Mitochondrial depolarization and PS externalization in human platelets are not affected by actin depolymerization and polymerization.

A. Mitochondrial depolarization was measured in washed human platelets using the JC-1 dye. Bar graph depicts the loss of mitochondrial membrane potential in platelets treated with DMSO vehicle alone (DMSO, white bars) or with 10 μM cytochalasin D alone (CytoD, black bars). Data are mean ± SEM, analyzed by t-test, and represent a minimum of 3 independent experiments using blood from different donors. B. Phosphatidylserine (PS) exposure was measured in washed platelets using the Annexin V dye. Bar graph depicts PS exposure in platelets treated with DMSO vehicle alone (DMSO, white bars) or with 10 μM cytochalasin D alone (CytoD, black bars). Data are mean ± SEM, analyzed by t-test, and represent a minimum of 3 independent experiments using blood from different donors. C. Mitochondrial depolarization was measured in washed human platelets using the JC-1 dye. Bar graph depicts the loss of mitochondrial membrane potential in platelets treated with DMSO vehicle alone (DMSO, white bars) or with 1 μM jasplakinolide alone (Jasp, black bars). Data are mean ± SEM, analyzed t-test, and represent a minimum of 3 independent experiments using blood from different donors. D. Phosphatidylserine (PS) exposure was measured in washed platelets using the Annexin V dye. Bar graph depicts PS exposure in platelets treated with DMSO vehicle alone (DMSO, white bars) or with 1 μM jasplakinolide alone (Jasp, black bars). Data are mean ± SEM, analyzed by using t-test, and represent a minimum of 3 independent experiments using blood from different donors.</p

ABT-737-induced caspase-3 cleavage in human platelets is not affected by actin depolymerization.

A. Washed human platelets were incubated in the absence (-) or presence (+) of the actin depolymerizing drug cytochalasin D, and in the absence or presence of 1 μM ABT-737, for the indicated times. Equal amounts of platelet lysate were resolved by SDS-PAGE; immunoblots were probed for pro-caspase-3 and cleaved caspase-3. Beta-actin (β-actin) is shown as a loading control. B. Bar graph depicts caspase-3 cleavage (normalized to beta-actin levels) induced by 1 μM ABT-737 in the absence (ABT-737, white bars) or presence (ABT-737+CytoD) of cytochalasin D. Data are mean ± SEM, analyzed by 2-way ANOVA and Bonferroni post-hoc multiple comparison tests, and represent a minimum of 3 independent experiments using blood from different donors. C. Immunoblots represent pro-caspase-3 and cleaved caspase-3 levels in platelets treated with 10 μM ABT-737, in the presence or absence of cytochalasin D. Beta-actin (β-actin) is shown as a loading control. D. Bar graph depicts caspase-3 cleavage (normalized to beta-actin levels) induced by 10 μM ABT-737 in the absence (ABT-737, white bars) or presence (ABT-737+CytoD) of cytochalasin D. Data are mean ± SEM, analyzed by 2-way ANOVA and Bonferroni post-hoc multiple comparison tests, and represent a minimum of 3 independent experiments using blood from different donors.</p

ABT-737-induced mitochondrial depolarization and PS externalization in human platelets are not affected by the induction of actin polymerization.

A. Mitochondrial depolarization was measured in washed human platelets using the JC-1 dye. Bar graph depicts the loss of mitochondrial membrane potential in platelets treated with DMSO vehicle alone (DMSO, white bars), with 1 μM ABT-737 (ABT-737, blue bars) or with 1 μM ABT plus the actin polymerizing agent jasplakinolide (ABT-737+Jasp, pink bars). Data are mean ± SEM, analyzed by 2-way ANOVA and Bonferroni post-hoc multiple comparison tests, and represent a minimum of 3 independent experiments using blood from different donors. B, C. Phosphatidylserine (PS) exposure was measured in washed platelets using the Annexin V dye. Bar graphs depict PS exposure in platelets treated with DMSO vehicle alone (DMSO, white bars), with 1 μM ABT-737 (ABT-737, blue bars) or with 1 μM ABT plus the actin polymerizing agent jasplakinolide (ABT-737+Jasp, pink bars). Data are expressed both in terms of mean fluorescence intensity (B) and as a percentage (%) of PS-positive platelets (C). Data are mean ± SEM, analyzed by using 2-way ANOVA and Bonferroni post-hoc multiple comparison tests, and represent a minimum of 3 independent experiments using blood from different donors. D. Bar graph depicts the loss of mitochondrial membrane potential in platelets treated with DMSO vehicle alone (DMSO, white bars), with 10 μM ABT-737 (ABT-737, blue bars) or with 10 μM ABT plus the actin polymerizing agent jasplakinolide (ABT-737+Jasp, pink bars). Data are mean ± SEM, analyzed 2-way ANOVA and Bonferroni post-hoc multiple comparison tests and represent a minimum of 3 independent experiments using blood from different donors. E, F. Bar graphs depict PS exposure in platelets treated with DMSO vehicle alone (DMSO, white bars), with 10 μM ABT-737 (ABT-737, blue bars) or with 10 μM ABT plus the actin polymerizing agent jasplakinolide (ABT-737+Jasp, pink bars). Data are expressed both in terms of mean fluorescence intensity (E) and as a percentage (%) of PS-positive platelets (F). Data are mean ± SEM, analyzed by 2-way ANOVA and Bonferroni post-hoc multiple comparison tests, and represent a minimum of 3 independent experiments using blood from different donors.</p

S1 Data -

(ZIP)</p

ABT-737-induced mitochondrial depolarization and PS externalization in human platelets are not affected by actin depolymerization.

