Provisional Matrix 1 Adhesive Proteins

Extracellular matrix proteins such as fibronectin, vitronectin and fibrinogen, play a major role in cell-matrix interactions (Brown and McFarland, 1996). These glycoproteins possess several domains within their structure, via which they can participate in a variety o f functions. These may include a cell binding domain, a heparin binding domain, and an extracellular protein (collagen/fibrin) binding domain. Each domain consists o f short amino acid sequences, the most documented being the Arg-Gly-Asp (RGD) sequence present in the cell binding domain o f proteins, such as fibronectin and vitronectin (refer to section 1.6.9) (Agrez et al. 1991). The extensive individual and combined properties of these proteins all contribute to the complex cellular behaviour observed during wound healing.

1.6.10.2 Fibronectin

Fibronectin is a well characterised, multifunctional cell adhesion glycoprotein that is found in the blood and in extracellular matrices (Mosher, 1989; Kiihn, 1997). Fibronectin present in the blood is synthesised predominantly by hepatocytes and is termed plasma fibronectin. Several cell types synthesise and secrete fibronectin into the extracellular

matrix, this form is termed cellular fibronectin. Cellular fibronectin is assembled as fibronectin fibrils and can be associated with or without collagen I (Kiihn, 1997). Fn is recognised by several integrins, including a 5 p i, a S p l, a 4 p l, a S p i, a v p i and avp3 (Eble, 1997) and has been widely documented to initiate and support cell attachment and migration (Mosher, 1989; Hynes, 1990; Nagai et al. 1991; Clark, 1995). Integrin-ligand binding through fibronectin occurs through integrin recognition sites present within the ligands, the most studied being the tripeptide cell binding motif, RGD (Boucaut et al. 1984). The RGD motif is present in many integrin binding ligands, others being vitronectin, tenascin, thrombin and laminin (Pfafif, 1997). Nagai et al. (1991) highlighted 2 distinct regions present in the cell binding domain o f Fn which were important in Fn mediated cell spreading and migration, as well as Fn fibril assembly. This site was found to fimction in synergy with the RGD sequence. Studies by Mould et al. (1991) identified 2 additional cell specific binding sites present on fibronectin that allowed interaction with the a 4 p i integrin (present on cells such as melanoma cells, peripheral blood lymphocytes (Mould et al. 1991) and human dermal fibroblasts (GaUit et al. 1993)). This interaction was independent o f the RGD sequence, however dependent on a different tripeptide ‘LDV’ (leucine-aspartate-valine) present in another spliced section o f the fibronectin molecule (Komoriya et al. 1991).

Elevated levels o f plasma fibronectin can be located in clots, where as cellular fibronectin is predominantly found in healing dermal wounds, for example in the fibrin clot where it serves as a scaffold for migrating cells (Clark, 1995). Other roles for fibronectin in tissue repair include monocyte chemotaxis, cell migration, cell adhesion, regulation o f cell growth and the synthesis o f extracellular matrix. It possesses a multi-domain structure through which it can exert its effect. It is capable o f interacting with a broad range o f cell types, extracellular matrix molecules and cytokines (Figure 17).

Figure 17. The fibronectin molecule

A. Schematic representation o f the fibronectin molecule showing its various domains, (courtesy o f G. Talas). 32kD 215kD -I I N PO. glu 32ko' ' 185kD PO, 37kD Heparin II I Snien^ 6 Factor X llla

Glucosamine containing carbohydrate

B. Three dimentional structure o f the fibronectin molecule showing the Arg-Gly-Asp (RGD) sequence, part o f the major cell binding site.

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The cell binding domain o f the fibronectin molecule contains the classic RGD (Arg-Gly- Asp) amino acid sequence (Pierschbacher and Ruoslahti, 1984; Yamada and Kennedy, 1984; Agrez et al. 1991; Cheresh and Mecham, 1994). Absence or dysfunction o f this peptide sequence leads to the loss o f virtually all adhesive properties o f the parent molecule (Obara et al. 1988). Interestingly, although impaired parent molecules may cease to function due to this alteration in peptide sequence, fi-agments o f the parent molecule can continue to provide additional cellular mechanism during wound healing. This is due to proteolytic enzymes present in the wound environment which fi-agment many intact parent molecules, including fibronectin (Grinnell et al. 1992). Many o f these protein fi*agments display properties that the intact parent molecule does not. For example the cell binding domain fi*agment is a potent chemotactic factor for monocytes, where as the intact fibronectin molecule is less so (Clark et al. 1988, Doherty et al. 1990).

