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PubMed2006

Fibrinogen and coronary heart disease: test of causality by 'Mendelian randomization'.

BACKGROUND: Blood concentrations of fibrinogen have been associated with coronary heart disease risk in epidemiological studies, but it is uncertain whether this association is causal or reflects residual confounding by other risk factors. We investigated the relationship between the single nucleotide polymorphism at position -148 in the beta-fibrinogen gene promoter (beta - 148C/T), blood fibrinogen levels, and risk of myocardial infarction (MI) in sufficiently large numbers of coronary disease cases to reliably address this question. METHODS: Genotyping and measurement of blood fibrinogen concentration were carried out in 4,685 cases of confirmed MI and 3,460 controls with no history of coronary disease. A meta-analysis of ISIS and 19 other studies of beta-fibrinogen genotypes involving a total of 12,220 coronary disease cases and 18,716 controls was conducted. RESULTS: Among the ISIS controls, mean plasma fibrinogen concentrations with the C/C, C/T and T/T genotypes were 3.34 (SE 0.015), 3.48 (0.022), and 3.60 (0.064) g/l, respectively, corresponding to an increase of 0.14 (0.024) g/l per T allele (trend P < 0.0001). In the case-control comparison, 0.14 g/l higher usual plasma fibrinogen concentration was associated with an age-adjusted and sex-adjusted risk ratio for MI of 1.17 [95% confidence interval (95% CI) 1.14-1.19; P < 0.0001]. But, after further adjustment for smoking, body mass index, and plasma apolipoprotein B/A(1) ratio, this risk ratio fell to 1.03 (95% CI 1.00-1.05; P = 0.05). Moreover, fibrinogen genotype was not significantly associated with MI incidence: risk ratio of 1.06 (95% CI 0.96-1.16) per higher-fibrinogen allele in ISIS alone and of 1.00 (95% CI 0.95-1.04) per allele in the meta-analysis. CONCLUSIONS: Genotypes that produce lifelong differences in fibrinogen concentrations do not materially influence coronary disease incidence. As these genotype-dependent differences in fibrinogen were allocated randomly at conception (Mendelian randomization), this association is not likely to be confounded by other factors. Consequently, these genetic results provide strong evidence that long-term differences in fibrinogen concentrations are not a major determinant of coronary disease risk.

Adult↗
PubMed1976

In vivo behaviour of homologous urea-soluble 131I-fibrin and 125I-fibrinogen in rabbits: the effect of fibrinolysis inhibition.

The in vivo behaviour of urea-soluble fibrin monomer (FM) was compared with that of fibrinogen in rabbits. Purified rabbit 125I-fibrinogen was injected into 36 unanaesthetized rabbits. Three days later the rabbits received either purified rabbit 131I-FM I mg/kg body weight, which corresponds to cI% of the circulating plasma fibrinogen pool 131I-fibrinogen, or buffered urea only. The distribution volume of FM was 43.4 +/- 6.9 ml/kg and of fibrinogen 43.5 +/- 7.7 ml/kg (mean +/- SD). The elimination curve of urea-soluble FM as represented by the clottable 131I-radioactivity. plotted on a semi-logarithmic paper, consisted of an initial steep decay within the first 6 h and a slow flattening of the slope 6 to 24 h after injection of FM. Although a terminal single-exponential slope, as observed in fibrinogen elimination, could not be computed for the 24 h following FM injection the mean half-life time of the last segment of the clottable-radioactivity curve, between 12 h and 24 h, was 12 h. Within 24 h a mean of 83-5% of the injected FM were removed from the circulating blood. The elimination characteristics of fibrinogen were not influenced by the injected FM. Control experiments showed that buffered 3.0 M urea, the solvent of FM, does not influence distribution volume and elimination of 125I-fibrinogen. The distribution of 131I as well as 125I-radioactivities in organs representing FM and fibrinogen respectively did not differ from each other. Elevated levels of 131I-radioactivity, however, were found in the urine after FM injection suggesting an accelerated elimination of FM in comparison to fibrinogen. Fibrinolysis inhibition with high doses of aprotinin did not significantly reduce the urinary excretion of 131I-radioactivity representing breakdown of FM. Furthermore, inhibition of the fibrinolytic system had no influence on the elimination characteristics of either fibrinogen or FM. In order to explain the results obtained in this study the following theory is proposed. Intravenously injected FM forms complexes with fibrinogen and fibrinolytic degradation products. The complexes continuously increase in size until they dissociate into fibrin oligomers and carrier proteins. The oligomers are eliminated whereas the carrier proteins recycle.

