Plasma fibronectin

Fibronectin (FN) is a glycoprotein (disulfite-bonded dimer of 200 to 220 Kd submits) found in a soluble form in blood (concentration 250--500 microg/ml), it can be removed from it by cryoprecipitation and affinity chromatography on gelatin or heparin-agarose. It is also found in an insoluble fibrillar form as a component of connective tissue matrix like collagen, proteoglycans... FN fundamentally forms molecular complexes with collagen, fibrinogen or fibrin, heparin, activated factor XIII, bacteria, cellular membranes..., these various proteins binding with now well known functional "domains" on subunits. Thus FN mediates adhesion of cells to cells as well to biomaterials or tissue, cell migration and chemotactic activity, tissue stromal organization... The transformed cultured cells in presence of oncogen virus loose ability to secrete FN which contribute to their invasive tendency. FN also interacts with hemostatic and fibrinolytic systems, as component of the subendothelium (secreted, like Willbrand factor, by endothelial cells) and of platelet alpha-granules released by stimulated platelets. FN could then provoke platelet spreading on the subendothelium surface after collagen-platelet adhesion, triggered by Willebrand factor, has happened. FN is a part of the fibrinous clot. It participates in anchorage of the clot to subendothelium and mediates its colonisation by fibroblasts, first step to wound reparation. Lastly FN probably has an important role in organism defence. It acts as a non-immunological opsonin, promoting phagocytosis by RES macrophages of bacteria, cellular or fibrin fragments, immune complexes... present in blood. Plasmatic FN concentration is strongly decreased in several ill patients following major trauma, extensive burns, shock, sepsis, with or not evidence of DIVC, of respiratory distress... SABA and various other authors have obtained good results after injections of FN (as cryoprecipitates or concentrated fractions). It is yet necessary to confirm therapeutic role of FN.     

Plasma fibronectin binds glucose

Equilibrium dialysis studies demonstrated that plasma fibronectin bound D-glucose with moderate affinity. The binding of glucose by plasma fibronectin caused the dissoclation of plasma fibronectin-gelatin complexes. Glucose and gelatin did not compete for the same binding sites on plasma fibronectin. The glucose-caused dissociation of plasma fibronectin from plasma fibronectin-gelatinized horse erythrocyte complexes destroyed the potent hemagglutination activity of these complexes against trypsinized, formalinized sheep erythrocytes.     

Plasma fibronectin in mammary and uterine carcinomas

Plasma fibronectin was determined in cancer patients and in age- and sex-matched controls and analyzed as a function of age, size of tumor, receptor content of the tumor, metastases and treatment. In the control population, plasma fibronectin increased with age exponentially. The age-dependent increase in plasma fibronectin was strongly attenuated in the cancer population. As normal and cancer curves intersect at about 40-46 years, below this age cancer plasmas have slightly higher values than normal, above this age the inverse is true. No correlation was found between estrogen or progesterone receptor levels and plasma fibronectin values, nor with plasma albumin. Tumor patients with distant metastases gave slightly but significantly higher values than those with local or no metastases. No significant difference was found between tumors when Bloom grading was taken as the second parameter instead of age. The size of the tumor or the type of treatment had no influence. Increased proteolytic activity, increased trapping of plasma fibronectin in tissues and especially in the stromal (desmoplastic) reaction and/or modifications in plasma fibronectin biosynthesis may well be responsible for these results.     

Plasma fibronectin levels in patients with glomerular proteinuria

Plasma fibronectin concentration was measured by means of rocket immunoelectrophoresis in 20 cases of glomerular proteinuria of various origins, and correlated with urinary protein loss, serum albumin, cholesterol and plasma alpha 1 antitrypsin and alpha 2 macroglobulin. Plasma fibronectin was significantly increased in the patient's group as compared to the controls (1.91 +/- 0.659 U/ml, 1.01 +/- 0.193 U/ml respectively, p less than 0.001) and correlated with cholesterolaemia (r = 0.662, p less than 0.001). Increased plasma fibronectin may be an additional risk factor for thrombotic tendency in NS.     

Plasma fibronectin levels in acute and recovering malnourished children

58 malnourished children (mean age 18 months) with a clinical diagnosis of marasmus or kwashiorkor were studied with respect to plasma fibronectin levels, plasma total solids, spun hematocrits, heights, weights, mid-arm circumferences, and head circumferences. Bimodal distributions were demonstrated for plasma fibronectin versus weight deficits, total solids, hematocrits, and mid-arm circumference in children 12 months of age and older (p less than 0.003 for all). The mean plasma fibronectin level for controls was 253 micrograms/ml. The mean level for the malnourished group was 96 micrograms/ml (p less than 0.0001). Malnourished children with initial plasma fibronectin levels above 100 micrograms/ml had a higher survival rate than those with levels less than 100 (92 versus 69%). With successful therapy, plasma fibronectin levels rose quickly in most children often before detectable changes were noted in clinical and other laboratory parameters. An overshoot of the mean normal levels was observed with successful treatment wherein the mean levels rose to 315 micrograms/ml (p less than 0.05). Plasma fibronectin determinations on malnourished children can serve as an important prognostic marker as well as a reliable indicator of successful therapy and recovery.     

