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PubMed1995

[Clinical significance of the cardiovascular risk factor fibrinogen in secondary prevention].

Seven prospective, epidemiological studies indicate plasma fibrinogen levels (over 300-350 mg/dl) as an important, independent cardiovascular risk factor for subsequent myocardial infarction and stroke. Furthermore, several clinical studies revealed an association between fibrinogen and both the angiographic and clinical degree of coronary heart disease. In addition, a significant relation of fibrinogen with the number of occluded coronary vessels was found. The following pathophysiologic mechanism are of particular importance: Fibrinogen is a main determinant of plasma viscosity and red cell aggregation. Both phenomena deteriorate blood fluidity especially in the microcirculation. Fibrinogen plays a central role in platelet aggregation and performs an essential substrate in the coagulation cascade. Thus, high fibrinogen levels may favor a hypercoagulable state resulting in final thrombotic events of cardiovascular disease. Fibrinogen is also involved in atherogenesis by stimulating proliferation and migration of smooth muscle cells. Several determinants of fibrinogen levels are known. Smoking is the strongest one in healthy persons. This clinically important effect is dose related. Consequently, cessation of smoking is a major step to lower fibrinogen and subsequently the individual cardiovascular risk. Reduction of overweight and maintenance of regular physical activity are further nonpharmacologic means. Fibrates decrease fibrinogen about 10-30% on an average. Finally, intermittent low-dose Urokinase for end-stages of coronary artery disease and LDL-apheresis (HELP) represent additional approaches to reduce fibrinogen.

Adult↗
PubMed1975

Partial chemical characterization of rat fibrinogen.

Rat fibrinogen has been purified and compared with bovine and human fibrinogen with respect to a number of chemical characteristics, including molecular size, charge distribution, NH2-terminal amino acids, total amino acid composition, and interspecies immunological cross-reactivity. Although human and bovine fibrinogen demonstrated three nonidentical polypeptide chains by sodium dodecyl sulfate gel separations and by CM-cellulose separations, rat fibrinogen Aalpha and Bbeta chains exhibited identical molecular weight sizes as well as identical charges. The presence of two nonidentical chains in these preparations was shown by qualitative NH2-terminal sequence analyses. The gamma chain of rat fibrinogen was also shown to be quite distinct from the gamma chains of human and bovine fibrinogen in its elevated content of cysteinyl and methionyl residues. Rat fibrinogen possesses the first reported blocked gamma chain NH2-terminal amino acid of any species. It is concluded that, although many chemical properties of rat fibrinogen are unique, the basic molecular structure has remained consistent when compared with that of fibrinogen from the vertebrates studied thus far. Moreover, the inducibility of this system, together with the partial chemical characterization of the fibrinogen molecule, provides important information for the use of rat fibrinogen as a model system in studying the biosynthesis and assembly of this complex molecule.

Amino Acid Sequence↗
PubMed1994

Overexpression of any fibrinogen chain by Hep G2 cells specifically elevates the expression of the other two chains.

Earlier studies showed that overexpression of B beta fibrinogen chains, by transfection of Hep G2 cells with B beta cDNA, specifically enhanced the synthesis of all three fibrinogen chains (Roy, S. N., Mukhopadhyay, G., and Redman, C. M. (1990) J. Biol. Chem. 265, 6389-6393). To determine whether overexpression of any of the three component chains of fibrinogen affects the synthesis of the other two chains, we developed stable Hep G2 cell lines transfected with individual fibrinogen chain cDNAs. As a control, cells were also transfected with expression vector, which did not contain fibrinogen cDNA. Transfection with any fibrinogen cDNA increased the synthesis of all three fibrinogen chains but not of other plasma proteins. Hep G2 cells transfected with B beta cDNA produced 3-4-fold more fibrinogen than control cells, and cells transfected with A alpha or gamma cDNA made about 2-fold more fibrinogen. Northern blot analyses showed that levels of all 3 fibrinogen mRNAs were increased and were highest in Hep G2-B beta cells. Nuclear run-on transcription assays demonstrated that increased expression of the chains was due to increased transcriptional activity. These studies show that transcription of the three fibrinogen chains is tightly linked, and increased expression of any chain specifically leads to increased synthesis of the other two chains.

Blood Proteins↗
PubMed1998

Disparate chondrocyte metabolism in three-dimensional fibrin cultures derived from autogenous or commercially manufactured fibrinogen.

