Direct interaction between COMP and thrombin. (A) Representative surface plasmon resonance analysis of different concentrations of thrombin injected through a biosensor chip conjugated with COMP. The altered response unit (ΔRU) is recorded in real time after perfusion. (B) The best fitting of plot data in thrombin concentrations and their corresponding ΔRUs yields a K_D of 1.38 ± 0.24 μM for thrombin based on a 1:1 Langmuir model (n = 3). Thrombin (1 μM) in the presence of hirudin (C), HD1 (D), heparin (E), and HD22 (F) at different concentrations was injected over a COMP-linked biosensor chip. The percentage of ΔRU to the ΔRU in thrombin (1 μM) injection alone (thrombin bound) regressed according to the concentration of hirudin, HD1, heparin, or HD22 (n = 3). Concentrations corresponding to 50% of thrombin bound are the IC₅₀. (G) Western blot of protein fractions precipitated with amylase beads from a mixture of thrombin (3 μg) and different purified MBP-fused COMP protein fragments. Fractions before precipitation were applied as input for loading control. Thrombin (0.1 U/mL)-cleaved chromogenic substrate S2238 (50 μM) (H) and fluorogenic substrate FluCa (2.5 mM) (I) were measured with absorbance at 405 nm and fluorescence intensity (excitation, 390 nm; emission, 460 nm), respectively. Hirudin (0.1 mg/mL) was applied as a positive control. n = 3, *P < .05 vs 0 ng/mL. IP, immunoprecipitation; OD₄₀₅, optical density at 405 nm; WB, western blot.