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PSG6 inhibits platelet function without inducing cytotoxicity or impairing hemostasis . (A) Platelet cytotoxicity by LDH release. (B) Platelet apoptosis by Annexin-V (phosphatidylserine externalization). (C) Platelet viability by Calcein-AM. (D) Platelet aggregation induced with TRAP-6 at 5 μM of gentisic acid linked to TPP by different chain lengths. (E) P-Selectin expression (PE-CD62p). (F) Activated IIb/IIIa (FITC-Fibrinogen) (G) Representative image of clot retraction assay and clot weight after 2 h. <t>(H–I)</t> <t>PFA-200</t> closure times in collagen/ADP and collagen/epinephrine cartridges, respectively. Eptifibatide (EPT) 10 μM was used as a control for G–I. (J) Representative fluorescence microscopy images of thrombus formation on 3 different areas of collagen-coated channels under arterial shear conditions, showing vehicle (DMSO 0.2%) versus PSG6 at 10 μM. Thrombus coverage is expressed as the percentage of surface area occupied by fluorescently marked platelets. (K) Quantification of thrombus area based on fluorescence intensity of platelet-specific markers, using a defined threshold to exclude non-platelet signals. For A-F, data is shown as mean ± SEM (n = 6). One-way ANOVA – Bonferroni was performed as a statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 vs vehicle (DMSO 0.2%). For G-K, data are -presented as mean ± SEM (n = 3). One-way ANOVA with Bonferroni's post hoc test was used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. vehicle (DMSO 0.2%).
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PSG6 inhibits platelet function without inducing cytotoxicity or impairing hemostasis . (A) Platelet cytotoxicity by LDH release. (B) Platelet apoptosis by Annexin-V (phosphatidylserine externalization). (C) Platelet viability by Calcein-AM. (D) Platelet aggregation induced with TRAP-6 at 5 μM of gentisic acid linked to TPP by different chain lengths. (E) P-Selectin expression (PE-CD62p). (F) Activated IIb/IIIa (FITC-Fibrinogen) (G) Representative image of clot retraction assay and clot weight after 2 h. <t>(H–I)</t> <t>PFA-200</t> closure times in collagen/ADP and collagen/epinephrine cartridges, respectively. Eptifibatide (EPT) 10 μM was used as a control for G–I. (J) Representative fluorescence microscopy images of thrombus formation on 3 different areas of collagen-coated channels under arterial shear conditions, showing vehicle (DMSO 0.2%) versus PSG6 at 10 μM. Thrombus coverage is expressed as the percentage of surface area occupied by fluorescently marked platelets. (K) Quantification of thrombus area based on fluorescence intensity of platelet-specific markers, using a defined threshold to exclude non-platelet signals. For A-F, data is shown as mean ± SEM (n = 6). One-way ANOVA – Bonferroni was performed as a statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 vs vehicle (DMSO 0.2%). For G-K, data are -presented as mean ± SEM (n = 3). One-way ANOVA with Bonferroni's post hoc test was used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. vehicle (DMSO 0.2%).
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PSG6 inhibits platelet function without inducing cytotoxicity or impairing hemostasis . (A) Platelet cytotoxicity by LDH release. (B) Platelet apoptosis by Annexin-V (phosphatidylserine externalization). (C) Platelet viability by Calcein-AM. (D) Platelet aggregation induced with TRAP-6 at 5 μM of gentisic acid linked to TPP by different chain lengths. (E) P-Selectin expression (PE-CD62p). (F) Activated IIb/IIIa (FITC-Fibrinogen) (G) Representative image of clot retraction assay and clot weight after 2 h. <t>(H–I)</t> <t>PFA-200</t> closure times in collagen/ADP and collagen/epinephrine cartridges, respectively. Eptifibatide (EPT) 10 μM was used as a control for G–I. (J) Representative fluorescence microscopy images of thrombus formation on 3 different areas of collagen-coated channels under arterial shear conditions, showing vehicle (DMSO 0.2%) versus PSG6 at 10 μM. Thrombus coverage is expressed as the percentage of surface area occupied by fluorescently marked platelets. (K) Quantification of thrombus area based on fluorescence intensity of platelet-specific markers, using a defined threshold to exclude non-platelet signals. For A-F, data is shown as mean ± SEM (n = 6). One-way ANOVA – Bonferroni was performed as a statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 vs vehicle (DMSO 0.2%). For G-K, data are -presented as mean ± SEM (n = 3). One-way ANOVA with Bonferroni's post hoc test was used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. vehicle (DMSO 0.2%).
