Cold-insoluble Globulin Research Articles

Actions of the functional upstream domain of protein F1 of Streptococcus pyogenes on the conformation of fibronectin

Fibronectin (Fn), discovered by Harvard's Plasma Protein Program as plasma “cold-insoluble globulin” in the 1940s, has attracted much interest over the past three decades. One of the most interesting features of Fn is its ability to change shape in response to various environmental conditions and interactions with other substances found in the extra-cellular space. Here we examine the potential of the functional upstream domain (FUD) of Streptococcus pyogenes protein F1 to bring about changes in structure of Fn. In particular, we investigate the accessibility of Fn's 10th type III module that contains the integrin binding RGD motif. By use of monoclonal antibodies in a competitive ELISA assay, we found that FUD interacts with the amino-terminal type I modules of Fn to unveil the cell-binding region of Fn. This conformational change was achieved at sub-equimolar ratios of FUD/Fn monomer. We discuss the functional relevance of the interaction for both Fn and S. pyogenes and correlate the results with a conformational model of Fn that arose out of a collaboration between our laboratory and that of John Ferry.

Therapeutic cryogel removal in autoimmune disease: what is cryogel. 1982.

Guest Editor's Introduction: This paper was presented as a keynote address of the 2nd congress of the Japanese Society for Therapeutic Plasmapheresis on June 5, 1982. It was printed in Therapeutic Plasmapheresis II (ed) T. Oda, and published by F.K. Schattauer Verlag, Stuggart, Germany in 1982 on pages 15–25. In this paper, attempts were made to clarify the formation of the cryogel, composition of cryogel, and cryogel and its temperature dependence by cryofiltration procedures. Clinical effects of cryogel removal or cryofiltration procedures were described from macromolecular changes, pre and post procedures, and the clinical effects of rheumatoid arthritis patients were assessed from these macromolecular kinetics. Basically, cryogel is a fibrinogen‐heparin‐fibronection complex and contains various types of pathological macromolecules.Abstract: Cryofiltration is the on‐line technique of plasma treatment consisting of plasma cooling and its membrane filtration. In cryofiltration, cryogel is formed and removed by the cryofilter. The formation of cryogel on‐line at temperatures near 4°C and the removal of cryogel by the membrane filter has been shown to produce positive clinical results in the treatment of rheumatoid arthritis and other autoimmune diseases. Cryogel as generated in cryofiltration is distinct from cryoprecipitate (cryoprotein, cryoglobulin) as isolated by classical laboratory tests or those substances removed by off‐line techniques as cryopheresis and cryoglobulinpheresis. Cryogel is formed under conditions of relatively fast cooling (10–20 minutes) and is removed during membrane filtration of the plasma. Cryogel concentrates the pathological molecules, and therefore, is selective for their removal. Clinical and in vitro studies of cryofiltration indicate the primary importance that temperature has on the bulk properties of the solution and the filtration process. While clinical studies to date have been carried out at 4°C, cryogel can form at temperatures below 37°C. In general we can remove more cryogel from pathological plasma than from normal plasma. Mechanisms involved in the formation of cryogel are complex and involve multiple plasma solutes. Cold‐induced precipitation of macromolecules from plasma is most likely due to the interaction of heparin, fibronectin (cold insoluble globulin), and fibrin/fibrinogen under cooling conditions. Plasma subjected to the cooling procedures contains sufficient amounts of heparin to produce heparin‐fibrinogen complex. This complex acts as a nucleus for cryogel formation and the binding of macromolecular weight plasma solutes which are then trapped on the cryofilter.

