Proteolytic cleavage of recombinant vWF expressed in COS-7 cells. (A) Autoradiograph of a 1.5% SDS agarose gel probed with ¹²⁵I-labeled anti-vWF. Lane N, vWF in normal plasma. Lanes 1 and 2, recombinant WT vWF and R834W vWF mutant in the culture media of COS-7 cells. Lanes 3 through 5 and lanes 9 through 11, WT vWF (lanes 3 through 5) and R834W vWF mutant (lanes 9 through 11) after 60 minutes of incubation at 37°C with the plasma proteinase at 0.1 U/mL (lanes 3 and 9), 0.2 U/mL (lanes 4 and 10), and 0.4 U/mL (lanes 5 and 11). Lanes 6 through 8 and lanes 12 through 14, same as lanes 3 through 5 and lanes 9 through 11, respectively, except that the incubation was performed without prior recalcification of the vWF samples. (B) Representative densitometric tracings of lanes 1, 2, 5, and 11 in (A). (▴) The sample starting point; (▾) the peaks of the tracings. (C) The normalized peak distances of the lanes shown in (A) for recalcified WT vWF (▵) or R834W mutant (▴) and nonrecalcified WT vWF (□) or R834W mutant (•) were plotted against the proteinase concentration that the vWF sample was exposed to.