A new function of calphobindin I (annexin V)

Abstract

Calphobindin I (CPB-I, annexin V) is an anticoagulant protein purified from human placenta; it is a member of the annexin family that binds phospholipids in a calcium-dependent manner. In this study, we discovered and examined a new function of CPB-I: promotion of both the migration and the urokinase-type plasminogen activator (uPA) activity of normal human keratinocytes (NHK). While the treatment of NHK with a 10-micrograms/ml concentration of CPB-I for 24 h or 48 h caused an approximate 30% increase in the migration of NHK (compared with the no treatment), migration was inhibited when anti-CPB-I monoclonal antibodies (i.e. A46 and A180) were added along with the CPB-I. Moreover, while the treatment of NHK with a CPB-I concentration greater than 10 micrograms/ml caused a significant increase in the activity of secreted uPA, reflected in an approximately 40% increase in cell migration, uPA activity was inhibited both by cycloheximide and by monoclonal antibodies. This significant increase of secreted uPA was seen 8 h after the addition of CPB-I. Specific binding of CPB-I to NHK had a Kd value of 95.2 nM (equivalent to a CPB-I had no effect on NHK proliferation. Furthermore, CPB-I enhanced reepithelialization when it was applied locally twice a day to full-thickness cutaneous wounds made in male rats. Our results show that, during an injury, CPB-I helps reepithelialization through the promotion of both uPA synthesis and migration of keratinocytes without stimulating their proliferation.