Animals↗
PubMed1986

Expression of intracellular fibrinogen on the surface of stimulated platelets.

Participation of fibrinogen in platelet aggregation is contingent upon the capacity of various stimuli to induce specific receptors for the molecule on the surface of the cell. The interaction of fibrinogen with this receptor results directly in platelet aggregation, and dissociation of fibrinogen is associated with disaggregation. While the role of exogenous fibrinogen in this process has been fully documented, the mechanisms which control the surface exposure of platelet fibrinogen are less understood. In the present study Fab fragments of antibodies monospecific for fibrinogen have been used to examine the surface expression of intracellular fibrinogen and its involvement in platelet aggregation. Radiolabelled Fab fragments did not interact with non-stimulated platelets but significant binding was observed when the cells were stimulated by ADP, thrombin, collagen and Ca ionophore A23187. Binding was specific for fibrinogen, was not observed with thrombasthenic platelets and was dependent upon the presence of extracellular calcium. With all stimuli tested, the binding of the Fab probe to platelets correlated with platelet secretion. At the following concentrations of stimuli: 30 microM ADP, 4 micrograms/ml collagen, 3 microM A23187 and 0.05 U/ml thrombin, the immune Fab fragments inhibited platelet aggregation. A monoclonal antibody to glycoprotein IIb/IIIa complex and a synthetic peptide gamma 400-411, that inhibited the interaction of plasma fibrinogen with platelets, did not inhibit the binding of 125I-FAB fragments. Taken together these results support the hypothesis that endogenous fibrinogen becomes surface-expressed during stimulation of the cell and can support platelet aggregation, particularly that induced by low concentrations of stimuli. The mechanism for the surface expression of platelet fibrinogen may be distinct from that for the binding of plasma fibrinogen.

Antibody Specificity↗
PubMed2004

Fibrinogen binding potentiates FGF-2 but not VEGF induced expression of u-PA, u-PAR, and PAI-1 in endothelial cells.

Endothelial cell responses at sites of injury occur in a fibrin matrix and are regulated by growth factors including those of the FGF and VEGF families. The pericellular proteolytic balance is important in these responses, and FGF-2 and VEGF up-regulate endothelial cell u-PA, u-PAR and PAI-1. Because both VEGF and FGF-2 bind to fibrinogen, we have examined the capacity of fibrinogen to modulate the up-regulation of these proteins by FGF-2 and VEGF. Confluent cultures of endothelial cells were exposed to FGF-2, VEGF, and fibrinogen or to combinations of growth factors with fibrinogen. Changes in mRNA levels of u-PA, u-PAR and PAI-1 were measured by Northern blot. FGF-2 increased u-PA, u-PAR, and PAI-1 mRNA, but there was a significantly greater induction when fibrinogen was added to FGF-2 at all concentrations. The potentiation by fibrinogen was particularly evident at an FGF-2 concentration of 0.1 ng mL(-1), which resulted in non-significant change in transcript levels by itself, but significantly increased up to 2.6-fold with fibrinogen. VEGF also increased endothelial cell expression of u-PA, u-PAR and PAI-1, but this effect was not potentiated by fibrinogen. Addition of LM609, a monoclonal antibody to alphaVbeta3, significantly inhibited induction of u-PA mRNA and activity by fibrinogen-bound FGF-2 compared to FGF-2. A monoclonal antibody to FGFR1 also inhibited u-PA mRNA expression induced by fibrinogen-bound FGF-2. We conclude that fibrinogen increases the capacity of FGF-2, but not of VEGF, to up-regulate u-PA, u-PAR, and PAI-1 in endothelial cells and that fibrinogen-bound FGF-2 requires alphaVbeta3 binding to up-regulate endothelial cell u-PA.

Base Sequence↗
PubMed2004

Variable hypocoagulant effect of fish oil intake in humans: modulation of fibrinogen level and thrombin generation.

