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PubMed1992

Changes in fibrinogen adsorbed to segmented polyurethanes and hydroxyethylmethacrylate-ethylmethacrylate copolymers.

Fibrinogen adsorption from blood to biomaterials may regulate platelet adhesion and thrombus formation because of fibrinogen's central role in the coagulation cascade and its ability to bind specifically to the platelet membrane glycoprotein (GP) IIb-IIIa. Adsorption of fibrinogen from blood plasma to many materials exhibits a maximum with respect to plasma dilution and exposure time (the Vroman effect). In this study fibrinogen adsorption to several polymers was examined to ascertain the influence of controlled changes in surface chemistry on the Vroman effect. The materials included hydroxyethylmethacrylate-ethylmethacrylate (HEMA/EMA) copolymers, Biomer, and a series of segmented polyurethanes (PEUs), two of which contained fluorinated chain extenders. Each material exhibited maximal adsorption of fibrinogen at intermediate plasma concentrations. Little effect of soft-segment type or molecular weight was observed and no significant differences in fibrinogen adsorption to the fluorinated PEUs were seen. Changes in the strength of fibrinogen attachment to these materials with time after adsorption were also assessed. Fibrinogen adsorbed for 1 min was displaced more readily by blood plasma than that adsorbed for 1 h, regardless of the material. The more hydrophobic polymers exhibited greater retention of adsorbed fibrinogen. In addition, the fraction of fibrinogen retained by polyethylene depended on the amount of fibrinogen adsorbed to the surface, being greatest when the surface loading was the least. These studies indicate that spreading or transition of adsorbed fibrinogen molecules from a weakly to tightly bound state is a general consequence of protein adsorption to solid surfaces.

Adsorption↗
PubMed1986

The influence of preadsorbed canine von Willebrand factor, fibronectin and fibrinogen on ex vivo artificial surface-induced thrombosis.

We have examined the effects of preadsorption of several canine plasma proteins on surface-induced thrombogenesis in a canine ex vivo model. Our technique allowed determination of initial deposition and subsequent embolization of 51Cr-labeled platelets and 125I-fibrinogen onto and from polymeric arterio-venous shunts in non-anticoagulated canines. Segments of the tubing were removed at various time points between 2 and 120 minutes of blood contact for examination of the morphology of the thrombus by scanning electron microscopy. Thrombus deposition was measured on uncoated plasticized poly(vinyl chloride) (PVC) and PVC precoated with canine von Willebrand factor (vWF), fibronectin, partially purified fibrinogen (fibrinogen which contained vWF and fibronectin as impurities), or purified fibrinogen (fibrinogen which had been further purified to remove fibronectin and vWF). Preadsorption of all proteins studied enhanced the thrombogenic response relative to that of the uncoated surface. Precoating with vWF or partially purified fibrinogen resulted in the deposition of the greatest number of thrombi, and embolization was slower than on shunts precoated with canine fibronectin or purified fibrinogen. The deposition-embolization profiles for the fibronectin and purified fibrinogen-coated surfaces were similar. The amount and time sequence of initial adhesion and spreading of platelets was related to the extent and time sequence of peak thrombus formation. The partially purified fibrinogen-coated and vWF-coated surfaces had more adhered and spread platelets at the earliest time points and a greater number of larger thrombi at the peak deposition times. The slowest rate of platelet adhesion and spreading was seen on the purified fibrinogen-coated surface. White blood cells were present very early on surfaces precoated with vWF and partially purified fibrinogen, and were present prior to embolization on all surfaces. Major conclusions from this work indicate that, although fibrinogen and fibronectin promote thrombogenesis when adsorbed to a surface, vWF is even more active in promoting platelet deposition and in anchoring thrombi to the surface of biomaterials. Thus, differences in vWF adsorption to biomaterials may be a determinant of surface-induced thrombogenesis.

Adsorption↗
PubMed1988

Factor XIII-induced crosslinking in solutions of fibrinogen and fibronectin.

