogen and homologous (125)I-fibrinogen prepared from healthy donors' plasma were carried out. The average value for the plasma volume was 42.1 +/- 8.8 ml/kg; for the plasma fibrinogen concentration, 349 +/- 90 mg/100 ml; for the intravascular fibrinogen, 144 +/- 32 mg/kg; for the interstitial fibrinogen, 30 +/- 11 mg/kg; for the slower half-life of (131)I-fibrinogen, 2.34 +/- 0.17 days; for the transcapillary transfer rate of fibrinogen, 109 +/- 37 mg/kg per day; and for the catabolic and synthetic rates of fibrinogen, 51.7 +/- 13.1 mg/kg per day. Comparison of these results with those of the previous study in healthy male subjects showed that in patients with hemophilia A the catabolic and synthetic rates of fibrinogen are markedly increased, whereas the plasma fibrinogen concentration, intravascular and interstitial fibrinogen, and the transcapillary transfer rate of fibrinogen are not significantly different. The simultaneous studies of autologous (131)I-fibrinogen and normal homologous (125)I-fibrinogen in two subjects revealed that the two preparations behaved very similarly. Based on these findings, we concluded that our present findings are not due to the qualitative difference between the hemophilia A and normal fibrinogens, but that they are due to the difference in the host condition with respect to the fibrinogen metabolism, which is either an increased rate of direct breakdown of fibrinogen or an increased rate of fibrinogen breakdown after fibrin formation, or both.

Adolescent↗
PubMed1989

High molecular weight kininogen inhibits fibrinogen binding to cytoadhesins of neutrophils and platelets.

Fibrinogen inhibited 125I-high molecular weight kininogen (HMWK) binding and displaced bound 125I-HMWK from neutrophils. Studies were performed to determine whether fibrinogen could bind to human neutrophils and to describe the HMWK-fibrinogen interaction on cellular surfaces. At 4 degrees C, the binding of 125I-fibrinogen to neutrophils reached a plateau by 30 min and did not decrease. At 23 and 37 degrees C, the amount of 125I-fibrinogen bound peaked by 4 min and then decreased over time because of proteolysis of fibrinogen by human neutrophil elastase (HNE). Zn++ (50 microM) was required for binding of 125I-fibrinogen to neutrophils at 4 degrees C and the addition of Ca++ (2 mM) increased the binding twofold. Excess unlabeled fibrinogen or HMWK completely inhibited binding of 125I-fibrinogen. Fibronectin degradation products (FNDP) partially inhibited binding, but prekallikrein and factor XII did not. The binding of 125I-fibrinogen at 4 degrees C was reversible with a 50-fold molar excess of fibrinogen or HMWK. Binding of 125I-fibrinogen, at a concentration range of 5-200 micrograms/ml of added radioligand, was saturable with an apparent Kd of 0.17 microM and 140,000 sites/cell. The binding of 125I-fibrinogen to neutrophils was not inhibited by the peptide RGDS derived from the alpha chain of fibrinogen or by the mAb 10E5 to the platelet glycoprotein IIb/IIIa heterodimer. Fibrinogen binding was inhibited by a gamma-chain peptide CYGHHLGGAKQAGDV and by mAb OKM1 but was not inhibited by OKM10, an mAb to a different domain of the adhesion glycoprotein Mac-1 (complement receptor type 3 [CR3]). HMWK binding to neutrophils was not inhibited by OKM1. These observations were consistent with a further finding that fibrinogen is a noncompetitive inhibitor of 125I-HMWK binding to neutrophils. Fibrinogen binding to ADP-stimulated platelets was increased twofold by Zn++ (50 microM) and was inhibited by HMWK. These studies indicate that fibrinogen specifically binds to the C3R receptor on the neutrophil surface through the carboxy terminal of the gamma-chain and that HMWK interferes with the binding of fibrinogen to integrins on both neutrophils and activated platelets.