1.6.10.3 Vitronectin

Vitronectin (serum spreading factor) is a glycoprotein that is synthesised by hepatocytes and is found in plasma, extracellular matrix and fibrin clots (Kiihn, 1997; Memmo and McKeown-Longo, 1998). Its main properties are o f cell adhesion, spreading and migration (Felding-Haberman and Cheresh, 1993; Memmo and McKeown-Longo, 1998). Vitronectin appears to colocalise with fibronectin in extracellular matrices and tends to be prevalent in adult tissue compared to immature tissue, where it is absent (Clark, 1995). Cell attachment to vitronectin appears to be dependent on the RGD (Arg-Gly-Asp) amino acid sequence, present in its cell binding domain (Agrez et al. 1991; Cheresh and Mecham, 1994). There are a number o f vitronectin integrin receptors that have a tendency to bind to other proteins, particularly fibronectin (a v p l, avp3 and alip3). However, only avp5 and avp8 integrins selectively recognise Vn (Eble, 1997). In culture vitronectin integrin-ligand complexes are positioned at focal contacts, and are usually associated with intracellular cytoskeletal elements and actin cables (Burridge et al. 1988; Dejana et al. 1988; Singer et al. 1988).

1.6.10.4 Fibrinogen

Fibrinogen is synthesised by hepatocytes and is involved early on in wound healing, during coagulation (platelet aggregation and clotting) (Kiihn, 1997). Platelets express the

major fibrinogen integrin receptor allbp3 immediately after injuiy, this ensures platelet- extracellular matrix protein interaction, platelet activation, aggregation and results in stable clot formation (Eble, 1997; Gailit et al. 1997). Next, thrombin converts fibrinogen into fibrin. Stabilisation o f this fibrin polymer occurs through intermolecular covalent crosslinking, a reaction which is catalysed by the enzyme transglutamine (Kiihn, 1997). In

nonactivated platelets, allbp3 interacts specifically with surface bound fibrinogen,

however, upon activation allbp3 binds non-specifically to other matrix proteins,

including fibronectin and vitronectin (Kiihn, 1997). This allows the fibrin clot to fiarther interact with platelets and matrix proteins (fibronectin and vitronectin) in order for the clot to be firmly established within the wound, and thus providing cells with a scaffold through which they can migrate and proliferate (Gailit et al. 1997).

1.6.10.5 Thrombospondin

This large glycoprotein is released upon platelet activation and is secreted by a number o f different cell types (Brown and McFarland, 1996). As well as interacting with a number o f cells, thrombospondin can bind to different extracellular matrix molecules, including proteoglycans, fibronectin and collagen (Adams and Lawler, 1993). Cellular action of thrombospondin includes the initiation or inhibition o f cell adhesion, cell spreading, cell migration and the regulation o f cell growth (Clark, 1995; Kiihn, 1997). Action of thrombospondin is dependent on the cell type, for example it can bind to appropriate cell surface receptors and enhance cell spreading (eg: kératinocytes) or cell aggregation (eg: platelets). It can also inhibit cellular adhesion by the disruption o f focal contacts. TGFp bound to thrombospondin appears to regulate certain cell growth (eg: endothelial cells), indeed it is the binding interaction between TGFp and thrombospondin which serves to activate TGFp (Schultz et al. 1994).

1.6.10.6 Tenascin

Tenascins are a family o f large extracellular matrix molecules whose synthesis is stimulated by serum, as well cytokines such as TGFp (Erickson, 1993). Tenascin is expressed early on in adult wound healing and is located predominantly at the wound edge. It is also often found at sites o f tissue remodelling, however, appears to be undetectable in scar tissue (Betz et al. 1993). It has been suggested that tenascin serves to

reduce cell-matrix interactions (Brown and McFarland, 1996). Chiquet-Ehrismann et al. (1988) showed how adhesion and spreading o f fibroblasts on fibronectin could be inhibited by the addition o f tenascin in culture. This would effectively weaken tractional forces on migrating cells, thus enhancing the rate o f forward cell movement (Halfter et al.

1989).