OBJECTIVE: To compare chondrocyte proliferation and metabolism in three-dimensional fibrin cultures formed from polymerized autogenous fibrinogen with that of commercially manufactured fractionated fibrinogen. ANIMALS: Fibrinogen and chondrocytes for in vitro experimentation derived from 2 horses, ages 12 and 14 months, donated for reasons unrelated to skeletal or hematologic abnormalities. PROCEDURE: Fibrinogen was isolated from whole blood, using plasma cryoprecipitation and centrifugation, and fractionated fibrinogen was purchased. Each was mixed with 10 x 10(6) chondrocytes/0.5 ml of fibrinogen, and was polymerized by addition of 0.5 ml of calcium-activated thrombin. Thirty 1-ml fibrin-chondrocyte disks were formed from each fibrinogen source and cultured for 0 (n = 6), 7 (n = 12), or 14 (n = 12) days. Chondrocyte metabolism and cell proliferation in each fibrin type were objectively assessed by assays for total proteoglycan content, [35S]proteoglycan accumulation, proteoglycan monomer size, and total DNA. Cell morphology and cartilage-specific cell function was evaluated by routine histologic, alcian blue histochemical, type-II collagen immunohistochemical, and type-II collagen in situ hybridization methods. RESULTS: Histologic examination indicated better retention of chondrocyte morphology in autogenous composites. Autogenous fibrinogen also stimulated greater chondrocyte proliferation (DNA content increased 1.4-fold on day 14) and supported higher proteoglycan accumulation (increased 1.4-fold on day 14), compared with commercial, fractionated fibrinogen. Abundant intracellular type-II procollagen mRNA was detected in autogenous fibrin cultures by in situ hybridization, and translation was confirmed by extensive pericellular type-II collagen accumulation. CONCLUSIONS: Autogenous fibrinogen has an inherent capacity to maintain chondrocyte phenotypic metabolism that is reduced or absent in commercially prepared fibrinogen. Enhanced, differentiated cell function may be useful for in vivo applications, but represents an added variable that may confound in vitro experiments, and should be considered when designing studies of chondrocyte function.

Analysis of Variance↗
PubMed1990

Interactions between the contact system, neutrophils and fibrinogen.

Since plasma kallikrein activates human neutrophils, and in plasma prekallikrein (PK) circulates complexed with high molecular weight kininogen (HK), we determined whether HK could mediate kallikrein's association with neutrophils. Human neutrophils were found to possess surface-membrane binding sites for HK but no internalization was detected at 37 degrees C. 125I-HK binding to neutrophils was dependent upon Zn++, specific, saturable and reversible with a Kd of 9-18 nM and 40,000-70,000 sites per cell. Furthermore, HK found in neutrophils (240 ng/10(7) neutrophils) also served as a cofactor for HNE secretion since neutrophils deficient in HK have reduced HNE secretion when stimulated in plasma deficient in HK or with purified kallikrein. Thus, neutrophil surface HK may serve as a receptor for kallikrein. Fibrinogen inhibited 125I-fibrinogen bound specifically and reversibly to human neutrophils. Zn++ (50 microM) was required for binding of 125I-fibrinogen to neutrophils and the addition of Ca++ (2 mM) increased the binding 2-fold. Excess HK completely inhibited binding of and displaced labeled fibrinogen as well as unlabeled fibrinogen. Binding of 125I-fibrinogen was saturable with an apparent Kd of 170 nM and 140,000 sites/neutrophil. The binding of 125I-fibrinogen to neutrophils was not inhibited by the peptide RGDS derived from the alpha-chain of fibrinogen, nor by the monoclonal antibodies (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, a MAB to a different domain of the adhesion glycoprotein Mac-1 (CR3). HK binding to neutrophils was not inhibited by OKM1. These observations were consistent with a further finding that fibrinogen is a noncompetitive inhibitor of 125I-HK binding to neutrophils. These studies indicate that fibrinogen specifically binds to an integrin receptor (Mac-1) on the neutrophil surface through the carboxy terminal of the gamma-chain and that HK inhibits this interaction.

Adult↗
PubMed1985

Influence of fibrinogen on fibrin polymerization. Ultracentrifugation studies.