Plasma fibronectin in acute leukemia. Effects of the cytostatic therapy on plasma fibronectin level

Twice a week plasma (Pl.)-fibronectin was determined quantitatively in the course of disease with immunoelectrophoresis according to Laurell in 12 patients suffered from acute non-lymphoblastic leukemia (ANLL) and in 12 patients affected with acute lymphoblastic leukemia (ALL). At diagnosis Pl.-fibronectin concentration was found to be significantly lowered only in those patients affected with ANLL. During the induction therapy Pl.-Fibronectin could be observed to decline significantly in all patients: in acute non-lymphoblastic leukemia from mean 270 micrograms/ml, s 93 micrograms/ml, to mean 185 micrograms/ml, s 89 micrograms/ml (p less than 0.01), and in acute lymphoblastic leukemia from mean 290 micrograms/ml, s 98 micrograms/ml, to mean 180 micrograms/ml, s 94 micrograms/ml (p less than 0.01). After administering L-asparaginase there is a strong decline of Pl.-fibronectin. Pl.-fibronectin concentration could be observed to be significantly lower in patients without remission in comparison to those with remission. A correlation between Pl.-fibronectin concentration and tumour mass could not be identified.     

Plasma fibronectin is synthesized and secreted by hepatocytes

Primary cultures of hepatocytes of rats and hamsters were established and examined for the synthesis and secretion of fibronectin. Hepatocytes of both species secreted fibronectin as a soluble dimeric protein which could be purified by its affinity for gelatin and using specific antisera. Plasma and cellular fibronectins could be clearly resolved on two-dimensional gels. In both species, the majority of the fibronectin secreted by hepatocytes was of the plasma type, as shown by analyses on one- and two-dimensional gels. The secretion of plasma fibronectin increased with time in culture, both in absolute terms and relative to the secretion of albumin. Even during the first day of culture, the secretion of fibronectin relative to that of albumin appeared to be sufficient to account for the relative levels of these two proteins in plasma. Hepatocytes of both species secreted preferentially the chain of plasma fibronectin with higher apparent molecular weight, although the faster migrating chain was also secreted. In addition, hamster hepatocytes cultured for 2 or more days appeared to secrete a cellular form of fibronectin. Possible origins for the different chain types of cellular and plasma fibronectins are discussed.     

Plasma fibronectin: three steps to purification and stability.

Large amounts of soluble fibronectin were easily purified from cryoprecipitated or fresh citrated human blood plasma by a three-step combination of gelatin and heparin-cellufine affinity chromatography. The elution conditions were optimized to obtain a homogeneous fraction on SDS-PAGE and Western blot under reducing condition. No proteolytic activities were detected by zymography at acidic or neutral pH. Furthermore, the fibronectin preparation was stable over time up to 456 h at 37 degrees C in the presence of calcium, zinc, or mercury. This preparation of very stable fibronectin, called highly purified fibronectin (hpFN), gave a yield of 7.00 +/- 0.77 mg of fibronectin per gram of cryoprecipitated plasma and 0.16 mg of fibronectin per milliliter of fresh citrated, giving a yield of 32 to 53% (from presumed fibronectin concentration). This preparation may be useful for cellular tests and interaction analysis.     

Risk factors of pseudomonas infection in hemoblastosis patients

A total of 67 patients with diseases of the blood system and infectious complications, admitted to the Hematological Department of the Novosibirsk Regional Clinical Hospital, were examined. For this study only patients with etiologically established diagnosis were taken. The study revealed that Pseudomonas sp., whose strains were susceptible to Ceftazidime in 100% of cases and resistant to Cefepime and Imipenem in 15-17% of cases, was the etiological agent of 13.6% of all cases of infectious complications in hemoblastosis patients. Infectious lesions of pulmonary parenchyma, the presence of chronic diseases of the respiratory tract in the medical history, neutropenia, artificial ventilation of the lungs were found to be adverse prognostic factors with respect to a high risk of Pseudomonas infection in such patients. Therapy with glucocorticosteroids and cytostatics, preceding antibacterial prophylaxis were not linked with the isolation of Pseudomonas from the patients exhibiting the same levels of lethality and severity of infectious complications.     