In solutions containing fibrinogen and fibronectin, factor XIIIa catalyzes the formation of two types of crosslinked polymers: hybrid oligomers consisting of equimolar amounts of fibrinogen and fibronectin, and fibrinogen oligomers. The two types of oligomers are produced in amounts proportional to the starting concentration of fibronectin and fibrinogen in the reaction mixture. Increasing the fibronectin concentration relative to the fibrinogen concentration results in the production of more hybrid and less fibrinogen type oligomers. The lowest molecular weight hybrid oligomer, a dimer, is formed by ligation of one molecule of fibrinogen and fibronectin. The A alpha-chain of fibrinogen and one fibronectin subunit participate in the crosslinking. Larger size hybrid oligomers form by the joining of two hybrid dimers to each other via gamma-chain dimerization in the fibronectin moiety of the dimers. In fibrinogen oligomer formation, fibrinogen molecules are ligated by gamma-chain dimerization in a step-wise fashion producing fibrinogen dimers, trimers, tetramers, etc. without A alpha-chain crosslinking. The hybrid type and the fibrinogen type of oligomer grow in size and eventually become crosslinked to each other yielding large molecular weight complexes that interact to form a gel network.

Electrophoresis, Polyacrylamide Gel↗
PubMed2004

Purification and characterization of Atlantic salmon (Salmo salar) fibrinogen.

This study describes a purification protocol of salmon fibrinogen that gives a consumable and highly clottable fibrinogen. Some characteristics of salmon and human fibrinogen are compared. Fibrinogen was purified from barium sulphate adsorbed plasma of Atlantic salmon, using two steps of 25% ammonium sulphate precipitation followed by ultrafiltration. The clottability of the purified salmon fibrinogen was 91%. The Aalpha chains of salmon fibrinogen were heterogeneous with a molecular mass of 90-110 kDa, compared to approximately 67 kDa of human fibrinogen Aalpha chains. The Bbeta and gamma chains of salmon and human fibrinogen had molecular masses of approximately 55 and 50 kDa, respectively. Western blotting revealed that polyclonal rabbit anti-human fibrinogen antibodies had affinity for the gamma chains of salmon fibrinogen, making it possible to study factor XIII activity in purified salmon fibrinogen. Cross-linking of either gamma-gamma or gamma-alpha chains was not detected upon incubation of the purified fibrinogen with thrombin and calcium alone, but was detected when clotting was performed in plasma indicating absence of factor XIII activity in the purified product.

Animals↗
PubMed1998

Role of intracellular calcium ([Ca2+]i) and tyrosine phosphorylation in adhesion of cultured vascular smooth muscle cells to fibrinogen.

OBJECTIVE: Fibrinogen is an independent risk factor for cardiovascular disease. This study has investigated the role of intracellular Ca2+ ([Ca2+]i) and tyrosine phosphorylation in the attachment of human and rat-derived cultured vascular smooth muscle cells to fibrinogen. METHODS: Cells were cultured from human saphenous vein segments (HVSMC) and from an established rat aortic cell line (A7r5). [Ca2+]i was measured using fura-2 and adhesion was studied using pre-coated 96 well polystyrene plates. RESULTS: Fibrinogen increased [Ca2+]i in both cell types. In A7r5 cells fibrinogen-induced increases in [Ca2+]i were partially inhibited by a peptide containing the amino acid sequence Arg-Gly-Asp (RGD) which interferes with binding to integrins. In contrast RGD increased [Ca2+]i in HVSMC, but did not inhibit responses to fibrinogen. Ni2+, an inorganic calcium channel blocker largely abolished the rise in [Ca2+]i, but blockers of voltage-operated calcium channels failed to affect [Ca2+]i responses to fibrinogen in either cell type. Genistein, an inhibitor of tyrosine kinase inhibited fibrinogen-induced rises in [Ca2+]i, while daidzein, an inactive analogue, was without effect. Adhesion of cells to fibrinogen was concentration- and time-dependent. Cell adhesion to fibrinogen was partially inhibited by RGD peptide in both cell types. Adhesion of cell to fibrinogen was inhibited by chelation of [Ca2+]i with BAPTA-AM, inhibition of Ca2+ entry by Ni2+, and inhibition of tyrosine kinases by genistein, but heparin had no effect on adhesion. CONCLUSIONS: Vascular smooth muscle cells attach to fibrinogen in part through RGD-type interactions. Activation of tyrosine kinase(s) and a subsequent rise in [Ca2+]i appear to be important signals mediating the response to fibrinogen.

