Epidermal growth factor is resistant to plasmin

Epidermal growth factor is resistant to plasmin in vitro Tear fluid (TF) of eyes with corneal disease often contains proteolytic activity, e.g. plasmin (Tervo et al. 1988). This enzyme cleaves the glycoproteins laminin and fibronectin both of which are important for corneal wound healing. Destruction of these molecules is suggested to interfere with cellular attachment, thus delaying wound healing (Berman et al. 1988; Tervo 8c van Setten 1989). This idea is supported by the observation that topical application of fibronectin (Nishida et al. 1988) or the proteinase inhibitor aprotinin@ (Salonen et al. 1987; Tervo & van Setten 1989) accelerates corneal wound healing. Epidermal growth factor (EGF), a constant component of the TF in humans (van Setten et al. 1989), has also been shown to enhance corneal wound healing (Burstein 1987). In the TF of eyes with corneal disease both EGF and plasmin are present simultaneously. Therefore we tried with the present study to clarify whether plasmin might cleave the EGF in vitro. Plasmin (Sigma, St. Louis, MO, USA) was added to a 10 ng/ml low molecular weight EGF (6 kD) solution at final concentrations of 0.85, 0.17, 0.034, 0.0043 and 0.0 IU/1. Tris-HC1 buffer (pH 8.0) containing 0.1% I@ free BSA (Sigma, St. Louis, MO, USA) was used as the medium for incubation of the dilutions at 37°C. EGF concentrations were determined in duplicate after 3,24 and 48 h of incubation using the time-resolved immunofluorometric assay (TR-IFMA) described previously (Pesonen et al. 1986). It was shown that EGF concentration was not affected by the concentration of plasmin in the medium (Table 1). The present results imply that plasmin does not hydrolyse low molecular weight EGF in vitro. Investigations on the correlations between EGF concentration and plasmin activity in the TF of patients are under way. Prelinlinary results suggest that high plasmin activity does not lead to decreased EGF concentrations in TF (van Setten et al. in preparation). We conclude that, in contrast to the glycopro-