Coronavirus infection in immunodeficient patients with hemoblastosis and deficient hemopoesis

Coronavirus infection (CVI) was studied in 227 patients hospitalized in the clinic of the Research Institute of Hematology and Transfusiology in 1993-2003 with diagnosed acute and chronic leucosis, multiple myelogenic disease and aplastic anemia. Their blood sera and secretions of the nasal cavity were examined in the indirect hemagglutination (IHA) test with dried standard erythrocyte diagnostic preparations. CVI was shown to be activated in three year cycles in immunodeficient patients, which occurred, respectively, in 66.1, 56.9, 47.8 and 51.6% of cases in the above mentioned groups of patients. In 87% of cases CVI was associated with other respiratory pathogens, the following being prevailed: respiratory syncytial virus (37.9%), parainfluenza virus (32.2%) and Mycoplasma pneumoniae (36.8%). CVI was provoked by such factors as the course of the main disease and specific treatment, previous respiratory infections of other etiology with M. pneumoniae infection playing the leading role (60%). The most severe course of CVI was observed in patients with acute leucosis (in 75% of cases accompanied by lesions of lower respiratory tracts). The use of the highly sensitive IHA test made it possible to determine the potential for both serum and local antibodies production in the patients under observation.     

Plasma fibronectin (Fn) study in homozygous beta-thalassemia: relation to splenectomy and transfusion

The concentration of plasma Fn was determined in non-splenectomized and splenectomized patients with homozygous beta-thalassemia, before and 7-10 days after blood transfusion. The mean Fn concentration of non-splenectomized patients before transfusion did not differ from that of matched normal controls but appeared significantly decreased following blood transfusion. On the other hand, in splenectomized thalassemics, Fn levels were increased but were unrelated to transfusion. It is concluded that Fn plays some homeostatic function when RES activity of thalassemic patients is altered either as a result of splenectomy or blood transfusion.     

Plasma fibronectin promotes modulation of arterial smooth-muscle cells from contractile to synthetic phenotype

Isolated arterial smooth-muscle cells (SMCs) cultured in medium containing whole blood serum or plasma-derived serum undergo modulation from a contractile to a synthetic phenotype. This process includes the loss of myofilaments and cessation of the ability to contract. Instead, an extensive rough endoplasmic reticulum and a large Golgi complex are formed and, if properly stimulated, the cells start to proliferate actively and to produce extracellular-matrix components. In vivo, a similar change in the differentiated properties of SMCs appears to be an early key event in atherogenesis. The purpose of the present investigation was to try to identify plasma components that promote the modulation of the smooth-muscle phenotype. SMCs were enzymatically isolated from rat aorta and cultured in a defined, serum-free medium. The phenotypic state of the cells was determined by transmission electron microscopy, and their growth status was followed by 3H-thymidine autoradiography and cell counting. Under these conditions, Cohn fractions I (fibrinogen) and V (albumin) were found to partially support cell attachment and transition from the contractile to the synthetic phenotype, whereas fractions II-III and IV (globulins) were inactive in this respect. Analysis on adsorptive columns of gelatin Sepharose 4B indicated that Cohn fraction I, but not fraction V, contained fibronectin, an adhesive protein that is present in plasma and binds to fibrinogen. When seeded on a substrate of plasma fibronectin, the cells attached with high efficiency and modulated into the synthetic phenotype at a rate similar to that observed in serum-containing medium. In the absence of exogenous mitogens, the structural transformation of the cells was not accompanied by a proliferative response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of extracellular fibronectin matrix formation with fluoresceinated fibronectin and fibronectin fragments

Fluorescein isothiocyanate conjugated human plasma fibronectin, 70-kDa collagen-binding, 60-kDa central, 60-kDa heparin-binding, 180-kDa heparin, collagen-binding fibronectin fragments and gelatin were used to study extracellular fibronectin matrix formation. Exogenous fibronectin, gelatin, 70-kDa collagen-binding and 180-kDa heparin, collagen-binding fragments were shown to be able to bind specifically to preexisting extracellular matrix of living fibroblasts. The results suggest that: (i) Fibronectin matrix formation may occur through a self-assembly process; (ii) the NH2-terminal part of fibronectin is responsible for fibronectin-fibronectin interaction during fibronectin fibril formation; (iii) plasma fibronectin may be the source for tissue fibronectin.     