During the transformation of fibrinogen to fibrin, excess fibrinogen suppresses further polymerization of fibrin, thereby enabling the nascent fibrin to be transported in a soluble form in blood. The question of possible complex formation between fibrin and fibrinogen was addressed by analyzing fibrin/fibrinogen (1:20, mol/mol) mixtures in the presence of calcium ions in stable linear sucrose density gradients by ultracentrifugation at 37 degrees C. During the period of ultracentrifugation in independent experiments, 40% of desAA-fibrin and 30% of desAABB-fibrin, respectively, precipitated without the participation of fibrinogen. The desAABB-fibrin, recovered in the gradient fractions, appeared as high-molecular-weight polymers (22 S), whereas the recovered desAA-fibrin exhibited only a slight increase in molecular weight (9 S) compared to fibrinogen (8 S). In contrast to this finding, both types of fibrin were totally recovered in gradient fractions provided that fibrinogen was present in the gradient at a uniform concentration of 2 mg/ml. In addition, the presence of fibrinogen but not human serum albumin reduced the size of desAABB-fibrin polymers (17 S). However, stable fibrin-fibrinogen complexes could not be demonstrated, since cosedimentation of differently labelled desAABB-fibrin and fibrinogen was not detectable. These studies suggest a specific but weak interaction of the solubilizing fibrinogen with the soluble fibrin polymers as demonstrated by a rapid exchange of both macromolecules.

Centrifugation, Density Gradient↗
PubMed1979

Effect of fibrin degradation products and thrombin on fibrinogen synthesis.

The effect of intravenous administration of homologous fibrin degradation products and thrombin on fibrinogen synthesis was assessed in rabbits. The relative fibrinogen synthesis rate was calculated as a ratio of the amount of radiolabelled lysine incorporated into fibrinogen to the amount incorporated into albumin during the same measurement period. An increase in this ratio above control would indicate a relatively specific stimulation of fibrinogen synthesis as compared with albumin, which is not an acute-phase reactant. Injection of 45 mg of 'early' or 'late' fibrin degradation products failed to produce a significant increase in the relative fibrinogen synthesis rate, suggesting that fibrin degradation products play no feedback role in controlling fibrinogen synthesis. Infusion of small amounts of homologous thrombin (15--25 NIH u) was followed by a small but statistically significant elevation of the relative fibrinogen synthesis rate. This was not accompanied by any increase in the levels of fibrinogen degradation products in plasma, or by any decrease in plasma fibrinogen concentration, possibly suggesting that thrombin can stimulate fibrinogen synthesis by a mechanism independent of significant fibrinogenolysis or intravascular coagulation.

Animals↗
PubMed2000

Regulation by fibrinogen and its products of intercellular adhesion molecule-1 expression in human saphenous vein endothelial cells.

It has been reported that fibrinogen may act as a bridging ligand, binding to intercellular adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells and to Mac-1 on THP-1 cells (a monocytic cell line) to increase adhesion. In this study, we investigated whether fibrinogen altered the expression of ICAM-1 and, thus, increased the adhesion of THP-1 cells to cultured human saphenous vein endothelial cells (HSVECs). Incubation of HSVECs with 0.3 to 4 micromol/L fibrinogen caused a time- and concentration-dependent increase in ICAM-1, as determined by ELISA. The 4- to 5-fold increase in ICAM-1 protein concentration in HSVECs stimulated by 4 micromol/L fibrinogen for 6 hours was concomitant with a 4- to 5-fold increase in ICAM-1 mRNA. This fibrinogen-stimulated ICAM-1 upregulation was associated with a 2-fold increase in THP-1 cell adhesion to HSVECs. The fibrinogen-derived peptide Bbeta15-42 bound to HSVECs (K(d) 0.18 micromol/L). Preincubation of HSVECs with Bbeta15-42, a neutralizing antibody to urokinase plasminogen activator (uPA), or the F(ab)(1) fragment of a monoclonal antibody to vascular endothelial cadherin significantly attenuated the increase in ICAM-1 stimulated by fibrinogen. Capillary electrophoretic analysis indicated that anti-uPA prevented the release of any fibrinopeptide B (Bbeta1-14) in cultures of HSVECs incubated with 4 micromol/L fibrinogen for 6 hours. Moreover, incubation of HSVECs with either fibrin monomer (1 micromol/L) or monoclonal antibodies to vascular endothelial cadherin (25 microg/mL) increased ICAM-1 protein concentration 3- to 4-fold. These findings indicate that cleavage of fibrinopeptide B from fibrinogen by endothelial uPA permits the exposed Bbeta15-42 sequence of fibrinogen to bind to vascular endothelial cadherin on HSVECs and to upregulate the expression of ICAM-1.