Animals↗
PubMed2000

Nutritional status influences plasma fibrinogen concentration: evidence from the THUSA survey.

Nutritional status and risk factors for chronic diseases, including plasma fibrinogen and its determinants, of Africans in the Northwest Province of South Africa, have been studied in a cross-sectional survey. A representative sample of 1854 "apparently healthy" African men and women volunteers aged 15 years and older was recruited from 37 randomly selected sites throughout the Province and stratified for level of urbanisation. Information was collected using validated and culture-sensitive questionnaires. Fasting blood samples were drawn, and all measurements were done with standardised methodology using appropriate equipment, procedures, and controls. Fibrinogen concentration was measured in citrated plasma with the method of Clauss, using the ACL200 automated system and the international fibrinogen standard. The results revealed a population with a high mean plasma fibrinogen (3.17+/-1.10 g/L for HIV-negative men and 3. 64+/-1.12 g/L for HIV-negative women). Factors known to influence plasma fibrinogen, such as age, gender, smoking habit, and physical activity, were also observed in this population. Young rural men and women had the lowest fibrinogen level. Nasal snuff taking and HIV infection did not influence fibrinogen concentration. Multivariate analyses revealed that lower plasma fibrinogen was associated with low to normal body mass index in women, and with dietary intakes compatible with prudent dietary guidelines in men and women (low intakes of animal protein; trans fatty acids and higher intakes of plant protein; dietary fibre, vitamin E, and iron, and a high dietary P/S ratio). Subjects in the higher quartiles of plasma fibrinogen had significantly lower iron, vitamin E, and vitamin B6 (women) status. Increases in fibrinogen were associated with significant increases in serum lipids. Both under- and overnutrition seem to be associated with high plasma fibrinogen. It is concluded that overall nutritional status, possibly in addition to specific nutrients (and foods), influences plasma fibrinogen.

Adolescent↗
PubMed2001

Reactivity of human platelets with immobilized fibrinogen is dictated by the chemical character of the surface.

Quiescent platelets readily adhere to surface-immobilized fibrinogen. In contrast, platelets exposed to soluble fibrinogen do not demonstrate such activity. As part of an effort to characterize this phenomenon, a solid-phase reagent was prepared by adsorbing human fibrinogen to polystyrene-divinylbenzene microparticles. Using a suspension of human platelets, phase-contrast microscopy was used to quantitate directly platelets bound to fibrinogen-coated beads. This method is fast, straightforward, and requires minimal amounts of reagents and sample. An existing turbidimetric assay was modified to monitor optically the rate and extent of platelet-fibrinogen binding. When platelet-rich plasma was added to a stirred suspension of fibrinogen-coated beads, the rate of aggregation was related directly to the concentration of fibrinogen on the bead surface. This response could not be mitigated by the thrombin inhibitor, hirudin, indicating that any thrombin generated in the reaction has no role in bead aggregation. Conversely, the alpha(IIb)beta(3) antagonist, abciximab (ReoPro), completely prevented aggregation, implicating specific fibrinogen-alpha(IIb)beta(3) interactions as responsible for the observed effect. Beads coated with either albumin or a densely packed, pure film of the neutral phospholipid, phosphatidylcholine (lecithin), do not aggregate under identical conditions, nor do fibrinogen-coated beads aggregate when platelet-depleted plasma is added. When fibrinogen was coated to beads as a mixed film with lecithin, a striking increase in reactivity toward platelets was demonstrated, compared to unmodified beads. These studies indicate that the observed adhesion of platelets to beads is a direct result of platelet-fibrinogen interactions and platelets respond differently to fibrinogen when presented as a mixed film with lipid, compared to the protein alone at an interface.

Dose-Response Relationship, Drug↗
PubMed2002

Extracorporeal fibrinogen adsorption--efficacy, selectivity and safety in healthy subjects and patients with foot ulcers.