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PSG6 inhibits platelet function without inducing cytotoxicity or impairing hemostasis . (A) Platelet cytotoxicity by LDH release. (B) Platelet apoptosis by Annexin-V (phosphatidylserine externalization). (C) Platelet viability by Calcein-AM. (D) Platelet aggregation induced with TRAP-6 at 5 μM of gentisic acid linked to TPP by different chain lengths. (E) P-Selectin expression (PE-CD62p). (F) Activated IIb/IIIa (FITC-Fibrinogen) (G) Representative image of clot retraction assay and clot weight after 2 h. <t>(H–I)</t> <t>PFA-200</t> closure times in collagen/ADP and collagen/epinephrine cartridges, respectively. Eptifibatide (EPT) 10 μM was used as a control for G–I. (J) Representative fluorescence microscopy images of thrombus formation on 3 different areas of collagen-coated channels under arterial shear conditions, showing vehicle (DMSO 0.2%) versus PSG6 at 10 μM. Thrombus coverage is expressed as the percentage of surface area occupied by fluorescently marked platelets. (K) Quantification of thrombus area based on fluorescence intensity of platelet-specific markers, using a defined threshold to exclude non-platelet signals. For A-F, data is shown as mean ± SEM (n = 6). One-way ANOVA – Bonferroni was performed as a statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 vs vehicle (DMSO 0.2%). For G-K, data are -presented as mean ± SEM (n = 3). One-way ANOVA with Bonferroni's post hoc test was used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. vehicle (DMSO 0.2%).
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PSG6 inhibits platelet function without inducing cytotoxicity or impairing hemostasis . (A) Platelet cytotoxicity by LDH release. (B) Platelet apoptosis by Annexin-V (phosphatidylserine externalization). (C) Platelet viability by Calcein-AM. (D) Platelet aggregation induced with TRAP-6 at 5 μM of gentisic acid linked to TPP by different chain lengths. (E) P-Selectin expression (PE-CD62p). (F) Activated IIb/IIIa (FITC-Fibrinogen) (G) Representative image of clot retraction assay and clot weight after 2 h. <t>(H–I)</t> <t>PFA-200</t> closure times in collagen/ADP and collagen/epinephrine cartridges, respectively. Eptifibatide (EPT) 10 μM was used as a control for G–I. (J) Representative fluorescence microscopy images of thrombus formation on 3 different areas of collagen-coated channels under arterial shear conditions, showing vehicle (DMSO 0.2%) versus PSG6 at 10 μM. Thrombus coverage is expressed as the percentage of surface area occupied by fluorescently marked platelets. (K) Quantification of thrombus area based on fluorescence intensity of platelet-specific markers, using a defined threshold to exclude non-platelet signals. For A-F, data is shown as mean ± SEM (n = 6). One-way ANOVA – Bonferroni was performed as a statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 vs vehicle (DMSO 0.2%). For G-K, data are -presented as mean ± SEM (n = 3). One-way ANOVA with Bonferroni's post hoc test was used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. vehicle (DMSO 0.2%).
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PSG6 inhibits platelet function without inducing cytotoxicity or impairing hemostasis . (A) Platelet cytotoxicity by LDH release. (B) Platelet apoptosis by Annexin-V (phosphatidylserine externalization). (C) Platelet viability by Calcein-AM. (D) Platelet aggregation induced with TRAP-6 at 5 μM of gentisic acid linked to TPP by different chain lengths. (E) P-Selectin expression (PE-CD62p). (F) Activated IIb/IIIa (FITC-Fibrinogen) (G) Representative image of clot retraction assay and clot weight after 2 h. <t>(H–I)</t> <t>PFA-200</t> closure times in collagen/ADP and collagen/epinephrine cartridges, respectively. Eptifibatide (EPT) 10 μM was used as a control for G–I. (J) Representative fluorescence microscopy images of thrombus formation on 3 different areas of collagen-coated channels under arterial shear conditions, showing vehicle (DMSO 0.2%) versus PSG6 at 10 μM. Thrombus coverage is expressed as the percentage of surface area occupied by fluorescently marked platelets. (K) Quantification of thrombus area based on fluorescence intensity of platelet-specific markers, using a defined threshold to exclude non-platelet signals. For A-F, data is shown as mean ± SEM (n = 6). One-way ANOVA – Bonferroni was performed as a statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 vs vehicle (DMSO 0.2%). For G-K, data are -presented as mean ± SEM (n = 3). One-way ANOVA with Bonferroni's post hoc test was used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. vehicle (DMSO 0.2%).