Amino Acid Sequence↗
PubMed1999

Fibrinogen St. Gallen I (gamma 292 Gly--> Val): evidence for structural alterations causing defective polymerization and fibrinogenolysis.

Fibrinogen St. Gallen I was detected in an asymptomatic Swiss woman. Routine coagulation tests revealed a prolonged thrombin and reptilase time. Functionally measured fibrinogen levels were considerably lower than those determined immunologically. Polymerization of fibrin monomers derived from purified fibrinogen was delayed in the presence of either calcium or EDTA. Normal fibrinopeptide A and B release by thrombin was established. An abnormal degradation of fibrinogen St. Gallen I by plasmin was observed. Fragment D1 of normal fibrinogen was fully protected against further proteolysis in the presence of 10 mM calcium, whereas fibrinogen St. Gallen I was partially further degraded to fragments D2 and D3. In the presence of 10 mM EDTA, the conversion of variant fragment D1 to D2 was accelerated whereas the degradation of fragment D2 to D3 was delayed in comparison to degradation of fragments D1 and D2 of normal fibrinogen. Three high-affinity calcium binding sites were found in both normal and variant fibrinogen. Mutation screening with SSCP analysis suggested a mutation in exon VIII of the gamma-chain gene. Cycle sequencing of this gene portion revealed a single base substitution from G to T of the base 7527, leading to replacement of gamma 292 glycine by valine. The same mutation has already been described for the fibrinogen variant Baltimore I. Molecular modeling was performed of a part of the gamma-chain containing the mutation site, based on recently published X-ray crystal structures of human fibrinogen fragment D and of a 30 kD C-terminal part of the gamma-chain. Significant structural alterations due to the substitution of glycine by valine at gamma 292 were observed, e.g. spreading of the protein backbone, probably leading to a modified accessibility of the plasmic cleavage sites in the gamma-chain at 356 Lys and 302 Lys. A shift of gamma 297 Asp that is involved in interactions of fragment D with the Gly-Pro-Arg-Pro-peptide was noted by molecular modeling. The latter observation is compatible with delayed polymerization of fibrin monomers.

Abortion, Spontaneous↗
PubMed2003

Measurement of soluble fibrin monomer-fibrinogen complex in plasmas derived from patients with various underlying clinical situations.

We previously reported a monoclonal antibody named IF-43 that specifically recognizes thrombin-modified fibrinogen (desAA- and desAABB- fibrin monomer) bound with fibrinogen or other D(1) domain-containing plasmic fragments such as fragments X,Y, and D(1), but not intact fibrinogen or cross-linked fibrin degradation products (XDP). Here, we tentatively named such complexes, soluble fibrin monomer (FM) -fibrinogen complex. By utilizing IF-43, we have developed a kit to measure soluble FM-fibrinogen complex and compared the profiles with those of two established molecular markers for thrombo-embolic disorders: i.e. the thrombin-antithrombin complex (TAT) and the D-dimer in plasma of patients who underwent surgery without any thrombo-embolic complications. The result indicated that soluble FM-fibrinogen complex is a distinct entity from the two established molecular markers. We have also attempted to observe their profiles in patients with the disseminated intravascular coagulation syndrome (DIC). Although the pro-files of soluble FM-fibrinogen complex in individual patients appeared to vary from one patient to the other, the plasma level of soluble FM-fibrinogen complex was found to be increased at the initial phase of disseminated intravascular coagulation syndrome. Thus, the soluble FM-fibrinogen complex may serve as an independent molecular marker for the detection of thrombin generation and the diagnosis of thrombosis. The soluble FM-fibrinogen complex may also serve as a risk factor for thrombosis, because it may precipitate as insoluble complexes beyond its threshold in plasma, or when it is modified by thrombin.

Antithrombin III↗
PubMed1986

Characterization of fibrinogen Milano I: amino acid exchange gamma 330 Asp----Val impairs fibrin polymerization.