OBJECTIVE: The beneficial effect of dietary fish oil, rich in omega-3 polyunsaturated fatty acids (PUFAs), on cardiovascular disease is multifactorial and may partly rely on their anticoagulant action. We studied how fish oil intake influenced thrombin generation in plasma and which factors were involved herein. METHODS AND RESULTS: Twenty-five healthy males with borderline overweight received 3.0 g omega-3 PUFAs daily for 4 weeks. Fish oil intake reduced plasma triglycerides and lowered platelet integrin activation, as well as plasma levels of fibrinogen and factor V, but had no effect on vitamin K-dependent coagulation factors. Before fish oil intake, thrombin generation (reflecting the coagulant potential) considerably varied between plasmas from individual subjects, which were partly explained by variation in prothrombin, antithrombin, fibrinogen, and factor V levels. Fish oil intake reduced thrombin generation in the presence and absence of platelets. This reduction correlated with the fish oil effect on fibrinogen and factor V levels. Interestingly, the lowering effect of fish oil on thrombin generation and fibrinogen clustered around subjects with high fibrinogen carrying a structural fibrinogen alpha-chain polymorphism. CONCLUSIONS: Dietary omega-3 PUFAs provoke a hypocoagulant, vitamin K-independent effect in humans, the degree of which may depend on fibrinogen level. Intake of fish oil reduced fibrinogen and factor V levels as well as thrombin generation in plasma. The effects on thrombin generation and fibrinogen clustered around subjects with high fibrinogen carrying alpha-chain fibrinogen polymorphism. Thus, dietary fish oil can provoke a hypocoagulant effect depending on the fibrinogen level.

Adult↗
PubMed1994

Intraindividual variability of fibrinogen levels and cardiovascular risk profile.

Prospective population studies have established that fibrinogen is an independent predictor for ischemic heart disease and stroke. These study conclusions have prompted recommendations that fibrinogen determinations be included in the cardiovascular risk profile. The routine availability of fibrinogen measurements may result in widespread screening prior to establishing the validity of a single fibrinogen level as an accurate descriptor for individual subjects. The objectives of this study were to describe the methodological and intraindividual components of variability in fibrinogen measurements determined by using the Clauss method; to establish the usefulness of a single fibrinogen measurement on risk stratification and retest reproducibility; and to determine the influence of intraindividual fibrinogen variability on sample size estimates. Fibrinogen levels were measured by a modification of the Clauss method. Three cohorts of apparently healthy, nonsmoking volunteers were recruited. The single-day intra-individual component of fibrinogen variability was determined in 39 subjects. For the 5-day intraindividual component of fibrinogen variability, 32 subjects were recruited, and in the 6-week intraindividual study, 28 subjects were included. The coefficient of variation for the methodological component of fibrinogen variability was 5.8% as determined from batch analyses, but the intraindividual coefficient of variation for replicate measures on a single day was 10.7%. The 5-day intraindividual coefficient of variation was 14.2%, and for the 6-week period it was 17.8%. Based on the 6-week data, an average of four fibrinogen measures is required to reduce misclassification error to less than 10%. Sample size estimates were made based on predetermined levels of statistical power and the 6-week intraindividual and interindividual variability estimates.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1996

Association between serum fibrinogen concentrations and HDL and LDL subfraction phenotypes in healthy men.

Hyperfibrinogenemia and a dyslipoproteinemia characterized by reduced HDL2 cholesterol and elevated levels of small, dense LDL particles are risk factors for coronary artery disease. However, the relationship between fibrinogen and lipoproteins, in particular LDL subfractions, is uncertain. We therefore measured serum fibrinogen levels and serum concentrations of cholesterol and apolipoproteins of VLDL, IDL, six LDL, and two HDL subfractions by using the technique of density-gradient ultracentrifugation in 132 nonsmoking men without evidence of coronary artery disease or infection. Dividing the individuals into quartiles according to their fibrinogen values showed that men within the highest fibrinogen quartile (fibrinogen 2.90 to 4.34 g/L) had significantly higher concentrations of small, dense LDL (d > 1.044 g/mL) apolipoprotein B and cholesterol and lower concentrations of HDL2 cholesterol than men within the lower fibrinogen quartiles (fibrinogen < 2.55 g/L). Multivariate regression analysis revealed that the association between fibrinogen and small, dense LDL particles was independent of serum triglycerides, cholesterol, body mass index, and age. In contrast, the relationship between fibrinogen and HDL2 cholesterol was primarily influenced by triglycerides and cholesterol and not independently influenced by fibrinogen. There were no significant differences between the quartiles in terms of insulin, glucose, insulin resistance, free fatty acids, lipoprotein(a), and blood pressure. This study showed that fibrinogen is associated with the expression of a more atherogenic LDL subfraction phenotype independent of body mass index, age, other serum lipids, and insulin resistance in a healthy male nonsmoking population. The reason for this association is uncertain. These findings reinforce the evidence that fibrinogen should be determined when assessing coronary risk.