Antibodies, Monoclonal↗
PubMed1996

Modulation of plasma fibrinogen levels by ticlopidine in healthy volunteers and patients with stable angina pectoris.

Elevated plasma fibrinogen levels are associated with an increased risk for cardiac events. Ticlopidine is a drug that inhibits the ADP-induced aggregation of blood platelets and it also has been described that ticlopidine can decrease the plasma fibrinogen level in patients with vascular diseases. The mechanism of this decrease has not yet been elucidated and therefore mechanisms that are known to affect fibrinogen levels were studied, viz, the acute phase reaction, total fibrin plus fibrinogen degradation (TDP) levels and the polymorphisms of the fibrinogen beta-gene. The fibrinogen lowering effect of ticlopidine was studied in 26 healthy volunteers, selected on genotype of the Bcl] polymorphism of the fibrinogen beta-gene, and in 26 patients with stable angina pectoris in a double blind, randomized cross-over study. Functional plasma fibrinogen levels were measured with the Clauss assay. Fibrinogen antigen, C-reactive protein (CRP) and TDP levels were measured using an enzyme immuno assay (EIA). In the healthy volunteers the functional fibrinogen levels had decreased by 0.20 g/l (9%, p = 0.005 using the paired Student l-test) after 4 weeks of 250 mg bid ticlopidine administration, whereas fibrinogen antigen, CRP and TDP levels were not significantly changed. In the stable angina pectoris patients the pre-treatment fibrinogen, CRP and TDP levels were significantly higher than in the volunteer group. After four weeks 250 mg bid ticlopidine administration the functional fibrinogen levels had decreased by 0.38 g/l (11%, p < 0.005), whereas the fibrinogen antigen, CRP and TDP levels were not significantly changed. The levels of functional and antigen fibrinogen, CRP and TDP did not change significantly during the placebo period in the volunteers or the patients. Neither in the volunteers nor in the patients was the effect of ticlopidine on the fibrinogen levels associated with the fibrinogen beta-gene polymorphisms. Therefore, the fibrinogen lowering effect of ticlopidine is likely to be a modulation of the functionality of the molecule and unlikely to be modulated by the acute phase reaction, TDP-levels or the fibrinogen beta-gene polymorphisms.

Adult↗
PubMed1993

Influence of acute myocardial infarction and rt-PA therapy on circulating fibrinogen.

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)

Aged↗
PubMed1979

Reduced fibrinogen survival in diabetes mellitus. A reversible phenomenon.

Fibrinogen survival and turnover were examined in 15 adult-onset diabetic patients. (125)I-labeled fibrinogen was prepared from each patient during the period of poor carbohydrate control, or hyperglycemic period, and fibrinogen survival determined. Improved control was established in each patient and during this euglycemic period, fibrinogen survival was determined simultaneously with (125)I-fibrinogen saved from the hyperglycemic period and (131)I-labeled fibrinogen prepared from the patient during the euglycemic period. The results confirm reduced fibrinogen survival in hyperglycemic diabetic patients and demonstrate reversal of the fibrinogen abnormality when euglycemia is achieved. The results of the double-label experiments in the euglycemic period suggest that the fibrinogen molecule is not altered functionally and that an abnormal plasma or vascular environment is a more likely basis for reduced fibrinogen survival during hyperglycemia. Electrophoretic and chromatographic experiments demonstrated no gross chemical differences between the fibrinogens prepared from the hyperglycemic and euglycemic periods and normal fibrinogen. Fibrinogen survival gave a better correlation with serial glucose measurements than with correction of hemoglobin A(Ic) levels indicating that the reduced fibrinogen survival noted in diabetics is a rapidly reversible phenomenon. During the hyperglycemic period, pharmacological intervention with aspirin and dipyrimadole was attempted to examine the role of platelets in reduced fibrinogen survival. No significant change in fibrinogen survival was observed. Heparin infusion during hyperglycemia normalized the fibrinogen kinetics of hyperglycemic diabetic patients, suggesting that reduced fibrinogen survival during hyperglycemia is secondary to an effect on thrombin or one of its antagonists.