Cryofibrinogenemia: An addition to the differential diagnosis of calciphylaxis in end-stage renal disease

Cryofibrinogenemia is a disorder characterized by cryoprecipitation with variable clinical presentation that was first described by Korst and Kratochvil in 1955. Cryofibrinogen is a cold insoluble complex of fibrin, fibrinogen, and fibrin split products with albumin, cold insoluble globulin, factor VIII, and plasma proteins. Cryofibrinogenemia is associated with metastatic malignancies, collagen vascular diseases, and thromboembolic disorders and may be clinically asymptomatic or present with thromboembolic phenomena of skin and viscera. The pathogenesis of cryofibrinogenemia is unknown. It may be caused by the inhibition of fibrinolysis, leading to an accumulation of cryofibrinogen. Treatment of cryofibrinogenemia may include Stanozolol, plasmapheresis, and fibrinolytics. Cryofibrinogenemia simulates calciphylaxis clinicopathologically, because both may present with skin necrosis. Calciphylaxis has been reported in end-stage renal disease, but we report the first case of cryofibrinogen in a chronic dialysis patient. We suggest that in the appropriate clinical setting, cryofibrinogenemia should be considered in the differential diagnosis of calciphylaxis, and serum cryofibrinogen levels should be measured in end-stage renal disease patients presenting with skin necrosis. (Am J Kidney Dis 1998 Sep;32(3):494-8)

Plasma fibronectin levels during cardiopulmonary bypass

Plasma fibronectin, also called cold-insoluble globulin, is a cryoprecipitable glycoprotein with both opsonic and adhesive activities. It binds to collagen, actin, and heparin and can form soluble as well as cryoprecipitable complexes in the cold. Fibronectin augments particulate phagocytosis by the reticuloendothelial system and can influence lung vascular permeability. Plasma fibronectin deficiency is temporally associated with respiratory failure in septic surgical, trauma, and burn patients. We measured plasma fibronectin and albumin levels in nine adults undergoing elective cardiopulmonary bypass to determine whether dilution alone could account for the changes in plasma fibronectin. Plasma fibronectin concentration decreased 17% with the surgical trauma of opening of the chest and placement of the vascular cannulas. On heparinization and initiation of cardiopulmonary bypass, plasma fibronectin fell an additional 48% (P less than 0.001), whereas albumin concentration (corrected for albumin in the pump prime) fell only 25% (P less than 0.001), emphasizing that dilution was not the only mechanism contributing to the decline in plasma fibronectin. Fibronectin levels began to increase after discontinuation of cardiopulmonary bypass and in association with diuresis, but unexpectedly they remained subnormal until 4 days postoperation. Thus the decline in fibronectin concentration with cardiopulmonary bypass may be due to dilution as well as opsonic consumption and possible complexing with heparin in the cold.

Endothelial cell adhesion to vascular prosthetic surfaces

Improving the long term patency of small diameter prosthetic grafts remains an important but elusive objective in vascular surgery. In pursuit of a non-thrombogenic surface, we have cultured human-adult endothelial cells, examined their adhesive properties and their ability to colonize the inner surface of a Dacron® graft. To examine cell adhesion, endothelial cells were labelled with 111In-oxine and inoculated onto prosthetic wells previously prepared with either cold insoluble globulin (CIG), 1 % gelatin, alginate or left untreated as a control. At 100 min, mean percentage adhesion to CIG and gelatin precoated wells was 86.0 ± 9.9% (± sd) and 81.6 ± 2.9% respectively, whereas alginate at 57.5 ± 7.5% and the control well at 48.3 ± 9.9% showed significantly less adhesion. Further experiments examined the adhesion of Indiumoxine labelled endothelial cells to Dacron® graft (2 cm × 6 mm id) placed in an in vitro arterial circuit. An immediate loss of approximately 20% of the cells occurred within the first 30 s, whereafter, a stable population were adherent to the graft material. By 102 min, 73.4 ± 1.8% of cells remained attached when exposed to tissue culture medium, but only 64.1 ± 6.9% after exposure to blood. Cultured human adult endothelial cells adhere most effectively to prosthetic surfaces precoated with CIG or gelatin, and remain attached following exposure to shear forces.

Purification of human monocytes on gelatin-coated surfaces

Human peripheral blood monocytes secrete a cell membrane-associated glycoprotein, cold insoluble globulin (fibronectin). Since fibronectin binds to gelatin-coated surface, we developed a simple technique for separation of human peripheral blood monocytes from whole mononuclear cell preparation. These preparations are characterized by a high monocyte purity (more than 90%), low platelet contamination and excellent viability.