Aging↗
PubMed1971

Metabolism of fibrinogen in cirrhosis of the liver.

The metabolism of human fibrinogen labeled with radioactive iodine was studied in 50 patients with documented cirrhosis of the liver and in 35 healthy control subjects. Results in cirrhotic subjects were the following: plasma volume 47 +/- 10 ml/kg; plasma fibrinogen concentration 250 +/- 102 mg/100 ml; total plasma fibrinogen pool 118 +/- 59 mg/kg, representing 0.73 +/- 0.10 of the total body pool; fibrinogen half-life 2.99 +/- 0.59 days; fractional catabolic rate 0.34 +/- 0.09 of the plasma pool per day; absolute catabolic rate 39 +/- 20 mg/kg per day; fractional transcapillary efflux rate 0.82 +/- 0.30 of the plasma pool per day. Results in the control subjects were the following: plasma volume 42 +/- 7 ml/kg; plasma fibrinogen concentration 284 +/- 71 mg/100 ml; total plasma fibrinogen pool 119 +/- 40 mg/kg, representing 0.72 +/- 0.07 of the total body pool; fibrinogen half-life 4.14 +/- 0.56 days; fractional catabolic rate 0.24 +/- 0.04 of the plasma pool per day; absolute catabolic rate 28 +/- 9 mg/kg per day; fractional transcapillary efflux rate 0.60 +/- 0.26 of the plasma pool per day.A significant difference between cirrhotics and controls was observed for plasma volume, fibrinogen half-life, fractional and total catabolic rates, and transcapillary efflux rate. During heparinization of 10 cirrhotic patients the fibrinogen half-life was prolonged from 3.15 +/- 0.69 to 4.59 +/- 0.79 days. This was associated with a rise in plasma fibrinogen in six out of eight patients. Heparinization did not influence the fibrinogen half-life in five control subjects. Inhibition of the fibrinolytic system in 17 patients resulted in prolongation of the plasma radioactivity half-life of more than 1 day in only three patients, an incidence comparable with that in five control subjects. These results strongly support the concept of accelerated fibrinogen consumption by a process of disseminated intravascular coagulation in cirrhosis of the liver.

Adult↗
PubMed1981

A role for prostaglandins and thromboxanes in the exposure of platelet fibrinogen receptors.

Exposure of fibrinogen receptors by a variety of agonists is a prerequisite for platelet aggregation. Because the synthesis of prostaglandins and thromboxane A2 also occurs during platelet aggregation we wondered whether these agents participate in the exposure of platelet fibrinogen receptors. Therefore, we measured the binding of human 125I-fibrinogen to gel-filtered normal human platelets after prostaglandin and thromboxane synthesis had been inhibited by aspirin or indomethacin. The fibrinogen binding assay was performed at 37 degrees C but without stirring to prevent the formation of platelet aggregates. Platelet secretion, measured with [14C]serotonin, did not occur during the procedure. Aspirin or indomethacin inhibited fibrinogen binding stimulated by 10 microM epinephrine by 53%, and inhibited fibrinogen binding stimulated by 1-2 microM ADP by 37.1%. However, ADP at concentrations greater than 2 microM returned fibrinogen binding toward control values. Scatchard analysis demonstrated that aspirin decreased the number but not the affinity of the exposed fibrinogen receptors. To determine whether prostaglandins are capable of directly exposing fibrinogen receptors, prostaglandin H2 was used to stimulate platelets in the fibrinogen binding assay. Prostaglandin H2 exposed approximately 54,000 fibrinogen receptors/platelet and corrected the deficit in receptor exposure induced by aspirin. These studies demonstrate that platelet prostaglandins or thromboxane A2 can play a direct role in the exposure of platelet fibrinogen receptors. In addition, they suggest that the synthesis of prostaglandins and thromboxane A2 by stimulated platelets may be all that is required for optimal secondary platelet aggregation.

Adenosine Diphosphate↗
PubMed1988

Fibrinogen has a rapid turnover in the healthy newborn lamb.