Adult↗
PubMed1991

Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton.

Previous studies indicated a correlation between the formation of EDTA-resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 (Sigma Chemical Co, St Louis, MO) insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA-resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA-resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding. Examination of platelet cytoskeletons using monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting showed the presence of only traces of GP IIb-IIIa in the cytoskeletons of resting platelets, with no detectable increases after platelet activation or development of EDTA-resistant fibrinogen binding. These data suggest that GP IIb-IIIa-mediated fibrinogen binding to activated platelets is accompanied by time-dependent alterations in platelet-fibrinogen interactions leading to the GP IIb-IIIa independent association between bound fibrinogen and the platelet cytoskeleton.

Antibodies, Monoclonal↗
PubMed2005

Influence of HLA-DR genes on the production of rheumatoid arthritis-specific autoantibodies to citrullinated fibrinogen.

OBJECTIVE: Antibodies directed against citrullinated fibrinogen are highly specific for rheumatoid arthritis (RA). This study was undertaken to test whether RA-associated HLA-DR alleles are associated with anti-citrullinated fibrinogen in RA patient sera and whether replacement of arginyl by citrullyl residues on fibrinogen peptides modifies their binding to HLA-DR molecules and their recognition by T cells. METHODS: Antikeratin, antifilaggrin, and anti-citrullinated fibrinogen antibodies were assayed in RA patients who had undergone HLA-DR typing. Direct assays were performed to investigate binding of citrullinated or native fibrinogen peptides (encompassing the entire alpha- and beta-chains of fibrinogen) to purified HLA-DR molecules. T cell proliferative responses to citrullinated or native fibrinogen peptides were measured in RA patients and controls. RESULTS: HLA-DRB1*0404 was associated with anti-citrullinated fibrinogen in RA sera (P = 0.002). For the RA-associated alleles HLA-DRB1*0401 and HLA-DR1, there was a nonsignificant trend toward association (P = 0.07). Multiple peptides from the alpha- and beta-chains of fibrinogen bound many HLA-DR alleles; DRB1*0404 was the best fibrinogen peptide binder. Citrullination did not influence fibrinogen peptide binding to HLA-DR or fibrinogen peptide recognition by T cells. Peripheral blood T cells that recognized native or citrullinated fibrinogen peptides were common in RA patients but not in healthy controls. CONCLUSION: The RA-associated HLA-DRB1*0404 allele is also associated with production of antibodies to citrullinated fibrinogen. DRB1*0401 and DRB1*01 tend to be associated with anti-citrullinated fibrinogen, but this is not statistically significant. Citrullination of fibrinogen peptide does not influence peptide-DR-T cell interaction. Finally, T cell proliferation in response to citrullinated or uncitrullinated fibrinogen peptides is frequent in RA patients and very infrequent in controls.

Alleles↗
PubMed1998

Prothrombotic Consequences of the Oxidation of Fibrinogen and their Inhibition by Aspirin.