An abnormal fibrinogen was found in two asymptomatic members (father and daughter) of the same family, originating from northern Italy. Routine coagulation studies revealed prolonged thrombin and reptilase clotting times. Plasma fibrinogen levels, as determined by a functional assay, were markedly diminished, whereas the heat precipitation method indicated normal fibrinogen values. On the basis of these findings, a tentative diagnosis of dysfibrinogenemia was made, and according to the accepted nomenclature, this fibrinogen variant was called "fibrinogen Milano l." The time course of fibrinopeptide A and B release from fibrinogen Milano l was normal, but the aggregation of fibrin monomers was delayed. Two-dimensional electrophoresis of reduced variant fibrinogen chains showed a defective gamma-chain with increased cathodic mobility. An abnormal electrophoretic mobility was observed also for the gamma-chain remnants of fibrinogen fragments D1 and D2 derived from fibrinogen Milano l, whereas the charge anomaly was lost after a further digestion by plasmin to D3, suggesting that the structure abnormality of this variant is situated in the region gamma 303-356. An abnormal peptide was isolated after cyanogen bromide cleavage of intact fibrinogen Milano l. This fragment spans from position gamma 311 to gamma 336. Amino acid analysis of the abnormal peptide showed the presence of valine and a diminished content of aspartic acid. Sequence analysis demonstrated an amino acid exchange Asp----Val in the gamma-chain at position 330.

Adult↗
PubMed1986

Platelet aggregation by fibrinogen polymers crosslinked across the E domain.

There is evidence that platelet interactions with artificial surfaces are mediated by plasma proteins, especially fibrinogen, adsorbed on the surfaces. Multiple site interactions between fibrinogen molecules adsorbed in high concentration and receptors in the unactivated platelet may be sufficient for platelet adhesion and subsequent activation. To examine this hypothesis, we prepared soluble polymers of fibrinogen. Polymers produced by interaction of fibrinogen with Fab'2 fragments of antibodies against fibrinogen's E (central) domain (Fg-Fab'2(E] induced, in gel-filtered platelets, aggregation and serotonin release, which were blocked by monoclonal antibodies against the GPIIb/IIIa complex, by Fab fragments against the D domain, and by metabolic inhibitors; aggregation was attenuated but not abolished by enzymatic removal of ADP (with CP/CPK) or by blockage of ADP binding sites (with FSBA), and when secretion was inhibited by aspirin. Fg-Fab'2(E) also induced a dose-dependent elevation in cytoplasmic Ca2+ (measured by Aequorin luminescence) which was attenuated by CP/CPK and by FSBA, and was eliminated by metabolic inhibitors and by anti-IIb/IIIa antibody. Fibrinogen complexes crosslinked with dimethylsuberimidate or Factor XIII neither aggregated gel-filtered platelets nor inhibited platelet aggregation by ADP and fibrinogen, probably because of inaccessibility of lysine residues in the D (terminal) domain of fibrinogen, which are thought to be required for platelet binding. Thus, soluble complexes of fibrinogen having multiple available platelet receptor recognition sites activate gel-filtered platelets and may provide a useful model for platelet-surface interactions mediated by adsorbed fibrinogen.

Adenosine Diphosphate↗
PubMed1984

Complex formation of platelet membrane glycoproteins IIb and IIIa with the fibrinogen D domain.

Glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) form a macromolecular complex on the activated platelet surface which contains the fibrinogen-binding site necessary for normal platelet aggregation. To identify the specific region of the fibrinogen molecule responsible for its interaction with the GPIIb-GPIIIa complex, purified fragment D1 (Mr = 100,000) and fragment E (Mr = 50,000) were prepared from plasmin digests of purified human fibrinogen. In addition, the polypeptide chain subunits A alpha, B beta, and gamma of fibrinogen were prepared. Using an enzyme-linked immunosorbent assay we have demonstrated that isolated fragment D1 in a solid phase system forms a complex with a mixture of GPIIb and GPIIIa. The binding of the GPIIb-GPIIIa mixture to fragment D1-coated plates reached saturation at 8 nM and to fibrinogen-coated plates at 24 nM. Isolated A alpha, B beta, and gamma chains were not reactive with added glycoproteins. Fragment E coated directly on plastic plates or immobilized on antibody-coated plastic plates did not form a complex with GPIIb-GPIIIa. Only fluid phase fibrinogen and fragment D1 but not fragment E were inhibitory toward formation of a complex between solid phase fibrinogen and GPIIb-GPIIIa. Isolated A alpha, B beta, and gamma chains at concentrations equivalent to fluid phase fibrinogen were inactive. Binding of fragment D1 but not fragment E to the GPIIb-GPIIIa complex was also demonstrated by rocket immunoelectrophoresis of the membrane glycoprotein mixture through a gel containing the individual fragments and subsequent autoradiography of the complex following exposure to 125I-anti-fibrinogen. These observations with isolated platelet membrane glycoproteins provide strong evidence that each of the D domains of the fibrinogen molecule interacts directly with the GPIIb-GPIIIa complex on the activated platelet surface, thus allowing formation of a tertiary molecular "bridge" across the surface of two adjacent activated platelets.