The elimination of fibrinogen from plasma improves plasma viscosity and whole blood viscosity. For extracorporeal adsorption of fibrinogen the pentapeptide gly-pro-arg-pro-lys was coupled to sepharose CL-4B. Columns containing 100 ml of coupled sepharose CL-4B were used to eliminate fibrinogen from the plasma of 8 healthy male subjects (mean age 27.4 +/- 4.3 years, height 180.9 +/- 8.3 cm, weight 85.1 +/- 13.6 kg). Four treatments were performed in each proband (days 1, 2, 4 and 7). Plasma fibrinogen concentration was lowered from 221.1 +/- 39.0 to 123.5 +/- 21.7 mg/dl (2275 +/- 477 ml plasma treated) by the first treatment, from 172.8 +/- 42.3 to 105.6 +/- 16.5 mg/dl (1609 +/- 761 ml) by the second, from 140.5 +/- 13.8 to 98.8 +/- 8.6 mg/dl (1224 +/- 118 ml) by the third and from 160.2 +/- 23.6 to 106.4 +/- 9.7 mg/dl (1513 +/- 521) by the fourth. Plasma viscosity was improved from 1.40 +/- 0.18 mPa s before the first treatment to 1.23 +/- 0.06 mPa s after fourth treatment, whole blood viscosity from 4.49 +/- 0.36 mPa s to 3.83 +/- 0.27 mPa s (P < 0.01). No clinical side effects and no clinically relevant change of laboratory parameters including in vitro tests on thrombocyte function were observed. Seven men and three women (48-75 years old, 9 patients suffered from diabetes mellitus, one patient from peripheral arterial occlusive disease, 5 patients were on regular hemodialysis) were treated by fibrinogen adsorption. Each column contained 135 ml of coupled sepharose CL-4B. Treatments were scheduled on day 1, 2, 4, 6, 8, 10, 13, 16, 19, 22, 25 and 28. 144 treatments with fibrinogen adsorption were performed. No clinical side effects due to the fibrinogen-adsorption procedure were observed. In these 10 patients the fibrinogen concentration before the first treatment was 473.7 +/- 183.7 mg/dl. In the first treatment session it was lowered to 241.4 +/- 125.8 mg/dl by treating 4270 +/- 1180 ml of plasma. In the following 134 treatments the pre-treatment concentration of fibrinogen was 262.6 +/- 83.4 mg/dl, the post-treatment concentration was 120.6 +/- 37.2 mg/dl. The mean volume of plasma treated was 3737 +/- 1643 ml, the mean duration of a treatment session (except the first treatment) was 143.7 +/- 63.1 min. In 7 patients a mean post-treatment fibrinogen concentration of < or = 123 mg/dl was obtained, in the other patients 133, 177 and 184 mg/dl. Yet, the decrease of fibrinogen concentration was also pronounced in these 3 patients: -82%, -67%, and -73%, respectively. Accelerated wound healing was observed in 9 of the 10 patients. In conclusion, affinity chromatography using the pentapeptide gly-pro-arg-pro-lys is an effective, selective and safe procedure to lower fibrinogen concentration in plasma thereby improving blood viscosity. It could be a therapeutic option in severe blood vessel disease where drug therapy is not sufficient and invasive procedures like bypass or angioplasty cannot be applied.

Adolescent↗
PubMed2006

Modification of fibrinogen by homocysteine thiolactone increases resistance to fibrinolysis: a potential mechanism of the thrombotic tendency in hyperhomocysteinemia.

We have previously shown functional differences in fibrinogen from hyperhomocysteinemic rabbits compared to that in control rabbits. This acquired dysfibrinogenemia is characterized by fibrin clots that are composed of abnormally thin, tightly packed fibers with increased resistance to fibrinolysis. Homocysteine thiolactone is a metabolite of homocysteine (Hcys) that can react with primary amines. Recent evidence suggests that Hcys thiolactone-lysine adducts form in vivo. We now demonstrate that the reaction of Hcys thiolactone with purified fibrinogen in vitro produces fibrinogen (Hcys fibrinogen) with functional properties that are strikingly similar to those we have observed in homocysteinemic rabbits. Fibrinogen purified from homocysteinemic rabbits and Hcys fibrinogen are similar in that (1) they both form clots composed of thinner, more tightly packed fibers than their respective control rabbit and human fibrinogens; (2) the clot structure could be made to be more like the control fibrinogens by increased calcium; and (3) they both form clots that are more resistant to fibrinolysis than those formed by the control fibrinogens. Further characterization of human fibrinogens showed that Hcys fibrin had similar plasminogen binding to that of the control and an increased capacity for binding tPA. However, tPA activation of plasminogen on Hcys fibrin was slower than that of the control. Mass spectrometric analysis of Hcys fibrinogen revealed twelve lysines that were homocysteinylated. Several of these are close to tPA and plasminogen binding sites. Lysines are major binding sites for fibrinolytic enzymes and are also sites of plasmin cleavage. Thus, modification of lysines in fibrinogen could plausibly lead to impaired fibrinolysis. We hypothesize that the modification of lysine by Hcys thiolactone might occur in vivo, lead to abnormal resistance of clots to lysis, and thereby contribute to the prothrombotic state associated with homocysteinemia.