Fibronectin assays and their clinical application: a review.

Cold insoluble globulin (fibronectin) was discovered 30 years ago but recently there has been a remarkable growth of knowledge concerning its interaction with the cell cytoskeleton and its role in cell-cell and cell-matrix adhesion. The protein is also a major plasma opsonin with a role in regulating fixed macrophage activity and it is this area in which clinical applications are now beginning to develop. Methods are discussed for measuring the concentration of the protein and its opsonic function in vitro, and for the evaluation of fixed macrophage function in vivo. Also discussed are the metabolism of the protein, the implications of opsonin depletion in patients with serious injury or infection and the attempts to reverse this with plasma protein replacement therapy.

The role of fibronectin in hemostasis

Over the past 10-15 years, researchers in the field of experimental and clinical medicine have shown increased interest in fibronectin FN), a blood plasma protein with a diverse biological effect. FN is a glycoprotein found both in the blood and in other body fluids, and in an insoluble form - in connective tissue, in particular as part of the basement membrane [28, 30, 48, 69]. The main source of plasma FNL are endothelial cells and hepatocytes. FN ensures the convergence and adhesion of cells, contributing to the elimination of vascular endothelial defect in normal condition and after injury. Its molecular weight is about 4.4-105 daltons, the sedimentation constant is 12-14S, the isoelectric point is 5.5-6.2; it belongs to the class of mobile -globulins [44c]. The function of this protein in the human body is diverse. This can be judged, in particular, by the number of available synonyms, each of which reflects a certain biological property of the protein: cold-insoluble globulin, anti-gelatin factor, microfibrillary protein, protein with opsonin properties, fibroblast surface antigen, galactoprotein a, cell attachment factor, large external transformation-sensitive protein, cell surface protein, cell proliferation factor [28, 64]. The preferred term is "fibronectin", which means "fiber binding" (from Lat. fibra- fiber-nectere - binding) [23, 30, 41]. Plasma FN together with fibrinogen, factor XIII, and Willebrand factor is precipitated from plasma at 0 25% with ammonium sulfate [44a] or 8% ethanol [43]. Its concentration in the cryoprecipitate increases 5-10 times [43], in the plasma of healthy men is 180-720 mg / l, in women - 150-540 mg / l, in serum - 20-50% less than in plasma.

The involvement of fibronectin in molecular and cellular interactions

Among the proteins of the animal organism, fibronectin (FN) occupies a special place due to the exceptional diversity and importance of biological properties. In 1948, Morrison et al. [69] for the first time it was discovered as part of the I fraction of blood plasma by Cohn, but interest in FN increased rapidly only in the 70s after its identification with one of the proteins of the outer cell membrane. At various times, FN has been described under numerous names that determined any property of this protein or its localization, for example, large external transformation-sensitive (LETS) protein, cold-insoluble globulin, cell adhesion factor, opsonic a2-SB-glycoprotein, anti-gelatin factor, etc. Currently, these names are almost not used, and the term "fibronectin" (fibra fiber, nectere bind) is adopted to refer to all forms of this protein cellular and extracellular, soluble and membrane-associated cells that form a population of immunologically related molecules with some differences in physicochemical and biological properties.

Cryoprecipitate therapy in amniotic fluid embolization

Cryoprecipitate was administered to a patient with severe adult respiratory distress syndrome secondary to an amniotic fluid embolus, diagnosed cytologically. Following the administration of Cryoprecipitate, cardiopulmonary and hematologic status markedly improved, and the patient recovered without apparent sequela. She is the sixth surviving patient reported to have an amniotic fluid embolus confirmed cytologically. On the basis of accumulating data on the relationship between fibronectin levels and the integrity of the reticuloendothelial system, it is quite possible that fibronectin (cold-insoluble globulin), and not fibrinogen, played the key role in her dramatic improvement and may well have been responsbile for the clinical improvement in earlier patients treated with blood products containing fibronectin.