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PSG6 inhibits platelet function without inducing cytotoxicity or impairing hemostasis . (A) Platelet cytotoxicity by LDH release. (B) Platelet apoptosis by Annexin-V (phosphatidylserine externalization). (C) Platelet viability by Calcein-AM. (D) Platelet aggregation induced with TRAP-6 at 5 μM of gentisic acid linked to TPP by different chain lengths. (E) P-Selectin expression (PE-CD62p). (F) Activated IIb/IIIa (FITC-Fibrinogen) (G) Representative image of clot retraction assay and clot weight after 2 h. <t>(H–I)</t> <t>PFA-200</t> closure times in collagen/ADP and collagen/epinephrine cartridges, respectively. Eptifibatide (EPT) 10 μM was used as a control for G–I. (J) Representative fluorescence microscopy images of thrombus formation on 3 different areas of collagen-coated channels under arterial shear conditions, showing vehicle (DMSO 0.2%) versus PSG6 at 10 μM. Thrombus coverage is expressed as the percentage of surface area occupied by fluorescently marked platelets. (K) Quantification of thrombus area based on fluorescence intensity of platelet-specific markers, using a defined threshold to exclude non-platelet signals. For A-F, data is shown as mean ± SEM (n = 6). One-way ANOVA – Bonferroni was performed as a statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 vs vehicle (DMSO 0.2%). For G-K, data are -presented as mean ± SEM (n = 3). One-way ANOVA with Bonferroni's post hoc test was used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. vehicle (DMSO 0.2%).
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PSG6 inhibits platelet function without inducing cytotoxicity or impairing hemostasis . (A) Platelet cytotoxicity by LDH release. (B) Platelet apoptosis by Annexin-V (phosphatidylserine externalization). (C) Platelet viability by Calcein-AM. (D) Platelet aggregation induced with TRAP-6 at 5 μM of gentisic acid linked to TPP by different chain lengths. (E) P-Selectin expression (PE-CD62p). (F) Activated IIb/IIIa (FITC-Fibrinogen) (G) Representative image of clot retraction assay and clot weight after 2 h. <t>(H–I)</t> <t>PFA-200</t> closure times in collagen/ADP and collagen/epinephrine cartridges, respectively. Eptifibatide (EPT) 10 μM was used as a control for G–I. (J) Representative fluorescence microscopy images of thrombus formation on 3 different areas of collagen-coated channels under arterial shear conditions, showing vehicle (DMSO 0.2%) versus PSG6 at 10 μM. Thrombus coverage is expressed as the percentage of surface area occupied by fluorescently marked platelets. (K) Quantification of thrombus area based on fluorescence intensity of platelet-specific markers, using a defined threshold to exclude non-platelet signals. For A-F, data is shown as mean ± SEM (n = 6). One-way ANOVA – Bonferroni was performed as a statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 vs vehicle (DMSO 0.2%). For G-K, data are -presented as mean ± SEM (n = 3). One-way ANOVA with Bonferroni's post hoc test was used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. vehicle (DMSO 0.2%).
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PSG6 inhibits platelet function without inducing cytotoxicity or impairing hemostasis . (A) Platelet cytotoxicity by LDH release. (B) Platelet apoptosis by Annexin-V (phosphatidylserine externalization). (C) Platelet viability by Calcein-AM. (D) Platelet aggregation induced with TRAP-6 at 5 μM of gentisic acid linked to TPP by different chain lengths. (E) P-Selectin expression (PE-CD62p). (F) Activated IIb/IIIa (FITC-Fibrinogen) (G) Representative image of clot retraction assay and clot weight after 2 h. <t>(H–I)</t> <t>PFA-200</t> closure times in collagen/ADP and collagen/epinephrine cartridges, respectively. Eptifibatide (EPT) 10 μM was used as a control for G–I. (J) Representative fluorescence microscopy images of thrombus formation on 3 different areas of collagen-coated channels under arterial shear conditions, showing vehicle (DMSO 0.2%) versus PSG6 at 10 μM. Thrombus coverage is expressed as the percentage of surface area occupied by fluorescently marked platelets. (K) Quantification of thrombus area based on fluorescence intensity of platelet-specific markers, using a defined threshold to exclude non-platelet signals. For A-F, data is shown as mean ± SEM (n = 6). One-way ANOVA – Bonferroni was performed as a statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 vs vehicle (DMSO 0.2%). For G-K, data are -presented as mean ± SEM (n = 3). One-way ANOVA with Bonferroni's post hoc test was used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. vehicle (DMSO 0.2%).