Oxidant stress leads to covalent oxidative modification of several plasma proteins, chief among which is fibrinogen. Aspirin can nonenzymatically acetylate fibrinogen's lysine residues, the functional groups most susceptible to oxidative modification. Because oxidation of fibrinogen may occur in the atheromatous environment, we studied the effects of oxidative modification on fibrinogen function and the consequences of acetylation by aspirin on fibrinogen's susceptibility to oxidation and functional properties. We exposed fibrinogen to Fe(3+) ascorbate for 1 hour and showed that the carbonyl/protein molar ratio increased from 0.71 +/- 0.18 to 2.86 +/- 0.50 mol carbonyl/mol protein (P < 0.02) with an accompanying reduction in the alpha-helical content of the protein from 34% to 29%. Exposure of fibrinogen to aspirin led to acetylation of lysine residues and inhibition of oxidation. Oxidized fibrinogen was more readily able to form fibrin, and acetylation prevented this enhancement of clot formation. Oxidized fibrinogen also supported platelet aggregation better than did native, unoxidized fibri ogen, and aretylation of fibrinogen prior to oxidation prevented the enhanced platelet aggregation. Oxidized fibrinogen was less effective in stimulating plasminogen activation by tissue-type plasminogen activator (tPA), with a catalytic efficiency that was reduced by 88% compared with native, unoxislized fibrinogen; metylated fibrinogen, by contrast, enhanced plasminogen activation by t-PA with a catalytic efficiency that was increased by 18% compared with native, anoxidized fibrinogen (P < 0.05) and was increased by 51% compared with oxidized fibrinogen (P < 0.05). Acetylation prevented the reduction in catalytic efficiency induced by oxidation. These data show that oxidized fibrinogen manifests prothrombotic effects that can be prevented by acetylation and suggest that inhibition of fibrinogen oxidation may be an additional antithrombotic benefit of aspirin therapy.

Journal Article↗
PubMed1982

Evidence for interaction between platelet fibrinogen receptors.

Previous analysis of fibrinogen binding to human aspirin-treated gel-filtered platelets yielded upwardly concave Scatchard plots. To ascertain whether this was due to the presence of independent heterogeneous receptor populations binding fibrinogen with different affinities, the dissociation of purified 125I-fibrinogen from ADP-treated gel-filtered platelets was evaluated as a function of receptor occupancy. Dissociation of bound labeled fibrinogen was measured after 50-fold dilution with buffer containing O, 0.2, 0.8, and 2.0 mg/ml unlabeled fibrinogen. Dissociation of labeled fibrinogen increased with increasing receptor occupancy and was biphasic. With buffer alone, 76.0% +/- 5.8% (SD) of labeled fibrinogen dissociated in 30 min, with an initial rate of 0.392 +/- 0.175 min-1; with 0.2 mg/ml fibrinogen, 83.7% +/- 3.9% dissociated, with an initial rate of 0.589 +/- 0.044 min-1; with 0.8 mg/ml, 91.8% +/- 1.3% of the labeled fibrinogen dissociated, with an initial rate of 0.910 +/- 0.028 min-1; and with 2.0 mg/ml fibrinogen, 97.3% +/- 2.3% of label dissociated, with an initial rate of 1.06 +/- 0.257 min-1 (n=5). The final rates of fibrinogen dissociation were unaffected by unlabeled fibrinogen in the dilution buffer and were not statistically different from the final dissociation rate of 0.015 +/- 0.10 min-1 observed following dilution with buffer alone. These results were neither an artifact of aspirin treatment or gel filtration, as similar observations were made using non-aspirin-treated washed platelets, nor were they an artifact of the purified fibrinogen preparations, because binding studies using whole plasma as the major source of fibrinogen also yielded upwardly concave Scatchard plots. Since the data demonstrate that the initial rate and extent of fibrinogen dissociation are dependent on fibrinogen receptor occupancy, they suggest receptor interactions possibly resulting from receptor clustering or crosslinking. Because the dissociation was biphasic, the results also suggest some heterogeneity among platelet-fibrinogen interactions.

Aspirin↗
PubMed1983

Reevaluation of some properties of fibrinogen, purified from cord blood of normal newborns.

