Expression and purification of the FVIII C1C2 domain. (A) Lanes 1 and 2 show the insoluble and soluble fractions of an E coli cell lysate (15% SDS-PAGE, Coomassie Blue staining; marker proteins on far left). (B) Refolded C1C2 run on a 15% acrylamide gel, with 5 μg protein in lane 1 and 10 μg in lane 2; the left panel shows Coomassie blue staining (marker proteins on far left), and the right panel shows a Western blot using ESH8. (C) Removal of the amino-terminal peptide: lanes 1 and 2 show C1C2 before and after thrombin cleavage (10% SDS-PAGE, Coomassie blue staining; marker proteins on far left). The image was cropped below the C1C2 band because the lower percentage of acrylamide resulted in the loading dye running much closer to the C1C2 band than in the 15% gels; additional Coomassie-stained gels and Western blots showed no evidence of proteolytic degradation or lower molecular weight contaminants (not shown). The thrombin-cleaved C1C2 (containing a 16–amino acid amino-terminal extension) was used for all binding and inhibition studies.