The Role of Fibrin Adhesive in Vascular Surgery

Abstract Fibrin Seal (FS) (a natural adhesive material composed of fibrinogen, cold insoluble globulin, factor XIII, platelet growth factor, antiplasmin thrombin, and calcium chloride) was utilized in the construction of vascular anastomosis of the femoral artery of mongrel dogs. A significant decrease in the number of sutures necessary to achieve adequate anastomosis and minimize the likelihood of postoperative bleeding was noted. There was no long-term increase in tissue reaction or decrease in arterial patency. On the basis of these preliminary results the role of FS as a hemostatic agent in vascular and especially microvascular surgery warrants further investigation.

Growth characteristics of A431 human epidermoid carcinoma cells in serum-free medium: inhibition by epidermal growth factor.

A431 human epidermoid carcinoma cells in culture may be grown in the absence of serum in a one to one mixture of Ham's F12 and Dulbecco-modified Eagle's medium supplemented with 10 micrograms/ml bovine insulin, 10 micrograms/ml human transferrin, 5 micrograms/ml human cold-insoluble globulin and 0.5 mM ethanolamine. Growth rate of the cells in this serum-free medium is essentially identical to that of cells in medium containing 10% fetal calf serum. Addition of epidermal growth factor at concentrations which are stimulatory for the growth of other cell types in culture causes and inhibition of A431 cell growth in both serum-free and serum-containing medium. The inhibitory effect of epidermal growth factor is reversible upon replacement of epidermal growth factor-containing medium with medium containing no epidermal growth factor. Simultaneous addition to the medium of antibody to epidermal growth factor along with epidermal growth factor prevents the inhibitory effect.

Plasma fibronectin levels after splenectomy and splenic autoimplantation in rats with and without dietary ascorbic acid supplementation

The critical role of the spleen in protecting subjects from systemic bacterial infection is well known. Plasma fibronectin (PF), cold-insoluble globulin, and opsonic alpha2 surface binding glycoprotein, has regulatory influence on reticuloendothelial system clearance activity and it is important for maximal phagocytosis. Ascorbic acid (AA) also appears to play an important role in phagocytic cell function. The purposes of this study were to determine the effects of splenectomy and splenectomy with autoimplantation, with and without dietary AA supplementation, on PF levels. Six-week-old male Sprague-Dawley rats were divided into four experimental groups of 20 animals each: Nonoperated controls, sham-operated controls, totally splenectomized animals, and splenectomized animals with intraperitoneal autoimplantation. Each group was further randomly divided into two subgroups of 10 animals, those receiving dietary AA supplementation and those not receiving AA. PF levels were measured with a colorimetric assay immediately prior to and at 4 and 8 weeks after operation. Plasma AA determinations documented the effectiveness of dietary AA supplementation. PF levels in nonoperated and sham-operated controls increased significantly during the 8 weeks of experimentation. In contrast, PF levels decreased significantly following total splenectomy from 328 +/- 46 mcg/mL (mean +/- S.D.) to 285 +/- 46 at 4 weeks and rose to 303 +/- 77 at 8 weeks postoperatively in non-AA supplemented animals. Splenic autoimplantation eliminated this decrease in PF levels at 4 weeks. AA supplementation also protected against the decrease in PF levels in the splenectomized group. In the intragroup comparisons, AA supplementation did not produce a significant elevation of PF.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of the tissue adhesive fibrin seal (FS) in esophageal anastomoses.

Following surgical removal of esophageal tumors, leakage and mediastinitis is a frequent and often fatal complication. A new method has been developed to seal suture lines in the esophagus with preparations containing fibrinogen, cold insoluble globulin, factor XIII, antiplasmin, platelet growth factor, thrombin, and calcium chloride. In experimental animals operated on by standard methods, esophageal leakage developed in 50% of the animals and death in 40%. By contrast, in treated animals, esophageal leak and death developed in only 20%. More adhesions were found in treated animals than in control animals.

The role of fibrin adhesive in vascular surgery.