Amino Acid Sequence↗
PubMed1998

The relative power of heat-precipitation nephelometric and clottable (Clauss) fibrinogen in the prediction of ischaemic heart disease: the Caerphilly and Speedwell studies.

The Caerphilly and Speedwell studies have previously reported the predictive power of heat-precipitation, nephelometric, fibrinogen for 10-year incidence of ischaemic heart disease. A Clauss, clotting time, fibrinogen was also measured at baseline, but has not previously been reported. The predictive power of the two assays is compared. Both methods were employed on fasting blood samples from a total of 4391 men aged 45-63 years. Over the following 10 years 533 (12%) developed major ischaemic heart disease. Nephelometric fibrinogen was higher by 0.33 g/l among the men who developed disease; clottable fibrinogen was higher by 0.20 g/l. This difference is statistically significant (P=0.01). Relative odds of developing heart disease increased steadily to 3.53 (P<0.0001) in the 20% of men with the highest nephelometric fibrinogen; for clottable fibrinogen the corresponding relative odds increased to 2.24 (P<0.0001). When both measures of fibrinogen were included in logistic regression models together with age and smoking habit, the trend for incidence to increase with increasing nephelometric fibrinogen remained highly significant (P<0.0001), whereas for the Clauss fibrinogen the trend almost entirely disappeared (P = 0.37). We conclude that functional assays of clottable fibrinogen may not reflect all of the mechanisms which mediate the association between fibrinogen and cardiovascular disease and that assays of both 'heat precipitable' and 'clottable' fibrinogen should be included in all future prospective studies.

Aged↗
PubMed1999

Fibrinogen fragments and platelet dysfunction in uremia.

BACKGROUND: The uremic state is characterized by subnormal platelet aggregation. Fibrinogen fragments, usually absent in normal human blood, but present in uremic plasma, may play a role in uremic platelet dysfunction. METHODS: To examine this hypothesis, we investigated the availability and function of fibrinogen receptors [glycoprotein (GP) IIb-IIIa] on uremic and normal platelets, as well as the effect of fragments obtained from chymotrypsin digestion of human fibrinogen on normal platelets. The availability of fibrinogen receptors was examined using anti-GP IIb-IIIa antibodies and flow cytometry, whereas receptor function was assessed by the receptor's ability to mediate fibrinogen binding and platelet aggregation. RESULTS: Platelet aggregation and the availability of GP IIb-IIIa were lower in uremic patients when compared with normal controls. Flow cytometric analysis showed that fibrinogen fragments decreased the binding of anti-CD61, an activation-independent anti-GP IIIa monoclonal antibody, to resting normal platelets. These fragments also reduced the binding of PAC-1, an activation-dependent anti-GP IIb-IIIa monoclonal antibody, to adenosine diphosphate (ADP)-activated normal platelets. In addition, the binding of radiolabeled fibrinogen to activated normal platelets and platelet aggregation in response to ADP were both decreased by fibrinogen fragments. CONCLUSIONS: These findings suggest that fibrinogen fragments impair platelet function by occupying fibrinogen receptors prior to cell activation, thus preventing the binding of intact fibrinogen to platelets after subsequent stimulation. These observations also suggest a plausible mechanism by which endogenous fibrinogen fragments present in uremic plasma may contribute to platelet dysfunction.

Adenosine Diphosphate↗
PubMed2001

Effects of dietary protein restriction on fibrinogen and albumin metabolism in nephrotic patients.