In this study we compared some properties of fibrinogens, obtained from normal adult and cord plasma. Fibrinogen preparations were made under conditions, which minimize proteolytic breakdown in vitro. We were not able to demonstrate any significant differences between both purified fibrinogens as to the effects of pH and ionic strength on its clotting properties, the Km for thrombin, SDS polyacrylamide gelelectrophoresis behaviour or carbohydrate content. However, the phosphorus content of cord fibrinogen was 3-4 times higher than that of adult fibrinogen. The accelerating effect of calcium on the thrombin clotting time was more pronounced for newborn cord plasma and for purified cord fibrinogen preparations as compared with adult fibrinogen. This might be explained by the higher phosphorus content of the cord fibrinogen molecule. The thrombin clotting time of both purified adult and cord fibrinogen was markedly prolonged, when increasing amounts of fibrinogen degradation product fragment X were added to the fibrinogen solutions under conditions with high pH or high ionic strength. At high pH the effect of adding fragment X was more pronounced in cord fibrinogen preparations. Therefore, mixtures of purified fibrinogen and fragment X have several properties in common with fetal fibrinogen. These observations show, that some of the properties that have been attributed in the literature to a distinct fetal fibrinogen can be caused by the presence of fragment X in the cord fibrinogen preparations.

Adult↗
PubMed1989

Decreased accessibility of platelet-bound fibrinogen to antibody and enzyme probes.

The binding of fibrinogen to platelets is a multiphasic process leading to apparently nonreversible associations between fibrinogen and stimulated platelets. To further investigate changes in platelet-fibrinogen interactions, the present study examined the accessibility of platelet-bound fibrinogen and its GPIIb-IIIa receptor to antibody and enzyme probes as a function of time after platelet stimulation with adenosine diphosphate (ADP). Whereas only minimal changes in fibrinogen and 10E5 binding were observed within 60 minutes after platelet stimulation and equilibrium fibrinogen binding, the binding of polyclonal antifibrinogen antibodies decreased significantly (75% +/- 13%, mean +/- SD, n = 9). Similar decreases were noted with rabbit antifibrinogen Fab and F(ab')2 fragments. In addition, plasmin (32 mU/mL) added to platelets five minutes compared with 60 minutes after equilibrium fibrinogen binding dissociated 52% +/- 12% compared with 33% +/- 7% of platelet-bound fibrinogen in five minutes, and 83% +/- 15% compared with 66% +/- 14% of bound fibrinogen in 15 minutes. No difference in plasmin cleavage products was observed, however, by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE). Complete fibrinogen dissociation occurred 30 minutes after plasmin addition, confirming that fibrinogen was not internalized. In contrast, dissociation of platelet-bound fibrinogen by chymotrypsin was less affected by time after equilibrium fibrinogen binding, and minimal changes in antifibrinogen antibody recognition and plasmin-induced dissociation of fibrinogen bound to stimulated but glutaraldehyde-fixed platelets were observed. The data suggest that ADP-induced fibrinogen binding to fresh platelets is accompanied by progressive rearrangements of fibrinogen on the platelet surface.

Antibodies↗
PubMed1995

Platelet and monoclonal antibody binding to fibrinogen adsorbed on glow-discharge-deposited polymers.

The state of fibrinogen adsorbed on untreated and glow-discharge-treated surfaces was examined by measuring platelet adhesion, monoclonal antibody (mAb) binding, the amount of fibrinogen adsorbed, and the amount of adsorbed fibrinogen which could be eluted with sodium dodecyl sulfate (SDS). Tetrafluoroethylene (TFE) glow-discharge-treated polymers have a lower surface free energy (in air) and retain a larger fraction of adsorbed fibrinogen than untreated surfaces after SDS elution. Platelet adhesion was lowest on the TFE-treated surfaces which retain the highest amounts of fibrinogen after SDS elution. Fibrinogen may undergo unfolding or spreading on the TFE-treated surfaces to minimize interfacial free energy (in water) and maximize protein-surface interactions. When it is adsorbed on the TFE-treated surfaces, fibrinogen evidently assumes a state which somehow prevents its recognition and binding by platelet receptors. Monoclonal antibodies that bind to the three regions in fibrinogen thought to be involved in platelet adhesion were therefore used to detect changes in adsorbed fibrinogen. These regions and the antibodies which bind to them are: the COOH-terminal of the gamma-chain, mAb M1; the RGD peptide sequence at A alpha 95-98, mAb R1; the RGD sequence at A alpha 572-575, mAb R2. For fibrinogen adsorbed on the untreated or TFE-treated surfaces, M1 and R2 binding was relatively high compared to background, while R1 binding was low. However, the amount of binding of each mAb to fibrinogen adsorbed on the TFE-treated surfaces was equal to or greater than fibrinogen adsorbed to the untreated surfaces. Therefore, antibody-detectable changes in the platelet binding regions of adsorbed fibrinogen that might have been caused by conformational or orientational rearrangements were not observed for the TFE-treated surfaces. The data suggest that the tight binding of fibrinogen on a surface may directly affect the ability of the fibrinogen to interact with the platelet receptors--i.e., that fibrinogen must be loosely held to facilitate maximal interaction with platelet receptors.