Isolation rate of gram negative microflora and its sensitivity to antibiotics in hemoblastosis patients

A total of 67 patients with blood system diseases and infectious complications were examined. During the period of the examination 139 microorganisms were isolated. Of these gram negative microorganisms constituted 51%, gram positive microorganisms--34.8% and fungal flora--14.2%. Most frequently the following gram negative microorganisms were isolated from the patients: Pseudomonas sp. (including P. aeruginosa), Klebsiella pneumoniae, Escherichia coli, Haemophilus influenzae. All isolated microorganisms retained sensitivity to imipenem, with the exception of individual strains of Pseudomonas sp.; the latter exhibited sensitivity to amicacin and ceftazidim. Cefotaxime was active with respect to 75% of K. pneumoniae strains and all E. coli strains, ciprofloxacin was active with respect to 43% of E. coli strains, 80% of K. pneumoniae strains and 83.4% of Pseudomonas sp. strains, cefepim was active with respect to 85.7% of Pseudomonas sp. strains and all E. coli strains, ceftazidim was active with respect to all Pseudomonas sp. and E. coli strains. 75% of K. pneumoniae strains, 77.8% of Pseudomonas sp. strains and 86% of E. coli strains retained sensitivity to amicacin. 25% of K. pneumoniae strains required testing for ESBL production.     

Co-operative domains in fibronectin

The melting of human plasma fibronectin and its proteolytic fragments has been studied by scanning microcalorimetry to reveal co-operative structural domains in the molecule. It has been established that each of the two similar polypeptide chains of fibronectin has at least 12 structural domains, which differ in stability, size and function. Many of the domains in the N-terminal half of the polypeptide chains appear to be composed of two homologous repeat modules that co-operate to form a single co-operative unit. In the intact fibronectin molecule, the C-terminal regions of both chains seem to interact forming a stable co-operative block.     

Multiple binding sites in fibronectin and the staphylococcal fibronectin receptor.

The binding of fibronectin to Staphylococci exhibits the properties of a ligand-receptor interaction and has been proposed to mediate bacterial adherence to host tissues. To localize staphylococcal-binding sites in fibronectin, the protein was subjected to limited proteolysis and, of the generated fragments, Staphylococci appeared to preferentially bind to the N-terminal fragment. Different fibronectin fragments were isolated and tested for their ability to inhibit 125I-fibronectin binding to Staphylococci. The results indicate that only the N-terminal region effectively competed for fibronectin binding. However, when isolated fragments were adsorbed to microtiter wells, we found that two distinct domains, corresponding to the N-terminal fragment and to the heparin-binding peptide mapping close to the C-terminal end of fibronectin, promoted the attachment of both Staphylococcus aureus Newman and coagulase-negative strain of Staphylococcus capitis 651. These same domains were recognized by purified 125I-labeled staphylococcal receptor, either when immobilized on microtiter wells or probed after adsorption onto nitrocellulose membrane. The heparin-binding domain is comprised of type-III-homology repeats 14, 15 and 16. To determine which repeats participate in this interaction, we isolated and tested repeats type III14 and type III16. We found that the major staphylococcal binding site is located in repeat type III14. The staphylococcal receptor bound the N-terminal domain of fibronectin with a KD of 1.8 nM, whereas the dissociation constant of the receptor molecule for the internal heparin-binding domain was 10 nM. Since the fusion protein ZZ-FR, which contains the active sequences of fibronectin receptor (D1-D3) bound only to the N-terminus, it is reasonable to assume that the bacterial receptor may have additional binding sites outside the D domains, capable of interacting with the internal heparin-binding domain of fibronectin.     

Involvement of gangliosides and glycoprotein fibronectin receptors in cellular adhesion to fibronectin

We have used a rat neural cell line, B65, to investigate the relative contributions of gangliosides and glycoprotein receptors in adhesion to fibronectin. Monoclonal antibodies against two neuroectoderm-associated gangliosides, D1.1 and GD3, inhibit the rate of B65 attachment to fibronectin, suggesting that these gangliosides are involved in the adhesion process. Adhesion to fibronectin is not affected by a third monoclonal antibody against a separate, unidentified cell-surface component of B65 cells. Furthermore, B65 cells lacking D1.1 adhere to fibronectin at a slower rate than B65 cells that express D1.1. The involvement of glycoprotein receptors in adhesion is demonstrated by the ability of antibodies against human fibronectin receptor to inhibit B65 attachment to fibronectin. In addition, adhesion is blocked by a hexapeptide containing the Arg-Gly-Asp fibronectin sequence which is necessary for binding to the receptor. Trypsin treatment of B65 cells in the absence of divalent cations results in proteolysis of the fibronectin receptor with an accompanying loss of ability of the cells to attach to fibronectin. D1.1 and GD3 expression is not affected by this trypsinization, indicating that the gangliosides alone are incapable of mediating attachment. The glycoprotein receptors must be primarily responsible for adhesion to fibronectin with the gangliosides playing a secondary role as enhancers or modulators.