Edward Pearlstein

Fibronectin: A review of its structure and biological activity

In 1948, MORRISON and co-workers demonstrated the co-precipitation of a high molecular weight plasma protein with cryofibrinogen following incubation in the cold 1. This protein was designated cold insoluble globulin (CIG). In 1973-1974 several laboratories using various techniques, made the independent observation that fibroblasts growing in tissue culture possessed a high molecular weight protein on their cell surface 2-7. CIG and this cell surface associated protein were established by immunological criteria to be identical a and subsequently by biochemical analysis to be very similar but to possess subtle differences in structure and function (see below). Fibronectin (Fn) is a widely accepted designation for both plasma and cell associated forms of this protein 9 and will be used throughout this review to represent both states of the protein. Differences between the two forms will be indicated where appropriate. Several reviews 1° 13 have extensively covered Fn and some degree of overlap has been unavoidable. However, the rapidly expanding attention and research effort devoted to this protein has allowed us to focus on more recent information and insights.

Role of Platelets in Tumor Cell Metastases

Abstract Platelets may have a role in the development of animal tumor metastases. Ultrastructural studies in vivo have shown arrested tumor emboli surrounded by platelets. Several tumor cell lines ...

ROLE OF PLATELETS IN TUMOR CELL METASTASES

Platelets may have a role in the development of animal tumor metastases. Ultrastructural studies in vivo have shown arrested tumor emboli surrounded by platelets. Several tumor cell lines induce thrombocytopenia in vivo. Certain tumor cells aggregate platelets in vitro. Correlations exist between the ability of some tumor cells to aggregate platelets in vitro and their metastatic potential in vivo. Antiplatelet agents have impaired or altered the spread of certain tumor metastases. It is suggested that platelets have a role in the sequestration, adherence, and penetration of tumor cells through the blood vessel endothelial cell barrier, thus preventing their rapid clearance from the circulation and allowing extravascular formation of nests of cells. Antiplatelet agents, particularly prostaglandins, may prove useful in preventing experimental animal metastases when administered before the inoculation of tumor cells. Their potential in human malignancy, where the patient presents with an established tumor, remains to be established.

Fibronectin-mediated cellular adhesion to vascular subendothelial matrices

Abstract Although fibronectin has been well established as a mediator of cell adherence in vitro and has therefore been presumed to play a role as such in vivo, the ability of fibronectin, laid down in vivo, to mediate cell attachment, has not been demonstrated. We have therefore developed a method for quantitating the attachment of cells to vascular endothelium and subendothelial matrix and have used it to assess the role of fibronectin in promoting the attachment of a fibronectin-deficient transformed cell to this site. Whereas only 10% of cells adhered to intact endothelial linings, this attachment increased 8–10-fold when the lining cells were removed by procedures which permitted the retention of subendothelial fibronectin. When lining cells were removed by more drastic procedures which also removed subendothelial fibronectin, adherence of transformed cells was decreased to 6%. Adherence of transformed cells to the basal lamina was not, however, exclusively dependent upon fibronectin. Treatment with antifibronectin antiserum which completely inhibited adherence of these cells to the extracellular matrices of endothelial cells or fibroblasts grown in vitro could inhibit attachment to basal lamina by only 60%. The data suggest that cellular adhesion to microexudates produced by cells in culture may not be an adequate model with which to study cell adhesion in vivo. The method we describe here may provide a more realistic model and could be used to study a variety of cell-substrate interactions.

Non-lytic, non-ionic detergent extractions of plasma membrane constituents from normal and transformed fibroblasts

A technique has been developed for the selective extraction of plasma membrane protein constituents from normal and transformed cell employing non-ionic detergents. The extraction procedure does not damage cells as judged by cell viability, 51Cr release, and trypan blue staining. Lactoperoxidase-catalyzed iodination followed by detergent extraction permits demonstration of a 100,000 dalton protein which is found on the surface of normal but not transformed hamster and mouse fibroblasts.

Isolation and partial characterization of plasma membrane glycoproteins from normal and transformed mammalian cells employing plant lectin affinity chromatography.

