=Type I

FN is a large dimeric glycoprotein whose two almost identical subunits are linked by two disulfide bonds near their carboxyl termini (Alberts, 1994) (Figure 1).

=Type III

=Type II

Figure 1. Diagram of plasma FN. Image adapted from Ingham.

Each 220-250 kDa unit consists of three types of homologous repeats, known as type I, type II, and type III. Functional domains, distinct segments of FN that interact with the surroundings, are composed of several homologous repeats. These domains are composed of internally stable regions, which are linked to each other by flexible, proteolytically susceptible linkers. These domains are generally categorized as cell- binding or matrix- binding. FN molecules may vary slightly due to variable transcriptional splicing, however they are all encoded within the same 50 kb gene (Alberts, 1994; Clark, 1996). Cellular FN may contain extra regions known as EDB, EDA. The type III connecting segment (IIICS) is found in only half of plasma FN dimers within the major heparin-binding domain (Hep II), whereas they are found in almost all cellular FN dimers (Magnusson, 1998).

The cell-binding domain (CBD) is centrally located from FN type III repeat 1-11. The domain consists of repeats presenting the Arginine-Glycine-Aspartine-Serine (RGDS) sequence, and the synergy site presenting the Proline-Histidine-Serine-Arginine-Asparagine (PHSRN). These segments are recognized by recognition motifs on cell integrins a3b1, a5b1, and avb3 integrins and bind the FN to the cell surface. The Hep II domain and the IIICS region are also cell- binding, reacting with cell integrins a4b1 (Yamada et.al.,1996b).

The matrix binding domains consist of the heparin-binding domains, the collagen-binding domain, and the fibrin-binding domain. The major heparin-binding domain binds to heparan sulfate proteoglycans in the extracellular matrix and on cellular surfaces. It therefore functions both as a matrix- and as a cell- binding domain. The Hep II domain is composed of FN type III repeats 12-14. The secondary heparin-binding site is toward the amino terminus of the protein coinciding with the fibrin- binding domain. There are two fibrin binding domains as well, the major one localized by the amino terminus of FN, on FN type I repeats 4-5. The second fibrin- binding domain is by the disulfide bridges by the carboxyl terminus on FN type I repeats 10-12. These domains are important for cellular interactions with fibrin in a clot. The collagen-binding domain of FN is located at type I repeats 7-9, as well as the protein’s only type II repeats.

It is important to note that the fibrin binding domains and the domains that bind to cells are on different parts of the molecule and the linkages between those domains are susceptible to lysis. In a highly proteolytic chronic wound environment, these linkages are broken. This causes the cell binding domains to become free from the rest of the molecule, which may still be tethered onto the fibrin clot. As the CBD, Hep II, and IIICS domains are awash in the wound fluid, they cannot provide traction to enable fibroblasts to enter the wound space. They may, however, competitively bind to the integrin receptors on fibroblasts, thus inhibiting migration.

It has been found that fibroblasts need three functional domains to support full migration. They are the central cell- binding domain, the heparin II domain and the IIICS domain (Clark et.al., 2002). Conversely, if the wound is lacking in these three FN domains, as is the case in the chronic wound described above, fibroblasts cannot migrate optimally.

Introduction

Product Design

Materials

Methods

Recombinant FN Fragment Purification

Cell Culture