The half-lives for coagulation factors in the healthy newborn infant are not known and may be different than for the adult. We measured the half-life for fetal sheep fibrinogen and compared it to the half-life of adult sheep fibrinogen. Fibrinogen was purified from adult and fetal sheep plasma and radiolabeled with either 125I or 131I. The half-lives for these fibrinogens were determined in the adult sheep and newborn lamb. In addition, the fetal and adult sheep fibrinogens were compared by reptilase time, thrombin clotting time, sialic acid content, and the behavior of the N-glycans derived from these fibrinogens on the immobilized lectin, Sepharose-concanavalin A. Finally, the in vivo response of coinjected radiolabeled fibrinogens to increasing doses of infused thrombin was determined. The fetal sheep fibrinogen differed from the adult as indicated by a prolonged reptilase time and an increased sialic acid content (fetal: 10-11 residues/340 Kd versus adult: 8-9 residues/340 Kd). The latter was also reflected in differing chromatographic profiles for the N-glycans on Sepharose-concanavalin A. The half-lives for both the adult and fetal fibrinogen were significantly more rapid in the newborn lamb (fetal: 47 +/- 2.0 h; adult: 46 +/- 2.4 h, mean +/- SEM) than in the adult (fetal: 116 +/- 6.5 h; adult: 121 +/- 6.9 h). Finally, the adult and fetal sheep fibrinogen responded to thrombin in an identical fashion in the intact animal. In summary, both adult and fetal fibrinogen have faster half-lives in the lamb compared tot he adult, despite a higher sialic acid content for the fetal fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed2005

Relationship among urinary albumin excretion rate, lipoprotein lipase PvuII polymorphism and plasma fibrinogen in type 2 diabetic patients.

Plasma fibrinogen level represents a strong cardiovascular risk factor and is regulated by an interplay of genetic and environmental factors. Hyperfibrinogenemia frequently occurs in cluster with dyslipidemia within the frame of insulin resistance syndrome (IRS) and type 2 diabetes mellitus. Genetic variants with a pleiotropic effect have been proposed to cause IRS features including hyperfibrinogenemia. We studied the influence of polymorphisms in lipoprotein lipase (LPL) gene, beta-fibrinogen gene (FIBB) and environmental factors on plasma fibrinogen levels in type 2 diabetes patients. 131 type 2 diabetes patients (mean age 62+/-10 years, 33% male) were genotyped for polymorphisms in LPL gene (intron 6 PvuII, intron 8 HindIII) and FIBB gene (-148C/T, -455G/A) by PCR-RFLP method. Fibrinogen was measured by thrombin coagulation method, albuminuria by immunoturbidimetric assay. Polymorphism LPL PvuII showed a gene-dose effect on fibrinogen levels, with the highest fibrinogen in P-P- homozygotes (p = 0.05, analysis of variance). P-carriers (P-P- and P+P- combined) had significantly higher fibrinogen levels compared with P+P+ homozygotes (3.74+/-1.40 g/l vs 3.06+/-1.20 g/l, p=0.03). Other studied polymorphisms were not significantly related to fibrinogen levels. Age- and sex-adjusted fibrinogenemia correlated significantly with albuminuria (r = 0.48, p=0.001), serum uric acid (r = 0.42, p=0.006) and serum creatinine (r = 0.32, p=0.04). Multiple stepwise linear regression identified interaction term of LPL PvuII and albuminuria as an independent predictor of fibrinogen level, explaining 18% of fibrinogen variance. Albuminuria thus appears to be the best predictor of fibrinogen plasma levels in type 2 diabetic patients. Relationship between albuminuria and fibrinogenemia may be modified by the genotype LPL PvuII, which also shows a weak association with plasma fibrinogen level in type 2 diabetes patients.

Albuminuria↗
PubMed2001

Fibrinogen biosynthesis. Assembly, intracellular degradation, and association with lipid synthesis and secretion.

