Kashiwagi H., Yuhki K., Kojima F., Kumei S., Takahata O., Sakai Y., Narumiya S. and Ushikubi F. The novel prostaglandin I2 mimetic ONO-1301 escapes desensitization in an anti-platelet effect due to its inhibitory action on thromboxane A2 synthesis in mice The Journal of Pharmacology and Experimental Therapeutics

Supplemental Figure 1

80 ONO-1301 Beraprost

60

40

20 Aggregation (%) Aggregation 0 C 10 9 8 7 6 5 -Log [Reagent] (M)

Effects of ONO-1301 and beraprost on U-46619-induced platelet aggregation Effects of ONO-1301 and beraprost on U-46619-induced aggregation of platelets prepared from WT mice. U-46619 was added at a concentration to induce platelet aggregation of 45-55% (2.5–3.0 µM). ONO-1301 or beraprost was added at indicated concentrations to PRP 1 min before the addition of U-46619. C of the horizontal axis represents control. Each value is the mean ± S.E.M. (n = 4). Kashiwagi H., Yuhki K., Kojima F., Kumei S., Takahata O., Sakai Y., Narumiya S. and Ushikubi F. The novel prostaglandin I2 mimetic ONO-1301 escapes desensitization in an anti-platelet effect due to its inhibitory action on thromboxane A2 synthesis in mice The Journal of Pharmacology and Experimental Therapeutics

Supplemental Figure 2

Vehicle ONO-1301 (20 mg/kg) ONO-1301 (100 mg/kg) *

Beraprost (0.3 mg/kg) * 90 Beraprost (3 mg/kg) *

70

50

30

Mean blood pressure (mmHg) pressure Meanblood 0 30 60 90 120 150 180 Time (min)

Effects of ONO-1301 and beraprost on blood pressure Effects of ONO-1301 and beraprost on mean blood pressure in mice. ONO-1301 (20 or 100 mg/kg) or beraprost (0.3 or 3 mg/kg) was administered orally to WT mice and mean blood pressure of conscious mice was measured by the tail-cuff method. Each value is the mean ± S.E.M. (n = 5). *P < 0.05 vs. vehicle. Kashiwagi H., Yuhki K., Kojima F., Kumei S., Takahata O., Sakai Y., Narumiya S. and Ushikubi F. The novel prostaglandin I2 mimetic ONO-1301 escapes desensitization in an anti-platelet effect due to its inhibitory action on thromboxane A2 synthesis in mice The Journal of Pharmacology and Experimental Therapeutics

Supplemental Figure 3

Vehicle ONO-1301 80 Beraprost

60

40 * * * 20 Aggregation (%) Aggregation 0 1 4 7 10 Days

Effects of repeated administration of ONO-1301 and beraprost on U-46619-induced platelet aggregation Inhibitory effects of ONO-1301 and beraprost on U-46619-induced platelet aggregation at indicated time points are presented. ONO-1301 (20 mg/kg/day) or beraprost (0.3 mg/kg/day) was administered orally to WT mice once a day, and PRP was prepared 2 hr after the administration. U-46619 (3.0 µM) was added to PRP to induce platelet aggregation. Each value is the mean ± S.E.M. Vehicle, n = 4; ONO-1301, n = 6; beraprost, n = 6. *P < 0.05 vs. beraprost at 1st day. Kashiwagi H., Yuhki K., Kojima F., Kumei S., Takahata O., Sakai Y., Narumiya S. and Ushikubi F. The novel prostaglandin I2 mimetic ONO-1301 escapes desensitization in an anti-platelet effect due to its inhibitory action on thromboxane A2 synthesis in mice The Journal of Pharmacology and Experimental Therapeutics

Supplemental Figure 4

150

100 α level(pg/mL) α 1 50 ketoPGF

- 0 6 Vehicle ONO-1301 Beraprost

Repeated administration

Effects of repeated administration of ONO-1301 and beraprost on PGI2 production Effects of repeated administration of ONO-1301 and beraprost on plasma 6-keto PGF1α levels were examined. ONO-1301 (20 mg/kg/day) or beraprost (0.3 mg/kg/day) was administered orally to WT mice once a day, and plasma was prepared 2 hr after administration on the 10th day. Each value is the mean ± S.E.M. Vehicle, n = 4; ONO-1301, n = 6; beraprost, n = 6. Kashiwagi H., Yuhki K., Kojima F., Kumei S., Takahata O., Sakai Y., Narumiya S. and Ushikubi F. The novel prostaglandin I2 mimetic ONO-1301 escapes desensitization in an anti-platelet effect due to its inhibitory action on thromboxane A2 synthesis in mice The Journal of Pharmacology and Experimental Therapeutics

Supplemental Figure 5

60 *

40 level (pg/mL)level

2 20 PGE 0 Vehicle ONO-1301

Repeated administration

An effect of repeated administration of ONO-1301 on PGE2 production An effect of repeated administration of ONO-1301 on plasma PGE2 level was examined. ONO-1301 (20 mg/kg/day) was administered orally to WT mice once a day, and plasma was prepared 2 hr after administration on the 10th day. Each value is the mean ± S.E.M. (n = 5). *P < 0.05 vs. vehicle.