Fibrin Seal (FS) (a natural adhesive material composed of fibrinogen, cold insoluble globulin, factor XIII, platelet growth factor, antiplasmin thrombin, and calcium chloride) was utilized in the construction of vascular anastomosis of the femoral artery of mongrel dogs. A significant decrease in the number of sutures necessary to achieve adequate anastomosis and minimize the likelihood of postoperative bleeding was noted. There was no long-term increase in tissue reaction or decrease in arterial patency. On the basis of these preliminary results the role of FS as a hemostatic agent in vascular and especially microvascular surgery warrants further investigation.

Microfibrillar protein and phospholipid in granular corneal dystrophy.

Keratoplasty specimens from eight patients with granular corneal dystrophy (GCD) and age-matched control subjects were examined by combinations of immunohistological stains, transmission electron microscopy (TEM), and sodium dodecyl sulfate gel electrophoresis. Fresh frozen sections from corneas with GCD stained positively with antibodies to microfibrillar protein by immunofluorescence. Routine TEM disclosed that the granules had central electron-dense areas partially surrounded by 9- to 10-nm tubular microfibrils. Material eluted from corneas with GCD showed denser peptide bands at 65 and 110 kilo than in normal corneas. Stains were negative for elastin, amyloid, neutral lipids, cholesterol, and glycosaminoglycan. Luxol fast blue MBSN stain was strongly positive in the granules in all cases examined. Immunofluorescent stains were negative with antibodies to plasma fibronectin (cold insoluble globulin), laminin, collagens I to V, basement membrane proteoglycan, tropoelastin, and keratin. In two corneas with GCD an increased lipid content was found in every phospholipid class, although cholesterol content was unchanged. Alterations in the fatty acid profiles of phospholipids were also observed.

Actin-induced reticuloendothelial phagocytic depression as mediated by its interaction with fibronectin

Circulating fibronectin, also known as opsonic α 2 surface binding glycoprotein or coldinsoluble globulin, modulates phagocytosis of tissue debris, fibrin microaggregates, and gelatin-coated colloids by the reticuloendothelial (RE) system. Opsonically active fibronectin has an actin binding site and a demonstrated in vitro affinity for actin. Since actin potentially released into blood and tissue fluids following tissue injury could complex with fibronectin, the present study evaluated the effect of actin on plasma opsonic activity and Kupffer cell phagocytosis. Intravenous injection of actin did not acutely decrease plasma immunoreactive fibronectin levels although fibronectin levels increased at 6, 12, and 24 hr postinjection. However, intravenous actin injection did depress RE phagocytic activity in vivo as measured by decreased blood clearance of test colloid and impaired hepatic uptake of colloid particles as well as retention of the particles in the circulation. In vitro, preincubation of plasma with actin depressed the opsonic activity of plasma with respect to its ability to support phagocytosis, but such treatment of plasma did not alter the detection of fibronectin by immunoassay. Utilizing purified fibronectin with demonstrated opsonic activity, it was also observed that actin interaction with fibronectin would block its biological ability to enhance phagocytosis. This effect appeared to be mediated at the humoral level, since no direct depressant effect of actin on Kupffer cell function was observed. Thus, actin, if released into the blood following injury, may contribute to bioassayable opsonic fibronectin deficiency and phagocytic dysfunction, but this disturbance would remain undetectable by immunoassay of fibronectin levels.

Entamoeba histolytica: Correlation between virulence and content of proteolytic enzymes

Intact trophozoites of the virulent Entamoeba histolytica strain HM-1:IMSS (HM-1) destroyed a monolayer of baby hamster kidney (BHK) cells at a higher rate and efficiency than trophozoites of the nonvirulent strain HK-9. The destructive effect could be partially attributed to the proteolytic activity of the amoeba, since quantitative differences were found in the enzymatic activity of the two strains tested. Crude extracts or secreted enzymes of HM-1 trophozoites digested Azocoll, as well as the bovine cold-insoluble globulin fraction, at a much higher rate than the corresponding preparations from HK-9. This proteolytic activity was found to be activated by free sulfhydryl groups. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of the BHK cell proteins of pre- and postamoebic activities showed patterns similar to the trypsin effect on the same target cells. These enzymes were found to digest the proteins participating in the attachment of the target cells to the substrate and, consequently, cause detachment of these cells.