Plasma fibrinogen is synthesized primarily in hepatocytes and assembly of the three component chains (A alpha, B beta, and gamma) into its final form as a six-chain dimer (A alpha, B beta, gamma)2 occurs rapidly in the lumen of the endoplasmic reticulum (ER). Assembly takes place in a stepwise manner with single chains interacting with each other to form A alpha-gamma and B beta-gamma complexes. The two-chain complexes then acquire another chain to form half-molecules (A alpha, B beta, gamma)1, which in a final step are linked to form the six-chain (A alpha, B beta, gamma)2 complex. As with other secreted glycoproteins, N-linked glycosylation of B beta and gamma chains commences in the ER and is completed in Golgi organelles. Sulfation and phosphorylation occur at post-ER stages of the secretory process. Since some ER chaperones coisolate with nascent fibrinogen chains they have been implicated in assisting chain assembly. Studies with recombinant systems, using deletion and substitution mutants, indicate that initial chain assembly depends on hydrophobic interactions present in the C-terminal half of the coil-coil domains and that inter- and intra-disulfide bonds that stabilize fibrinogen are needed to complete chain assembly. Not all the chains that are synthesized are assembled into fibrinogen and the unassembled chains are not secreted. HepG2 cells contain surplus A alpha and gamma chains that accumulate as free gamma chains and as an A alpha-gamma complex. A alpha-gamma is degraded by lysosomes whereas the gamma chain is degraded by the proteasome-ubiquitin system. Studies with expression of single chains by COS cells confirm that gamma and B beta are hydrolyzed by proteasomes and indicate that A alpha is degraded partially both by lysosomes and proteasomes. The role of surplus chains in regulating fibrinogen assembly is not understood but overexpression of any one chain, elicited by transfection of HepG2 cells, results in the upregulation of the other two genes, increased fibrinogen synthesis and secretion, and maintenance of surplus intracellular A alpha and gamma chains. HepG2 cells, programmed in this manner to increase basal fibrinogen expression, have higher HMG-CoA reductase mRNA levels, enhanced cholesterol and cholesterol ester synthesis, and increased secretion of apolipoprotein B (apoB). Overexpression of basal levels of fibrinogen does not affect synthesis of other acute phase proteins. Enhanced secretion of apoB is due to diminished degradation of nascent apoB by proteasomes and not to increased expression. Increased secretion of apoB is associated with increased basal expression of fibrinogen and is not affected when fibrinogen expression is stimulated by interleukin-6. In HepG2 cells, a feedback mechanism exists and extracellular sterols specifically downregulate expression of the three fibrinogen genes. These studies link, at the cellular level, basal fibrinogen expression with lipid metabolism.

Animals↗
PubMed2001

Protransglutaminase (factor XIII) mediated crosslinking of fibrinogen and fibrin.

Plasma factor XIII (plasma protransglutaminase) circulates as an A2B2 tetramer bound to the gamma' variant chains of fibrinogen "2". During clotting the A subunits of fXIII are cleaved by thrombin to form fXIIIa (transglutaminase) and in the presence of calcium ions, activated A2* subunits dissociate from the B subunits. When purified plasma fXIII or recombinant cellular factor XIII (A2) was incubated with fibrinogen in the presence of calcium ions (> or =50 microM) a non-synerizing gel formed concomitant with formation of gamma dimers, followed by Agamma polymers, and eventually gamma trimers and gamma tetramers. As is the case of fXIIIa, the fXIII-mediated crosslinking rate was enhanced in the presence of thiols. After an initial lag period, fXIII catalyzed fibrinogen crosslinking at approximately 75% of the rate of fXIIIa under typical crosslinking conditions (100 Loewy u/ml, 5 mM CaCl2 & 500 microM DTT). Fibrin was crosslinked about 8 times more rapidly by fXIII than was fibrinogen, and after an initial lag period fXIII crosslinked fibrin at nearly the same rate as fXIIIa. Substituting plasma for purified fXIII as the source for fXIII resulted in robust fibrinogen crosslinking activity. In contrast to the high level of fXIII-mediated crosslinking activity observed with fibrinogen or fibrin as substrates, when transglutamination was measured using cadaverine incorporation into casein, fXIII was 30-fold less active than fXIIIa. Thus, factor XIII displays constitutive enzymatic activity with respect to fibrinogen and fibrin. The results further indicate that uncleaved fXIII in plasma provides a potent source of readily available crosslinking activity in clotting blood. Fibrinogen 2, whose gamma'chains bind fXIII B subunits, was crosslinked 3.5 times more slowly by fXIII than was fibrinogen 1 (lacking gamma' chains), suggesting that complex formation between fibrinogen 2 and plasma fXIII plays a significant role in down-regulating potential plasma fXIII-mediated crosslinking activity. Since fibrin is a considerably better substrate for fXIII than is fibrinogen, the rate at which crosslinking takes place in a fibrinogen-containing plasma environment is much lower than it would be if fibrin were present.

Calcium↗
PubMed2003

Oxysterols suppress constitutive fibrinogen expression.

