![CS585 potently inhibits human platelet activity. (A) Aggregation of washed human platelets treated with vehicle or CS585 (0.2-100 nM), stimulated with thrombin (0.5 nM), collagen (0.5 μg/mL), or ADP (20 μM). Data represent mean ± SEM. One-way ANOVA with Dunnett comparison (n = 4-6). (B) Representative aggregation curves for panel A. (C) Aggregation of human PRP treated with vehicle or CS585 (0.25-5 μM), stimulated with collagen (1 and 2 μg/mL, closed and open boxes, respectively) (n = 4). Data represent mean ± SEM. Mixed effects analysis with Dunnett comparison. Collagen aggregation of 1 μg/mL response compared with that of the vehicle; ∗∗P < .01; ∗∗∗∗P < .0001. Collagen aggregation of 2 μg/mL response compared with that of the vehicle; ##P < .01; ####P < .0001. (D) Representative aggregation curves for panel C. (E) Aggregation of washed human platelets treated with CS585 (1 μM), stimulated with thrombin (0.5-5 nM), collagen (0.5-10 μg/mL), or ADP (10-80 μM) (n = 4). (F) Expression of VASP phosphorylation (Ser157 VASP, 50 kilodalton [kDa]; total VASP, 46 and 50 kDa), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (37 kDa) on washed human platelets treated with vehicle or CS585 (10 pM-100 nM) for 10 minutes (n = 4). Representative blots are included above the graph. Data represent mean ± SEM. One-way ANOVA and Dunnett comparison. Asterisks denote statistical differences between vehicle and treated groups. (G) Activation of integrin αIIbβ3, α-granule, and dense granule secretion of washed human platelets treated vehicle or CS585 (6.2-100 nM), stimulated with convulxin (25 ng/mL) (n = 6). Data represent mean ± SEM. One-way ANOVA with Dunnett comparison. Asterisks denote statistical differences between vehicle and treated groups. (H) cAMP and cGMP levels measured in washed human platelets treated with vehicle, CS585 (0.1-100 nM), or positive controls forskolin (10 μM) and PAPA-NONOate (PAPA-NO, 5 μM) (n = 4). Data represent mean ± SEM. One-way ANOVA with Dunnett multiple comparison test. (I) Expression of Ser157 VASP (50 kDa) and GAPDH (37 kDa) in washed human platelets treated with vehicle or PKA inhibitor H89 (100 μM) before incubation with vehicle or CS585 (100 nM) (n = 6). Data represent mean ± SEM. One-way ANOVA with Tukey multiple comparison test. (J) Representative blot of panel I. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/142/18/10.1182_blood.2023020622/2/blood_bld-2023-020622-gr2.jpeg?Expires=1735551800&Signature=AwOWfS6-cRj~1Nlp5xKz0IjOwldb1trQXZV2NsxsT9NWwwXeEuTqrM9AeiaSMKJaIEhRb5lyDIWeFFrwinRfN4Df-cbPoKI2D8rYgUMk5-JqSNlWXaLWKGNe-M5MBM~NmfHekXmH4sBfzRsLEpVVo6-Izpc4ytfdvuR91v4iVkuqewT2OxvPExJAHfYKjfmzk7Kf8f04KkaADFSnvo5qi5f9c6DtTRXm1R4bFh8FmVsyqpwuHqFFYm4wPoE2X-gJct5fyx44so698knCA8wLvRlrCirgnevyE-3IRYdMvHQFYqbD44ss4YOYIHbjjsd9Pu97XG5MFlHns63NOi0QWA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
CS585 potently inhibits human platelet activity. (A) Aggregation of washed human platelets treated with vehicle or CS585 (0.2-100 nM), stimulated with thrombin (0.5 nM), collagen (0.5 μg/mL), or ADP (20 μM). Data represent mean ± SEM. One-way ANOVA with Dunnett comparison (n = 4-6). (B) Representative aggregation curves for panel A. (C) Aggregation of human PRP treated with vehicle or CS585 (0.25-5 μM), stimulated with collagen (1 and 2 μg/mL, closed and open boxes, respectively) (n = 4). Data represent mean ± SEM. Mixed effects analysis with Dunnett comparison. Collagen aggregation of 1 μg/mL response compared with that of the vehicle; ∗∗P < .01; ∗∗∗∗P < .0001. Collagen aggregation of 2 μg/mL response compared with that of the vehicle; ##P < .01; ####P < .0001. (D) Representative aggregation curves for panel C. (E) Aggregation of washed human platelets treated with CS585 (1 μM), stimulated with thrombin (0.5-5 nM), collagen (0.5-10 μg/mL), or ADP (10-80 μM) (n = 4). (F) Expression of VASP phosphorylation (Ser157 VASP, 50 kilodalton [kDa]; total VASP, 46 and 50 kDa), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (37 kDa) on washed human platelets treated with vehicle or CS585 (10 pM-100 nM) for 10 minutes (n = 4). Representative blots are included above the graph. Data represent mean ± SEM. One-way ANOVA and Dunnett comparison. Asterisks denote statistical differences between vehicle and treated groups. (G) Activation of integrin αIIbβ3, α-granule, and dense granule secretion of washed human platelets treated vehicle or CS585 (6.2-100 nM), stimulated with convulxin (25 ng/mL) (n = 6). Data represent mean ± SEM. One-way ANOVA with Dunnett comparison. Asterisks denote statistical differences between vehicle and treated groups. (H) cAMP and cGMP levels measured in washed human platelets treated with vehicle, CS585 (0.1-100 nM), or positive controls forskolin (10 μM) and PAPA-NONOate (PAPA-NO, 5 μM) (n = 4). Data represent mean ± SEM. One-way ANOVA with Dunnett multiple comparison test. (I) Expression of Ser157 VASP (50 kDa) and GAPDH (37 kDa) in washed human platelets treated with vehicle or PKA inhibitor H89 (100 μM) before incubation with vehicle or CS585 (100 nM) (n = 6). Data represent mean ± SEM. One-way ANOVA with Tukey multiple comparison test. (J) Representative blot of panel I. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.