BACKGROUND: Nephrotic syndrome (NS) is characterized by profound changes in albumin and fibrinogen levels. Dietary protein restriction has been advocated in the treatment of patients with NS, but its effects on albumin and fibrinogen metabolism have not been fully elucidated. METHODS: We evaluated the effects of dietary protein restriction on endogenous leucine flux (ELF), fibrinogen and albumin metabolism in seven patients with NS who consumed either a normal protein diet (NPD; 1.20 +/- 0.06 g/kg/day), or a low protein diet (LPS; 0.66 +/- 0.04 g/kg/day) for four weeks. Seven normal subjects served as controls. The postabsorptive ELF value, fractional synthesis rate (FSR) and absolute synthesis rate (ASR) of both albumin and fibrinogen were evaluated during the last 120 minutes of a five-hour 5,5,5-D3-L-leucine infusion. RESULTS: During the NPD regimen. ELF was increased, serum albumin was reduced, plasma fibrinogen was increased, albumin FSR and ASR were both increased, fibrinogen FSR was normal, and fibrinogen ASR was greater in patients with NS compared to controls. In patients with NS the LPD regimen reduced proteinuria, ELF, albumin FSR and ASR, plasma fibrinogen levels, fibrinogen ASR, and increased serum ulbumin levels. Dietary-induced changes in albumin and fibrinogen synthesis were significantly correlated (r = 0.719, P < 0.05). CONCLUSIONS: Patients with NS treated with LPD show: (1) a reduction of proteinuria, albumin ASR and FSR, with an increase in serum albumin levels and its intravascular pool; (2) a decrease of fibrinogen ASR, with a reduction in both plasma fibrinogen levels and intravascular pool; and (3) a reduced rate of whole body proteolysis.

Adult↗
PubMed2003

Impact of albumin synthesis rate and the acute phase response in the dual regulation of fibrinogen levels in hemodialysis patients.

BACKGROUND: Fibrinogen is a risk factor for cardiovascular disease. It also is an acute phase protein (APP) and its plasma concentration increases with inflammation. Fibrinogen synthesis correlates with albumin synthesis in nephrotic patients and in patients with an expanded plasma volume even when serum albumin is normal and there is no inflammatory disease. The relationships among albumin synthesis, the acute phase response and plasma fibrinogen levels in hemodialysis patients are unknown. METHODS: In 74 hemodialysis patients, albumin synthesis, plasma volume (PV) and acute phase proteins (APPs) C-reactive protein (CRP), alpha1 acid glycoprotein (alpha1 AG), ceruloplasmin (Cer), and interleukin 6 (IL-6) were measured in serum and fibrinogen in plasma, and the results analyzed by multiple regression analysis. CRP, IL-6, alpha1 AG, Cer and fibrinogen were measured monthly, which enabled us to determine whether changes in these APPs correlated with the levels of and variability in plasma fibrinogen over time using a longitudinal modeling approach. Length of follow-up for the 74 patients ranged from 3.25 to 67.5 months. RESULTS: Baseline fibrinogen (548.6 +/- 106. 4 mg/dL) was significantly greater than levels reported for normal adults and correlated positively with albumin synthesis (P < 0.001), age (P < 0.001) and log CRP (P = 0.002) and negatively with PV (P < 0.001). Longitudinally, fibrinogen varied positively with long-lived APPs, Cer and alpha1 AG, as well as the short-lived APP, CRP. CONCLUSION: Plasma fibrinogen concentration is high in HD patients and directly correlates with increased albumin synthesis rates and the serum levels of APPs. Fibrinogen levels also correlate negatively with PV. Fibrinogen levels vary over time in synchrony with levels of other long-lived APPs, supporting the hypothesis that fibrinogen is regulated in part as a component of the acute phase response and in part by factors that increase albumin synthesis.

Acute-Phase Reaction↗
PubMed2001

Fibrinogen adsorption in the diabetic foot syndrome and peripheral arterial occlusive disease: first clinical experience.