Partial primary structure of bovine plasma fibronectin: three types of internal homology.

Approximately one-half of the amino acid sequence (911 amino acid residues out of 1,880 expected) for bovine plasma fibronectin (cold-insoluble globulin) has been determined. Three types of internal homology were identified, showing that a number of partial gene duplications (multiplications) have occurred during the evolution of this protein. Digestion of fibronectin with plasmin results in major fragments with molecular masses of 29, 170, 23, and 6 kilodaltons (kDal). The NH(2)-terminal 29-kDal fragment consists of 259 residues ordered as five mutually homologous domains (type I homology) with two disulfide bonds in each domain. The 170-kDal fragment shows two to three bands after NaDodSO(4) gel electrophoresis, indicating heterogeneity. This fragment contains the gelatin binding site and the strong heparin binding site present in fibronectin. Digestion of the 170-kDal fragment with chymotrypsin liberates a 45-kDal fragment that also binds to gelatin. This fragment contains at least one domain of type I homology and two domains of type II homology. Further digestion of the 170-kDal fragment with chymotrypsin results in the formation of a 30-kDal fragment that retains the heparin binding activity. This fragment contains sequences constituting type III homology. The 23-kDal fragment consists of 178 residues having three domains of type I homology. The 6-kDal fragment consists of two identical peptides of 26 residues, and these two peptides are linked to each other by two disulfide bonds that form the interchain bridges. Another one of the peptides for which the sequence was determined links the COOH-terminus of the 29-kDal fragment to the NH(2)-terminus of the 170-kDal fragment. This and the fact that the COOH-terminal residue of the 6-kDal fragment is a glutamic acid residue order the four plasmin-digestion fragments as 29-, 170-, 23-, and 6-kDal in the intact fibronectin molecule.

Fibronectin — Mediator between cells and connective tissue

Fibronectin, previously also termed LETS-protein, is a high-molecular-weight protein (mol. w. ca. 450,000) present in the form of thin fibrils in the pericellular space of fibroblasts and other adherent cells, as well as in distinct areas of the connective tissue. A soluble form, immunologically identical and chemically at least very similar to the cell-attached protein, is found in plasma in a concentration of about 300 micrograms/ml. It is also denominated cold-insoluble globulin. The protein has affinity both to cell surfaces and to various matrix substances such as fibrin and collagen and, therefore, is capable of mediating cell attachment to these substrates. In addition, it serves as an opsonin for the phagocytosis of gelatin-containing compounds and probably is essential for the removal of soluble fibrin from the circulating blood by the reticulo-endothelial system. Bacterial cell walls are also recognized by fibronectin. A conversion of soluble fibronectin to fibrils is achieved by heparin which also enhances the binding of soluble fibronectin to cells. Heparin or, as suggested, the related heparan sulfate present on the surface of various cells, appears to function as a cofactor in the formation of pericellular fibrils. The fibronectin fibrils precipitated with heparin, compared to soluble fibronectin, show a considerably improved affinity to native collagen, especially to type III. Hyaluronic acid has an antagonistic function which, at higher concentrations, prevents the fibronectin fibrils from interacting with collagen and cell surfaces. Masking of fibronectin fibrils was also achieved by sulfated proteoglycans of cartilage. Virus-transformed fibroblasts produce less fibronectin and are less capable of maintaining surface pericellular fibrils. A reasonable explanation is that they have an elevated secretion of hyaluronic acid. The transformed cells attach only weakly to a surface and exhibit a rounded shape in contrast to healthy ones. This phenotype can be corrected to a great extent with fibronectin. It is suggested that fibronectin also influences the formation of connective tissue by accumulating collagen precursors on the surface of fibroblasts and facilitating fibrillogenesis.