FIGURE 5.

Thrombin is capable of cleaving HC1 from either cell-surface HC-HA cables or from serum IαI. HA cell-surface layer extracts from untreated, poly(I·C)-treated, or poly(I·C) and thrombin-treated cells were compared by Western blotting. Confluent M-SMCs were treated without or with poly(I·C) for 18 h at 37 °C to form HC-HA cable complexes. Poly(I:C)-treated cells were incubated without or with thrombin (25 units/ml) for 1 h at 37 °C. Cells were rinsed three times with PBS and incubated with Streptomyces hyaluronidase (100 milliunits/ml) for 5 min to release HA-bound cell-surface material. The supernatant was collected and prepared for Western blotting analysis of IαI (A) and HC1 (B). Serum from healthy donors was diluted and incubated without or with thrombin (25 units/ml) for 3 h at 37 °C prior to Western blotting analysis for either IαI (C) or HC1 (D). The green ovals in the schematic models represent HCs attached to bikunin (blue rectangle) via a single CS chain (black line). The schematic with two HCs represents IαI. The schematic with one HC represents PαI. Blots are representative of experiments from three independent patient cell lines and three technical replicates.