Blood Platelets↗
PubMed1982

The binding of fibrinogen to its platelet receptor.

Specific receptors for fibrinogen can be induced on platelets by a variety of stimuli. In this study, two independent approaches have implicated the D domain of fibrinogen in its interaction with the platelet. Immunochemically purified Fab fragments of anti-fibrinogen and anti-D inhibited 125I-fibrinogen binding to platelets in a dose-dependent fashion and platelet aggregation. In contrast, Fab fragments of anti-E produced only a slight inhibition of fibrinogen binding and nonimmune Fab fragments had no effect. Fibrinogen was digested with plasmin in the presence of 5 mM calcium and fragment D and E of Mr 100,000 and 50,000, respectively, were isolated. This D fragment inhibited 125I-fibrinogen binding to platelets in a concentration-dependent fashion, whereas the E fragment was ineffective. With 125I-fibrinogen at 0.17 microM, nonlabeled fibrinogen inhibited binding by 50% at 0.7 microM, whereas 170 microM fragment D was required to produce 50% inhibition. D fragment of Mr 80,000, generated in the absence of calcium, was noninhibitory. These observations provide strong evidence for the participation of the D domain in the binding of fibrinogen by its platelet receptor and suggest that the recognition site is lost in the conversion of the Mr 100,000 to 80,000 D species.

Adenosine Diphosphate↗
PubMedDate not supplied

Fibrinogen adsorption to receptor-like biomaterials made by pre-adsorbing peptides to polystyrene substrates.

Two peptides from the ligand-binding site of the platelet receptor GPIIb/IIIa, residues 296-306 of GPIIb, designated B12 by D'Souza et al. (1991), and 300-311 of GPIIb, designated G13 by Taylor et al., (1992), as well as two control peptides, designated C14 and C20, were adsorbed to treated polystyrene substrates. Fibrinogen adsorption to the peptide-coated substrates was characterized. The specificity of I-125 labeled fibrinogen binding to the peptide-coated substrates was investigated by measuring the amount of fibrinogen adsorbed to each substrate and the inhibition of fibrinogen binding by RGDS peptide, bovine serum albumin, a divalent ion chelator (ethylene diamine tetra-acetic acid disodium salt), unlabeled fibrinogen and the B12 peptide. The results show that non-specific binding of fibrinogen to the B12-coated substrate is predominant under most conditions. Binding of monoclonal antibodies to fibrinogen adsorbed to the peptide coated substrates was characterized. The failure of several antibodies to bind fibrinogen adsorbed to the B12 substrate suggested that adsorption of fibrinogen to the B12-coated substrate alters its conformation relative to fibrinogen adsorbed to the bare substrate.

Adsorption↗
PubMed1980

Fibrinogen Chapel Hill: hypodysfibrinogenemia with a tertiary polymerization defect.

A patient with a functionally defective fibrinogen (fibrinogen Chapel Hill) has been investigated. Fibrinogen Chapel Hill is characterized by hypofibrinogenemia, with a plasma concentration about one third of normal, as measured both functionally and immunochemically. Fibrinogen survival is normal; so also is fibrinopeptide release. A polymerization defect in this fibrinogen results in the delay of fibrin fibrils in solution to form a normal three-dimensional gel. This defect is not associated with end-to-end aggregation or with lateral associations in solution. Delayed gelation results from an abnormality in a tertiary contact site involved in network branching, which is associated with the hydrophilic, carboxy-terminal segment of the alpha chain. Fibrinogen Chapel Hill exhibits two additional abnormal responses, which are also associated with the same region. The early plasmin cleavages of fibrinogen and fragment X are delayed, and there is a concomitant delay in the appearance of fragments Y, D, and E. This fibrinogen also has an unusual sensitivity to Ancrod proteolysis, whereby Ancrod cleaves a large carboxy-terminal segment of the alpha chain more rapidly than in normal fibrinogen. The abnormalities in fibrinogen Chapel Hill can be explained by a structural abnormality which is functionally related to an alpha chain associated polymerization domain.