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PSG6 inhibits platelet function without inducing cytotoxicity or impairing hemostasis . (A) Platelet cytotoxicity by LDH release. (B) Platelet apoptosis by Annexin-V (phosphatidylserine externalization). (C) Platelet viability by Calcein-AM. (D) Platelet aggregation induced with TRAP-6 at 5 μM of gentisic acid linked to TPP by different chain lengths. (E) P-Selectin expression (PE-CD62p). (F) Activated IIb/IIIa (FITC-Fibrinogen) (G) Representative image of clot retraction assay and clot weight after 2 h. (H–I) PFA-200 closure times in collagen/ADP and collagen/epinephrine cartridges, respectively. Eptifibatide (EPT) 10 μM was used as a control for G–I. (J) Representative fluorescence microscopy images of thrombus formation on 3 different areas of collagen-coated channels under arterial shear conditions, showing vehicle (DMSO 0.2%) versus PSG6 at 10 μM. Thrombus coverage is expressed as the percentage of surface area occupied by fluorescently marked platelets. (K) Quantification of thrombus area based on fluorescence intensity of platelet-specific markers, using a defined threshold to exclude non-platelet signals. For A-F, data is shown as mean ± SEM (n = 6). One-way ANOVA – Bonferroni was performed as a statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 vs vehicle (DMSO 0.2%). For G-K, data are -presented as mean ± SEM (n = 3). One-way ANOVA with Bonferroni's post hoc test was used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. vehicle (DMSO 0.2%).

Journal: Redox Biology

Article Title: PSG6: A mitochondrially-targeted gentisic acid derivative exerts antiplatelet action via mitochondrial complex I inhibition

doi: 10.1016/j.redox.2026.104200

Figure Lengend Snippet: PSG6 inhibits platelet function without inducing cytotoxicity or impairing hemostasis . (A) Platelet cytotoxicity by LDH release. (B) Platelet apoptosis by Annexin-V (phosphatidylserine externalization). (C) Platelet viability by Calcein-AM. (D) Platelet aggregation induced with TRAP-6 at 5 μM of gentisic acid linked to TPP by different chain lengths. (E) P-Selectin expression (PE-CD62p). (F) Activated IIb/IIIa (FITC-Fibrinogen) (G) Representative image of clot retraction assay and clot weight after 2 h. (H–I) PFA-200 closure times in collagen/ADP and collagen/epinephrine cartridges, respectively. Eptifibatide (EPT) 10 μM was used as a control for G–I. (J) Representative fluorescence microscopy images of thrombus formation on 3 different areas of collagen-coated channels under arterial shear conditions, showing vehicle (DMSO 0.2%) versus PSG6 at 10 μM. Thrombus coverage is expressed as the percentage of surface area occupied by fluorescently marked platelets. (K) Quantification of thrombus area based on fluorescence intensity of platelet-specific markers, using a defined threshold to exclude non-platelet signals. For A-F, data is shown as mean ± SEM (n = 6). One-way ANOVA – Bonferroni was performed as a statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 vs vehicle (DMSO 0.2%). For G-K, data are -presented as mean ± SEM (n = 3). One-way ANOVA with Bonferroni's post hoc test was used for statistical analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. vehicle (DMSO 0.2%).

Article Snippet: Subsequently, the sample was loaded into the Collagen/Epinephrine or Collagen/ADP cartridge, and the closure time was measured in the Innovance PFA-200 system (Siemens Healthcare Diagnostics Products, Munich, Germany).

Techniques: Expressing, Control, Fluorescence, Microscopy, Shear