Abstract Lectins covalently bound to Sepharose 4B were employed, in the presence of 1 % sodium deoxycholate, to purify glycoproteins from isolated plasma membranes of normal and transformed fibroblasts. Lectins used in this study were isolated from Lens culinaris (LcH) and Ricinus communis (RCA I ) and were specific for d -glucose and d -mannose (LcH) and d -galactose (RCA I ), respectively. Approx. 5% of the total plasma membrane protein applied to the LcH-containing column bound and was eluted specifically, whereas approx. 3% was bound and specifically eluted from the RCA I column. A comparison of bound glycoproteins from normal and transformed cells by SDS polyacrylamide gel electrophoresis demonstrated that a glycoprotein of 85 000 D was present in substantially greater amounts in normal cell isolates. Another glycoprotein of 130000 D was found in higher concentration in transformed cell plasma membranes. The 85 000 D protein was shown to be a subunit of a serum protein of greater than 300 000 D which has a specific affinity for the normal but not transformed cell surface. These results are discussed in relation to the differential agglutinability of normal and transformed cells by plant lectins.

Fibronectin binds to amyloid P component. Localization of the binding site to the 31,000 dalton C-terminal domain

Fibronectin has been shown to play an important role in reticuloendothelial system functioning as well as in neutrophil and fibroblast migration to tissue injury sites. Fibronectin binds several macromolecules including components of the acute phase response. We have studied the interaction of fibronectin with the amyloid P component (AP). This glycoprotein, closely related to C-reactive protein, is deposited together with amyloid fibrils and is also a normal constituent of human fibronectin, its whole tryptic digest, and isolated fragments; fibronectin was retained by immobilized AP in a molar ratio fibronectin:AP of 1:5.8. In this paper we localized the binding site for AP in a tryptic 31 kDa fragment, near the C-terminal end of the fibronectin molecule. A shorter fragment of 22 kDa starting at position 82 of the 31 kDa domain and containing all the disulfide bridges present in the 31 kDa domain did not bind to AP; therefore the active site appears to be located within the 81 N-terminal residues of the 31 kDa fragment. To further support this conclusion, reduction and alkylation of either fibronectin or the 31 kDa fragment had no effect on their binding properties.

Stability of fibronectin biological activity following chemical modification

Fibronectin isolated from human plasma functions in vitro as a mediator of adhesion and spreading of trypsinized fibroblasts on native or denatured collagen. As a means of elucidating structural characteristics which might contribute to fibronectins biological activity, we have modified and digested the protein with several chemicals. Following various treatments, the protein was utilized to mediate cell adhesion and spreading on collagen to determine which alteration disrupted its activity. Fibronectin remained functionally intact after partial or complete reduction and alkylation, oxidation of 59% of the carbohydrates with sodium periodate, citraconylation, carbodiimide-catalyzed amide formation, and oxidation of 35.2 residues of tryptophan/molecule with N-bromosuccinimide. Dinitrofluorobenzene treatment, which phenylated ten residues/molecule of fibronectin, successfully inactivated fibronectins in vitro biological function. Effective modification of the protein was determined by appropriate analytical procedures. Since fibronectin retained its biological function after several treatments that presumably affected its molecular conformation, we concluded that its secondary or tertiary structure appears not to be essential for its in vitro activity, or alternatively that the protein possesses a biologically active domain relatively resistant to chemical modification.

Primary structure of human plasma fibronectin--characterization of the 6,000 dalton C-terminal fragment containing the interchain disulfide bridges.

The carboxy terminal fragment of human plasma fibronectin has been isolated after tryptic digestion and separation by DEAE-cellulose chromatography and gel filtration on Sephadex G-50. It has a molecular weight of 6,000 which changes to 3,000 after reduction indicating that the fragment is a dimer. We have determined the amino acid sequence of the 6kDa fragment and showed that it contains 26 residues including two half-cystines which form two interchain disulfide bridges. The 6kDa fragment is not phosphorylated as in bovine fibronectin although its amino acid sequence is identical to that reported for bovine plasma fibronectin. When compared to the sequence deduced from a rat cDNA, one amino acid substitution can be found. It appears that the carboxyl end of fibronectin is highly conserved among species.

Heterogenous Mechanisms of Tumor Cell-Induced Platelet Aggregation with Possible Pharmacologic Strategy toward Prevention of Metastases

Recent reports from several laboratories have emphasized the role of platelets in the development of certain tumor metastases (for review see 1). Platelets interact with certain tumor emboli, and in so doing, appear to enhance their survival in vivo. Both platelets as well as tumor cells adhere to subendothelial basement membrane. Platelets secrete permeability factors (2, 3) as well as tumor cell growth factors (4–7), which theoretically should assist in tumor cell penetration of the vessel wall and development of extravascular secondary tumor colonies.