SS-RBCs bind to the 140-kD carboxy-terminal proteolytic fragment of TSP. (A) Schematic illustration of intact TSP with selected domains as described in the text (HBD, heparin-binding domain; type 1, type 1 repeats; type 2, type 2 repeats; Ca⁺⁺ Binding, Ca²⁺-binding domains or type 3 repeats; CBD, cell-binding domain). The approximate locations of the 25-kD, 140-kD, 120-kD, and 70-kD proteolytic fragments as identified by amino-terminal sequencing and size16,29,32are indicated by arrows. The 25-kD fragment is a monomer,16the 70-kD, 120-kD, and 140-kD fragments are trimers,32 and the 18- to 20-kD C-terminal fragment remains disulfide linked to the 120-kD fragment.31 (B) Proteolytic digestion and purification of TSP fragments. TSP purified from platelet releasate was treated with thermolysin and purified by heparin-Sepharose affinity chromatography, or chymotrypsin in the presence of Ca²⁺or EDTA and purified by gel-filtration FPLC as described in Materials and Methods. Intact TSP (TSP), proteolyzed TSP (Thermolysin, Chtpn), or purified proteolytic fragments (140 kDa, 25 kDa, 70 kDa, 120 kDa) were resolved by 4% to 12% SDS-PAGE and stained with Coomassie Blue. (C) Washed SS-RBCs were perfused through parallel plate flow chambers coated (2 μg/cm²) with intact TSP (TSP, N = 12) or purified 25-kD (25 kDa, N = 4), 140-kD (140 kDa, N = 4), 70-kD (70 kDa, N = 4), or 120-kD (120 kDa, N = 4) TSP fragments at a wall shear stress of 1 dyne/cm². After rinsing, adherent RBCs per unit area were counted by direct microscopic visualization. The results are shown as the mean ± SE of SS-RBC adhesion, normalized to SS-RBC adhesion to intact TSP.