Jian-ming Li, MDa, Charles S. C. Garnette, MDa, Mitchell Cahn, MDa, R

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Recombinant thrombomodulin inhibits arterial smooth muscle cell proliferation induced by thrombin Jian-ming Li, MDa, Charles S.C. Garnette, MDa, Mitchell Cahn, MDa, R.Brannon Claytor, MDa, Michael J. Rohrer, MDa, James G. Dobson, PhDb, Bruce Gerlitz, MSc, Bruce S. Cutler, MDa Journal of Vascular Surgery Volume 32, Issue 4, Pages 804-813 (October 2000) DOI: 10.1067/mva.2000.107992 Copyright © 2000 Society for Vascular Surgery and The American Association for Vascular Surgery, a Chapter of the International Society for Cardiovascular Surgery Terms and Conditions

Fig. 1 Effect of α-thrombin on SMC proliferation. SMC proliferation was quantified with MTT mitochontrial function assay on day 7. Results represent the percent growth as compared with buffer control (vehicle PBS) groups. Values are means ± SEM of four individual experiments. Each experimental group was run in quadruplicate. Asterisk represents significance from control groups, P <.006. Journal of Vascular Surgery 2000 32, 804-813DOI: (10.1067/mva.2000.107992) Copyright © 2000 Society for Vascular Surgery and The American Association for Vascular Surgery, a Chapter of the International Society for Cardiovascular Surgery Terms and Conditions

Fig. 2 Effect of thrombin, hirudin, recombinant human soluble thrombomodulin (TMD1, CS+) (rTM) on SMC proliferation. SMC proliferation was quantified with MTT mitochondrial function assay on day 7. Results represent the percent growth as compared with buffer control (vehicle PBS ± Tris buffer) groups. Values are means ± SEM of three individual experiments. Each experimental group was run in quadruplicate. * represents significance from control groups, P <.006. ** represents significance from thrombin alone group, P <.0005. Journal of Vascular Surgery 2000 32, 804-813DOI: (10.1067/mva.2000.107992) Copyright © 2000 Society for Vascular Surgery and The American Association for Vascular Surgery, a Chapter of the International Society for Cardiovascular Surgery Terms and Conditions

Fig. 3 Effect of recombinant human soluble thrombomodulin (TMD1, CS+) (rTM) on SMC proliferation induced by α-thrombin. SMC DNA synthesis was evaluated with BrdU-DNA incorporation assay on day 3. Results represent the percentage of buffer control (vehicle PBS with and without Tris-HCl buffer) groups. Values are means ± SEM of three individual experiments. Each experimental group was run in quadruplicate. * represents significance from control groups, P <.03. ** represents significance from the thrombin group value, P <.008. Journal of Vascular Surgery 2000 32, 804-813DOI: (10.1067/mva.2000.107992) Copyright © 2000 Society for Vascular Surgery and The American Association for Vascular Surgery, a Chapter of the International Society for Cardiovascular Surgery Terms and Conditions

Fig. 4 Effect of recombinant human soluble thrombomodulin (TMD1, CS+) (rTM) on SMC proliferation induced by α-thrombin. SMCs were cultured in media with thrombin at 1 U/mL alone (solid bar ), thrombin at 1 U/mL combined with rTM at 1 μg/mL (striped bar ), at 2 μg/mL (checkered bar ), and at 3 μg/mL (open bar ) of rTM. SMC proliferation was quantified with MTT mitochondrial function assay on days 3, 7, and 10, respectively. Results represent the percent growth as compared with buffer control (vehicle PBS with and without Tris-HCl buffer) groups. Values are means ± SEM of three individual experiments. Each experimental group was run in quadruplicate. * represents significance from thrombin-alone groups, P <.003 to <.05. Journal of Vascular Surgery 2000 32, 804-813DOI: (10.1067/mva.2000.107992) Copyright © 2000 Society for Vascular Surgery and The American Association for Vascular Surgery, a Chapter of the International Society for Cardiovascular Surgery Terms and Conditions

Fig. 5 Effect of thrombin receptor agonist peptide SPLLRNPNDSYEPF (SFLL) on SMC proliferation. SMCs were subjected to a serial concentration of SFLL (A). SMC DNA replication was evaluated with BrdU-DNA incorporation assay at 48 hours. Results represent the percent growth as compared with buffer control groups. Values are means ± SEM of three individual experiments. Each group was run in quadruplicate. Asterisk represents significance from control groups, P <.007 (A). Effect of rTM at 100 nmol/L on the stimulation effect of SMC DNA synthesis (B) and SMC proliferation (C) induced by SFLL at 20 μmol/L. SMC DNA synthesis was evaluated with BrdU-DNA incorporation assay, and SMC proliferation was evaluated with mitochondrial function assay at 48 hours. Results represent the percent growth as compared with buffer control groups. Values are means ± SEM of three individual experiments. Each group was run in quadruplicate. Asterisk represents significance from control groups, P <.003. There was no significant difference between SFLL group and SFLL with rTM group. Journal of Vascular Surgery 2000 32, 804-813DOI: (10.1067/mva.2000.107992) Copyright © 2000 Society for Vascular Surgery and The American Association for Vascular Surgery, a Chapter of the International Society for Cardiovascular Surgery Terms and Conditions