Elevated levels of both fibrinogen and cholesterol are risk factors in coronary artery disease. Previously we reported a metabolic link between fibrinogen and lipid metabolism in that HepG2 cells that were programmed by transfection of Bbeta-fibrinogen cDNA to overexpress fibrinogen exhibited increased synthesis of cholesterol and increased secretion of apolipoprotein B. In this study we demonstrate that oxysterols, which participate in maintaining cholesterol homeostasis, also down regulate fibrinogen expression. Treatment of HepG2 cells with 25-hydroxycholesterol lowered fibrinogen Aalpha, Bbeta and gamma mRNA levels and inhibited fibrinogen synthesis and secretion but had no effect on alpha1 -antitrypsin which, like fibrinogen, is an acute-phase protein. The inhibition of fibrinogen synthesis by oxysterols was maintained in interleukin-6 treated cells. Other oxysterols, that inhibit cholesterol synthesis by a feedback mechanism, also diminished fibrinogen expression in HepG2, rat H-4-II-E hepatoma cells and in primary human hepatocytes. Overexpression of SREBP-1 and SREBP-2 by transfection of HepG2 cells, or treatment with a synthetic LXRalpha agonist, which affect cholesterol metabolism, did not affect fibrinogen expression. We conclude that fibrinogen and cholesterol may share a novel common regulatory pathway.

Acute-Phase Reaction↗
PubMed1992

[Fibrinogen seen as an independent cardiovascular factor from the molecular aspect].

The blood protein fibrinogen is one of the main independent cardiovascular risk factors and it is very likely that an increased fibrinogen level is not the consequence of cardiovascular disease but its direct cause. Fibrinogen participates actively in many processes in the organism and undergoes various changes of the molecule but it is not known what is the molecular background, why even a slight increase of the fibrinogen level is so dangerous as regards increased risk of cardiovascular attacks. In the present work the authors compared, using monoclonal antibodies, the rate of release of fibrinopeptides A and B (FpA, FpB) by the action of thrombin from fibrinogen in solution and from fibrinogen adsorbed to a solid surface. The authors revealed that the rate of FpB breakdown from sorbed fibrinogen, contrary to fibrinogen in solution is comparable to the release rate of FpA and does not depend on the release of FpA (except for competitive inhibition). The release of FpB from sorbed fibrinogen thus takes place, contrary to fibrinogen in solution, at a significant recordable rate from the very onset of thrombin action. Fibrinogen adsorption to a solid surface is associated with conformation changes which cause this effect and which lead to the formation of a two-dimensional formation formed by fibrinogen after its interaction with the surface of activated platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiovascular Diseases↗
PubMed1991

Fibrinogen Ledyard (A alpha Arg16----Cys): biochemical and physiologic characterization.

Fibrinogen Ledyard was discovered in a 10-year-old boy with a mild bleeding history. His father had the same defect and a bleeding history after surgery. Both patients were heterozygous. The plasma fibrinogen concentration was normal immunologically (335 mg/dL) and very low functionally (52 mg/dL). Purified fibrinogen Ledyard had a prolonged polymerization, which was somewhat corrected by addition of Ca2+ ions. High performance liquid chromatography (HPLC) analyses of the fibrinopeptides released by thrombin showed 1 mol of fibrinopeptide A (FPA) and 2 mol of fibrinopeptide B (FPB) released per mole of fibrinogen Ledyard. Steady-state kinetic parameters were evaluated for release of FPA by thrombin. When the concentration of fibrinogen Ledyard was corrected to 50% of total protein, because only 50% of fibrinogen Ledyard can release FPA, the kinetic constants were similar to those of control fibrinogen (Km = 7.5 mumol/L for A alpha chain, kcat = 54 s-1). This finding indicates that the cleavage site of the A alpha chain in these abnormal molecules may not interact with the catalytic site of thrombin. The three chains of fibrinogen Ledyard were isolated on reverse-phase C4-HPLC. The sequence of the amino terminus of A alpha chain showed that Arg in position 16 was replaced by Cys in the abnormal molecules. Approximately half of fibrinogen Ledyard (52%) was clotted by reptilase, suggesting that fibrinogen Ledyard may consist of 50% normal homodimers (A alpha Arg16 . A alpha Arg16) and 50% abnormal homodimers (A alpha Cys16 . A alpha Cys16). Abnormal molecules could form disulfide bond between the A alpha Cys16 residues. Thus, the abnormal molecules have a different structure that does not bind to thrombin. Probably the abnormality of polymerization of fibrinogen Ledyard results from the interaction of the abnormal molecules with normal fibrin monomers, so that the growth of fibrin protofibrils is inhibited. This abnormal fibrinogen supports adenosine diphosphate-induced platelet aggregation in a normal manner.

