Time course of TSP chymotrypsin digestion and its effect on SS-RBC adhesion. (A) TSP was incubated in the presence of CaCl₂ (1 mmol/L) with control buffer containing PMSF-inactivated chymotrypsin (time = 0 minutes) or active chymotrypsin (1:100, wt:wt) for 5, 10, 30, or 60 minutes before stopping the reaction with PMSF. Treated TSP was coated (2 μg/cm²) on flow-chamber wells followed by perfusion of washed SS-RBCs as described in the legend to Fig 1. The results are shown as the mean ± SE of SS-RBC adhesion, normalized to SS-RBC adhesion to control-buffer–treated TSP (N = 3 to 9 for each time point). (B) TSP treated with control buffer (PMSF-inactivated chymotrypsin, 0) or chymotrypsin (5, 10, 30 or 60 minutes) was resolved by 4% to 20% SDS-PAGE and stained with Coomassie Blue. Note that the heparin-binding domain (25 kD) is cleaved during the incubation with the control buffer, leaving the fully active 140-kD TSP fragment.