The elimination of fibrinogen from plasma improves plasma viscosity and whole-blood viscosity. For extracorporeal adsorption of fibrinogen, the pentapeptide gly-pro-arg-pro-lys was coupled to sepharose CL-4B3. Adsorbers containing 135 ml of coupled sepharose CL-4B were used to eliminate fibrinogen from the plasma of 7 men and 3 women (48-75 years old). Nine patients suffered from diabetes mellitus, 1 patient from peripheral arterial occlusive disease, and 5 patients were on regular hemodialysis. Treatments were scheduled on Days 1, 2, 4, 6, 8, 10, 13, 16, 19, 22, 25, and 28. One hundred forty-four treatments with fibrinogen adsorption were performed. No clinical side effects due to the fibrinogen adsorption procedure were observed. In these 10 patients, fibrinogen concentration before the first treatment was 473.7 +/- 183.7 mg/dl. In the first treatment session, fibrinogen concentration was lowered to 241.4 +/- 125.8 mg/dl by treating 4,270 +/- 1,180 ml of plasma. In the following 134 treatments, the pretreatment concentration of fibrinogen was 262.6 +/- 83.4 mg/dl, and the posttreatment concentration was 120.6 +/- 37.2 mg/dl. The mean amount of plasma treated was 3,737 +/- 1,643 ml, and the mean duration of a treatment session (except first treatment) was 143.7 +/- 63.1 min. In 7 patients, a mean posttreatment fibrinogen concentration of < or = 123 mg/dl was obtained; in the other patients, concentrations of 133, 177, and 184 mg/dl were obtained. Yet, the decrease of fibrinogen concentration was also pronounced in these 3 patients: -82%, -67%, and -73%, respectively. During the treatment period of 28 days, wound healing was observed in 9 of the 10 patients. In conclusion, affinity chromatography using the pentapeptide gly-pro-arg-pro-lys is an effective, selective, and safe procedure to lower fibrinogen concentration in plasma. It could be a therapeutic option in severe blood vessel disease in which drug therapy is not sufficient and invasive procedures such as bypass or angioplasty cannot be applied. Yet, more information is needed, for example, about the fibrinogen concentration that has to be reached to get the maximal improvement of micro- and/or macrocirculation.

Adsorption↗
PubMed1984

Potential basis for regulation of the coordinately expressed fibrinogen genes: homology in the 5' flanking regions.

The three chains of fibrinogen are encoded by three separate genes whose transcription is coordinately regulated. The breakdown of fibrinogen during the acute-phase reaction leads to a simultaneous increase in alpha-, beta-, and gamma-fibrinogen mRNA in the liver. In a search for the basis of this coordinate increase in transcription, we have determined the sequences of the regions surrounding the points of transcriptional initiation of the three rat fibrinogen genes, 1490 nucleotides upstream and 730 nucleotides downstream. Two unique regions of homology have been found. One region consists of 15 nucleotides that have a common 6-nucleotide core lying between -116 and -160; the other is approximately equal to 100 nucleotides long and is in the -165 to -472 region. In this region, the beta- and gamma-fibrinogen genes are approximately equal to 65% homologous. alpha-Fibrinogen has somewhat less homology with both beta- and gamma-fibrinogen. In addition, the beta-fibrinogen gene has 22 nucleotides at position -480 that are homologous to sequences that have been noted to occur in glucocorticosteroid-regulated genes in a similar position. We feel that these areas of conserved sequences play a role in the regulation of the transcription of fibrinogen. The fibrinogen chains are synthesized as precursor peptides, and the amino-terminal portion, the so-called signal peptide, is removed during the translocation of the peptide chain across the endoplasmic reticulum. We have determined those sequences that encode the signal peptides. Homology in the amino acid sequence between the rat and human signal peptides varies between 52% for alpha-fibrinogen and 66% for beta-fibrinogen. This homology implies that there has been strong selective pressure on this portion of these genes.

Amino Acid Sequence↗
PubMed1995

Binding of fibrinogen A alpha 1-50-beta-galactosidase fusion protein to thrombin stabilizes the slow form.

The interaction of fibrinogen A alpha1-50-beta-galactosidase fusion protein with the slow and fast forms of thrombin was studied and compared to thrombin-fibrinogen interaction under identical solution conditions. At equilibrium, the affinity of the fusion protein for the slow form of thrombin is 3 times higher than its affinity for the fast form. The fusion protein and fibrinogen have the same affinity for the fast form. On the other hand, the affinity of the fusion protein for the slow form of thrombin is 40 times tighter than that of fibrinogen. In the transition state, binding of the fusion protein has the same properties as fibrinogen, with the fast form showing higher specificity. The N-terminal fragment of the fibrinogen A alpha chain thus contains residues that are responsible for the preferential binding of the fusion protein to the slow form at equilibrium and to the fast form in the transition state. If this fragment binds to thrombin in a similar way for fibrinogen and the fusion protein, then the N-terminal domains of the B beta and gamma chains of fibrinogen, that are not present in the fusion protein, must play a key role in the binding of fibrinogen to thrombin at equilibrium. These chains may destabilize binding to the slow form by nearly 2.4 kcal/mol, thereby favoring binding of fibrinogen to the fast form. We propose that the three chains of fibrinogen play different roles in the thrombin-fibrinogen interaction, with the A alpha chain containing residues for preferential binding to the fast form in the transition state and the B beta and gamma chains containing residues that destabilize binding to the slow form at equilibrium.

