A new function of calphobindin I (annexin V)

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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‐μg/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 μg/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 concentration of 3.2 μg/ml), and was also inhibited by monoclonal antibodies. However, 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.