Recycling of internalized VIIa.

Confluent monolayers of BHK(TF) cells were allowed to internalize 10 nM ¹²⁵I-VIIa or ¹²⁵I-FFR-VIIa for 1 hour. Thereafter, the cell surface–associated radioactivity was removed by a low-pH glycine wash. After the glycine wash, the monolayers were washed 3 times with buffer B and then incubated with buffer B for various times at 37°C for recycling of (A) ¹²⁵I-VIIa or (B)¹²⁵I-FFR-VIIa. Total radioactivity in the internalized pool measured after the first glycine wash was set to 100%. Subsequent measurement of radioactivity at various time points of the residual internalized pool (▪), recycled intact protein (▾), and recycled degraded protein (▴) was performed as described in “Materials and methods.” Data are the mean ± SD of 3 independent experiments in triplicate.