Adult↗
PubMed2001

Differential degradation of the three fibrinogen chains by proteasomes: involvement of Sec61p and cytosolic Hsp70.

HepG2 cells, which synthesize and secrete fibrinogen, accumulate surplus Aalpha and gamma chains. The nonsecreted fibrinogen chains are degraded both by proteasomes and lysosomes, with unassembled chains primarily degraded by proteasomes and an Aalpha-gamma complex by lysosomes. To further determine the mechanisms by which unassembled fibrinogen chains are degraded, and to explain the pools of Aalpha and gamma chains that occur in HepG2 cells, the association of fibrinogen chains with Sec61beta, a component of the translocon, and with a cytosol chaperone, Hsp70, was studied in both HepG2 cells and COS cells expressing single fibrinogen chains. Retrotranslocation from the lumen of the endoplasmic reticulum was shown by treatment with MG132, a proteasome inhibitor. MG132 caused glycosylated Bbeta to accumulate on Sec61beta in COS cells expressing Bbeta and acted similarly with all three fibrinogen chains in HepG2 cells. In HepG2 cells, Bbeta was associated with Sec61beta ahead of Aalpha and gamma chains, suggesting that pools of Aalpha and gamma chains may be caused by unequal rates of retrotranslocation. In COS cells, retrotranslocation into the cytoplasm was demonstrated by the ATP-sensitive association of ubiquitinylated Aalpha, Bbeta, and gamma chains bound to Hsp70. More Aalpha and gamma than Bbeta accumulated on Hsp70 of HepG2 cells, consistent with more rapid degradation of Bbeta. Overexpression of Hsp70 in HepG2 cells resulted in decreased secretion, but not synthesis, of fibrinogen. Decreased secretion may be due to enhanced degradation of unassembled fibrinogen chains, indicating that proteolysis by proteasomes might regulate both the intracellular pools of fibrinogen chains and fibrinogen secretion.

Adenosine Triphosphate↗
PubMed2004

Fibrinogen stimulates in vitro angiogenesis by choroidal endothelial cells via autocrine VEGF.

BACKGROUND: The purpose of this study is to investigate the effect of fibrinogen on angiogenesis in vitro formed by cultured bovine choroidal endothelial cells (BCECs) and the involvement of vascular endothelial growth factor (VEGF) in this mechanism. METHODS: For in vitro tube formation assay, BCECs were seeded on collagen gel containing fibrinogen (0-1.5 mg/ml). After 3 days of cultivation, the total length of the tubular structure was measured using Macscope Analyzer. Total RNA and conditioned media were collected after fibrinogen treatment and subjected to Northern and Western blot analyses, respectively. Transcription factor HIF-1alpha was also analyzed by Western blot analysis using cytosolic and nuclear fraction of BCECs. Involvement of VEGF in fibrinogen-dependent in vitro tube formation was evaluated using anti-VEGF neutralizing antibody or VEGF receptor 2-selective inhibitor (SU5416). RESULTS: Formation of the tubular structure was enhanced 20 to approximately 50 times in fibrinogen-containing gel in a concentration-dependent manner. The treatment of BCECs with fibrinogen resulted in a significant increase in VEGF gene and protein expression. Accumulation of HIF-1alpha protein in the nuclear fraction was also detected after the treatment with fibrinogen. Finally, fibrinogen-induced tube formation was significantly inhibited in the presence of anti-VEGF-neutralizing antibody (52.0% inhibition at the concentration of 1 microg/ml, P<0.05) or SU5416 (54.8% inhibition at the concentration of 3 microM, P<0.05). CONCLUSIONS: Extravasated fibrinogen might play an important role in the development of choroidal neovascularization associated with age-related macular degeneration, at least in part, through the function of VEGF in an autocrine manner. Transcription factor HIF-1 appears to be involved in fibrinogen-induced VEGF expression.

Angiogenesis Inhibitors↗