Identification of the thrombin-binding domain of SMOC1. (A) Schematic diagram of the SMOC1 protein and the SMOC1 antibody binding site. KZ, Kazal domain; SP, SPARC domain; TY, thyroglobulin domain. (B) Microscale thermophoresis assay for the interaction between fluorescently labeled thrombin and full-length (FL) SMOC1 (FL-SMOC1), SMOC1ΔKazal, (ΔKazal), or the SMOC1 Kazal (Kazal) domain (6 pmol/L-8 nmol/L). The dotted lines indicate the beginning and the end of the laser pulse; n = 4 or 5 independent experiments. (C) Thrombin activity assay in the presence of solvent (Sol; A431 cell supernatant), recombinant SMOC1 (rSMOC1), the overexpressed full-length SMOC1 protein (FL), SMOC1ΔKazal (ΔKazal), and the isolated Kazal domain (Kazal) (all 4 nmol/L); n = 6. ***P < .001, 1-way ANOVA and Bonferroni. (D) Thrombin-induced aggregation of washed human platelets in the presence of Sol (A431 cell supernatant), full-length SMOC1 (FL-SMOC1), the ΔKazal mutant, and the isolated Kazal domain (all 4 nmol/L); n = 5 donors. *P < .05, ***P < .001, 2-way ANOVA and Bonferroni test. (E) Fibrin clot formation (absorbance λ350 nm) in vitro induced by thrombin (0.1 U/mL) in the presence of Sol, full-length SMOC1 (FL), the ΔKazal mutant, and the isolated Kazal domain (all 4 nmol/L); n = 5 independent experiments. ***P < .001, 1-way ANOVA and Bonferroni test.
Figure 4.

Identification of the thrombin-binding domain of SMOC1. (A) Schematic diagram of the SMOC1 protein and the SMOC1 antibody binding site. KZ, Kazal domain; SP, SPARC domain; TY, thyroglobulin domain. (B) Microscale thermophoresis assay for the interaction between fluorescently labeled thrombin and full-length (FL) SMOC1 (FL-SMOC1), SMOC1ΔKazal, (ΔKazal), or the SMOC1 Kazal (Kazal) domain (6 pmol/L-8 nmol/L). The dotted lines indicate the beginning and the end of the laser pulse; n = 4 or 5 independent experiments. (C) Thrombin activity assay in the presence of solvent (Sol; A431 cell supernatant), recombinant SMOC1 (rSMOC1), the overexpressed full-length SMOC1 protein (FL), SMOC1ΔKazal (ΔKazal), and the isolated Kazal domain (Kazal) (all 4 nmol/L); n = 6. ***P < .001, 1-way ANOVA and Bonferroni. (D) Thrombin-induced aggregation of washed human platelets in the presence of Sol (A431 cell supernatant), full-length SMOC1 (FL-SMOC1), the ΔKazal mutant, and the isolated Kazal domain (all 4 nmol/L); n = 5 donors. *P < .05, ***P < .001, 2-way ANOVA and Bonferroni test. (E) Fibrin clot formation (absorbance λ350 nm) in vitro induced by thrombin (0.1 U/mL) in the presence of Sol, full-length SMOC1 (FL), the ΔKazal mutant, and the isolated Kazal domain (all 4 nmol/L); n = 5 independent experiments. ***P < .001, 1-way ANOVA and Bonferroni test.

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