Amino Acid Sequence↗
PubMed1990

Interaction of integrins alpha v beta 3 and glycoprotein IIb-IIIa with fibrinogen. Differential peptide recognition accounts for distinct binding sites.

Glycoprotein (GP) IIb-IIIa is the major fibrinogen receptor on platelets and participates in platelet aggregation at the site of a wound. Integrin alpha v beta 3, which contains an identical beta-subunit, is expressed on endothelial cells and also serves as a fibrinogen receptor. Here, we demonstrate by several criteria that purified GPIIb-IIIa and integrin alpha v beta 3 bind to distinct sites on fibrinogen. First, a plasmin-generated fragment of fibrinogen lacking the RGD sequence at residues 572-574 retained the ability to bind GPIIb-IIIa, but failed to bind integrin alpha v beta 3. Second, a monoclonal antibody which exclusively recognizes the RGD sequence at fibrinogen A alpha chain residues 572-574 abolished interaction between integrin alpha v beta 3 and fibrinogen, but had only a minimal effect on fibrinogen binding to GPIIb-IIIa. Finally, we show that the difference in recognition of sites on fibrinogen by these two integrins is probably a consequence of their remarkably different ligand binding properties. Peptides corresponding to fibrinogen gamma chain residues 400-411 effectively blocked RGD sequence and fibrinogen binding by GPIIb-IIIa, but had no effect on the ability of integrin alpha v beta 3 to bind these ligands. We also show that integrin alpha v beta 3 has a higher affinity than GPIIb-IIIa for a synthetic hexapeptide containing the RGD sequence. In fact, this RGD-containing peptide was 150-fold more effective at blocking fibrinogen binding to integrin alpha v beta 3 than to GPIIb-IIIa. Collectively, our results demonstrate that integrins alpha v beta 3 and GPIIb-IIIa display qualitative and quantitative differences in their ligand binding properties, as is evident by their ability to interact with synthetic peptides. The ultimate result of these differences is the recognition of distinct sites on fibrinogen by the two integrins. These observations may have relevance in the processes of hemostasis and wound healing.

Amino Acid Sequence↗
PubMed1988

Mechanism of transient adsorption of fibrinogen from plasma to solid surfaces: role of the contact and fibrinolytic systems.

The transient detection of fibrinogen on surfaces has been described (Vroman effect) and high-mol-wt kininogen (HK) has been shown to play a role in this reaction. In this study, we attempted to identify the form of HK responsible for preventing detection of the fibrinogen initially adsorbed from plasma to various artificial surfaces and to determine if other plasma components were involved. We compared 125I-fibrinogen adsorption in the presence of normal plasma to plasma deficient in specific proteins. On all surfaces tested, we found that fibrinogen was displaced from the surface. The extent of displacement was greatly reduced, however, but not eliminated in HK-deficient plasma. Factor XII-deficient plasma also showed reduced fibrinogen displacement. These data indicate that HK can actually displace fibrinogen; however, factor XII, or a factor XII-mediated reaction also appears to be necessary for this displacement to occur. Furthermore, when normal plasma was first subjected to extensive contact activation by dextran sulfate, during which the HK was extensively degraded to components smaller than the light chain (as assessed by Western blotting), we observed greatly reduced displacement of fibrinogen. Extensive contact activation of Factor XI-deficient plasma failed to show low-mol-wt derivatives, however, and displacement of fibrinogen was similar to normal plasma that had not undergone extensive activation. These data indicate that HKa (active cofactor produced during contact activation by factor XIIa or kallikrein) is primarily responsible for displacing fibrinogen, and that HKi (inactive cofactor generated by factor XIa) cannot displace fibrinogen. The fibrinogen from all plasma samples looked similar by Western blot analysis, suggesting that fibrinogenolysis was not a component of the Vroman effect. In addition, experiments performed with plasma prechromatographed on lysine agarose showed that a lysine-agarose adsorbable protein may be minimally involved in fibrinogen desorption and a synergism may exist between HK and that protein.

Adsorption↗