Enzyme Stability↗
PubMed2006

Fibrinogen-neutrophil interactions in response to fMLP and Porphyromonas gingivalis fimbrial peptides.

Porphyromonas gingivalis (P.g) is the primary bacterial agent in many forms of chronic periodontitis. Since polymorphonuclear leukocytes (PMNs) are first-line responders to P.g.- induced inflammation, and fibrinogen is important for in vivo PMN in this disease, we have studied the effect of N-formyl-methionyl-leucyl-phenylalanine (fMLP) (an inflammatory stimulus), P.g. fimbriae and fimbrial peptides (based on FimA, the main structural protein of P.g. fimbriae) on PMN-fibrinogen interactions. Freshly isolated human PMNs were allowed to react with FITC-Fibrinogen and various fimbrial peptides (denoted as FimA followed by amino acid number within whole FimA protein), and FITC-Fibrinogen binding was measured using flow cytometry. Freshly isolated neutrophils were also challenged with Fibrinogen and/or fimbrial peptides to measure IL-8 secretion using ELISA. Our studies show that fibrinogen binding to PMNs is enhanced (p < 0.01) in response to fMLP as well as fimbrial peptides (FimA 61-80) containing the motif LTTE (p < 0.01) in a dose dependent manner but not in response to peptides without that motif. We also observed that fMLP and FimA 61-80 have an additive effect on fibrinogen binding to PMNs (p < 0.05), and fMLP and FimA 171-185 significantly inhibit fMLP-induced fibrinogen binding (p < 0.01). To determine of the role of inflammatory cytokines, we examined IL-8 release from PMNs in response to combinations of P. gingivalis fimbriae, fMLP and fibrinogen. In all cases, IL-8 release increased in a dose-dependent manner (p < 0.05). fMLP-fibrinogen effect on IL-8 release from PMNs was synergistic while fimbriae-fibrinogen effect was additive. In summary, PMN priming by fimbrial peptides facilitates fibrinogen-PMN interaction and may increase inflammation.

Fibrinogen↗
PubMed1985

The effects of hepatocyte stimulating factor on fibrinogen biosynthesis in hepatocyte monolayers.

The biosynthesis of fibrinogen increased at least eightfold in primary hepatocytes when incubated in the presence of monocyte/macrophage-derived hepatocyte stimulating factor (HSF). The large increase in fibrinogen production is due to increased availability of the mRNAs for the protein since cytodot analysis of cellular RNA showed a 10-12-fold increase in each of the fibrinogen mRNAs. Pulse-chase experiments showed that the time for fibrinogen synthesis, assembly, and secretion was 40-50 min for both control and stimulating conditions. This indicates that the increased production was due principally to the presence of greater amounts of fibrinogen mRNA rather than translation or secretion-specific events. Three lines of evidence indicate that the increase in fibrinogen production was due to HSF effects on transcription: (a) analysis of cytoplasmic levels of each of the fibrinogen mRNAs showed that all three increased at the same rate and to the same extent, demonstrating that HSF affects the three gene products coordinately; (b) Northern gel analysis of cytoplasmic RNA isolated after very brief exposures to HSF showed increases in a large molecular weight fibrinogen RNA precursor; and (c) actinomycin D blocked the HSF-stimulated increase in fibrinogen mRNA species. Furthermore, experiments in which protein synthesis was inhibited by cycloheximide failed to inhibit the increase in fibrinogen mRNAs, indicating new protein synthesis is not required for the HSF stimulation of fibrinogen mRNA. These results are consistent with our hypothesis that HSF is exerting its control of fibrinogen at the level of gene transcription.

Animals↗