Figure 6

Thrombin inhibition activity of sculptin fragments generated by factor Xa. Sculptin (10 µM) was incubated without or with 1 µM of factor Xa in 50 mM phosphate buffer containing 150 mM NaCl, and 50 µM phosphatidylserine and phosphatidylcholine, pH 7.4 for 6 h at 37 °C. (A) The reaction mixtures were separated by reversed phase C-18 HPLC column. (B) The collected peaks (H1–H5) were subjected to MALDI-TOF MS and thrombin inhibition assay (see Table 1, for sequence of the corresponding peptides). (C) Typical progress curves for hydrolysis of 15 µM S-2238 chromogenic substrate by 0.1 nM thrombin in the absence (trace Ctrl) and presence of 100 pM of sculptin fragment (traces H1 and H3) or intact sculptin (trance Scpt) in 50 mM phosphate buffer containing 150 mM NaCl and 0.1% PEG 6000, pH 7.4 at 37 °C. (D) Percent thrombin inhibition by sculptin and its fragments. The reaction conditions of (D) are same as in (C). The results shown in (D) correspond to the mean ± standard deviation values acquired in three independent experiments; **p < 0.01 and *p < 0.05.