Adhesiveness↗
PubMed1984

Metabolism of fibrinogen in children with acute lymphoblastic leukaemia.

The metabolism and in vivo kinetics of fibrinogen were studied using homologous 125I-labelled fibrinogen in 21 children with acute lymphoblastic leukaemia (ALL). Ten patients were undergoing induction therapy, 11 children were in complete remission on maintenance therapy. Results in the patients undergoing induction therapy were: plasma fibrinogen levels were normal in all except one patient, the plasma fibrinogen pool was elevated in six cases, seven patients had a shortened fibrinogen half-life and increased fractional catabolic rate for fibrinogen. The absolute catabolic rate for fibrinogen was elevated in six cases. This shortened fibrinogen half-life together with the correcting effect of heparinisation on the fibrinogen turnover indicated that fibrinogen was consumed by chronic disseminated intravascular coagulation. Inhibition of the fibrinolytic system with epsilon-aminocaproic acid in five patients had no influence on the fibrinogen half-life in three of them but resulted in its prolongation in two patients. All except two children in complete remission had normal fibrinogen levels. Six patients had elevated plasma fibrinogen pools and in all of the cases survival and fractional catabolic rate of fibrinogen were normal. The absolute catabolic rate for fibrinogen was normal in eight, elevated in three of the patients. This observation indicates that fibrinogen synthesis remains accelerated in some cases of ALL in complete remission, but the cause of this is not known.

Adolescent↗
PubMedDate not supplied

Effect of shear stress on fibrinogen adsorption and its conformational change.

The composition and molecular organization of adsorbed protein films are strongly correlated with thrombogenesis on artificial surfaces. In particular, the antibody-detectable (that is, conformationally intact) bound fibrinogen, but not that the total amount of adsorbed fibrinogen, is correlated with platelet reactivity. In this work, the authors quantified the adsorbed plasma protein distribution inside the left ventricular assist device. They also evaluated the effect of wall shear stress on protein adsorption and conformational change of adsorbed fibrinogen. Conformational change of adsorbed fibrinogen was measured by exposing the fibrinogen preadsorbed polyurethane to three anti-fibrinogen monoclonal antibodies; the 134B-29 detectable alpha 566-580 domain of fibrinogen was increased with increasing concentration of adsorbed fibrinogen, whereas the other two fibrinogen domains were almost saturated when increasing the concentration of adsorbed fibrinogen. The adsorbed amounts of total fibrinogen and monoclonal antibody detectable fibrinogen was decreased with increasing shear rate. Results of in vivo plasma protein adsorption on polyurethane surfaces disclosed that the adsorbed amount of fibrinogen, as well as albumin and globulin, was also decreased with increasing shear rate. In conclusion, less protein was adsorbed in the higher shear region and the effect of shear level on fibrinogen adsorption and its conformational change was strongly dependent upon the surface characteristics of the biomaterials. The monoclonal antibody 134B-29 against the 566-580 domain of fibrinogen was the most reactive with the fibrinogen adsorbed on polyurethane surfaces in this experiment.

Adsorption↗
PubMed1990

Bacteroides gingivalis and Bacteroides intermedius recognize different sites on human fibrinogen.

Bacteroides (Porphyromonas) gingivalis and Bacteroides (Porphyromonas) intermedius have been implicated in the etiology of human periodontal diseases. These organisms are able to bind and degrade human fibrinogen, and these interactions may play a role in the pathogenesis of periodontal disease. In attempts to map the bacterial binding sites along the fibrinogen molecule, we have found that strains of B. gingivalis and B. intermedius, respectively, recognize spatially distant and distinct sites on the fibrinogen molecule. Isolated reduced and alkylated alpha-, beta-, and gamma-fibrinogen chains inhibited binding of 125I-fibrinogen to both Bacteroides species in a concentration-dependent manner. Plasmin fragments D and to some extent fragment E, however, produced a concentration-dependent inhibition of 125I-fibrinogen binding to B. intermedius strains but did not affect binding of 125I-fibrinogen to B. gingivalis strains. Radiolabeled fibrinogen chains and fragments were compared with 125I-fibrinogen with respect to specificity and reversibility of binding to bacteria. According to these criteria, gamma chain most closely resembled the native fibrinogen molecule in behavior toward B. gingivalis strains and fragments D most closely resembled fibrinogen in behavior toward B. intermedius strains. The ability of anti-human fibrinogen immunoglobulin G (IgG) to inhibit binding of 125I-fibrinogen to B. intermedius strains was greatly reduced by absorbing the IgG with fragments D. Absorbing the IgG with fragments D had no effect on the ability of the antibody to inhibit binding of 125I-fibrinogen to B. gingivalis strains. A purified staphylococcal fibrinogen-binding protein blocked binding of 125I-fibrinogen to B. intermedius strains but not to B. gingivalis strains.

Bacteroides↗
PubMed1969

Chromatographic, ultracentrifugal, and related studies of fibrinogen "Baltimore".

Chromatographic, ultracentrifugal, and related studies of the fibrinogen of a patient with a congenital disorder of fibrinogen (fibrinogen "Baltimore" have provided evidence of structural differences from normal.Diethylaminoethyl-cellulose (DEAE-cellulose) gradient elution chromatography demonstrated two major peaks in the elution pattern of fibrinogen Baltimore as was the case for normal fibrinogen. However, the first peak of fibrinogen Baltimore was somewhat broader and more symmetrical and was eluted significantly later in the chromatogram than the corresponding peak of normal fibrinogen. Additionally, in some elution patterns, a shoulder on the ascending limb of peak 1 was present, suggesting the presence of chromatographically "normal" fibrinogen. Thrombin time determinations of eluted column fractions from a chromatogram of propositus fibrinogen supported this conclusion by demonstrating that fibrinogen from the ascending portion of peak 1 behaved functionally more like normal than that later in the chromatogram. Chromatograms of mixtures of propositus and normal fibrinogen confirmed the ability of this technique to distinguish normal from Baltimore fibrinogen. Chromatograms of fibrinogen isolated from two affected daughters displayed the characteristic increased anionic binding of peak 1 fibrinogen. Sedimentation velocity experiments indicated that the S(o) (20, [unk]) of fibrinogen Baltimore was slightly greater (8.13S vs. 7.85S) than that of normal fraction I-4. Differences in concentration dependence (- 0.65 c vs. - 1.30 c for normal) of the sedimentation coefficient could be attributable in part to spatial conformational differences. Molecular sieving experiments in acrylamide gels indicated that the molecular weight of propositus fraction I-2 was about the same as that of normal fibrinogen of comparable solubility (i.e. I-4, mol wt 325,000). Studies of the UV spectra, tyrosine/tryptophan ratios, sialic acid and hexose content, and N-terminal amino acids demonstrated no consistent significant differences from normal fraction I-4.

Amino Acids↗