A. Mitochondrial depolarization was measured in washed human platelets using the JC-1 dye. Bar graph depicts the loss of mitochondrial membrane potential in platelets treated with DMSO vehicle alone (DMSO, white bars), with 1 μM ABT-737 (ABT-737, blue bars) or with 1 μM ABT plus the actin depolymerizing agent cytochalasin D (ABT-737+CytoD, pink bars). Data are mean ± SEM, analyzed by 2-way ANOVA and Bonferroni post-hoc multiple comparison tests, and represent a minimum of 3 independent experiments using blood from different donors. B, C. Phosphatidylserine (PS) exposure was measured in washed platelets using the Annexin V dye. Bar graphs depict PS exposure in platelets treated with DMSO vehicle alone (DMSO, white bars), with 1 μM ABT-737 (ABT-737, blue bars) or with 1 μM ABT plus the actin depolymerizing agent cytochalasin D (ABT-737+CytoD, pink bars). Data are expressed both in terms of mean fluorescence intensity (B) and as a percentage (%) of PS-positive platelets (C). Data are mean ± SEM, analyzed by using 2-way ANOVA and Bonferroni post-hoc multiple comparison tests, and represent a minimum of 3 independent experiments using blood from different donors. D. Bar graph depicts the loss of mitochondrial membrane potential in platelets treated with DMSO vehicle alone (DMSO, white bars), with 10 μM ABT-737 (ABT-737, blue bars) or with 10 μM ABT plus the actin depolymerizing agent cytochalasin D (ABT-737+CytoD, pink bars). Data are mean ± SEM, analyzed 2-way ANOVA and Bonferroni post-hoc multiple comparison tests and represent a minimum of 3 independent experiments using blood from different donors. E, F. Bar graphs depict PS exposure in platelets treated with DMSO vehicle alone (DMSO, white bars), with 10 μM ABT-737 (ABT-737, blue bars) or with 10 μM ABT plus the actin depolymerizing agent cytochalasin D (ABT-737+CytoD, pink bars). Data are expressed both in terms of mean fluorescence intensity (E) and as a percentage (%) of PS-positive platelets (F). Data are mean ± SEM, analyzed by 2-way ANOVA and Bonferroni post-hoc multiple comparison tests, and represent a minimum of 3 independent experiments using blood from different donors.</p

ABT-737-induced caspase-3 cleavage in human platelets is not affected by the induction of actin polymerization.

A. Washed human platelets were incubated in the absence (-) or presence (+) of the actin polymerizing drug jasplakinolide, and in the absence or presence of 1 μM ABT-737, for the indicated times. Equal amounts of platelet lysate were resolved by SDS-PAGE; immunoblots were probed for pro-caspase-3 and cleaved caspase-3. Beta-actin (β-actin) is shown as a loading control. B. Bar graph depicts caspase-3 cleavage (normalized to beta-actin levels) induced by 1 μM ABT-737 in the absence (ABT-737, white bars) or presence (ABT-737+Jasp) of jasplakinolide. Data are mean ± SEM, analyzed by 2-way ANOVA and Bonferroni post-hoc multiple comparison tests, and represent a minimum of 3 independent experiments using blood from different donors. C. Immunoblots represent pro-caspase-3 and cleaved caspase-3 levels in platelets treated with 10 μM ABT-737, in the presence or absence of jasplakinolide. Beta-actin (β-actin) is shown as a loading control. D. Bar graph depicts caspase-3 cleavage (normalized to beta-actin levels) induced by 10 μM ABT-737 in the absence (ABT-737, white bars) or presence (ABT-737+Jasp) of jasplakinolide. Data are mean ± SEM, analyzed by 2-way ANOVA and Bonferroni post-hoc multiple comparison tests, and represent a minimum of 3 independent experiments using blood from different donors.</p

Aggregation is impaired in starved platelets due to enhanced autophagy and cellular energy depletion

<p>Platelet hyperactivity is the hallmark of thrombosis and hemostasis disorders including atherosclerosis, diabetes, stroke, arthritis, and cancer causing significant mortality and morbidity. Therefore, regulating platelet hyperactivity is an ever growing interest. Very recently, basal autophagic process has been demonstrated to be essential for normal functioning of platelets. However, autophagy can be elevated above basal level under conditions like starvation, and how platelets respond in these settings remains to be elucidative. Therefore, in this study we demonstrate a substantial autophagy induction (above basal level) by starvation, which decreases platelet aggregation responses to various agonists. The decreased aggregation in starved platelets was restored in combination with autophagy inhibitors (3-methyladenine and NH₄Cl) and acetate supplementation. Starved platelets also showed decreased calcium mobilization, granule release, and adhesive properties. Furthermore, <i>ex vivo</i> platelets obtained from starved rats showed increased autophagy markers and decreased aggregation responses to various agonists. Our results distinctly explain that enhanced autophagy and cellular energy depletion are the cause for decreased platelet activation and aggregation. The study emphasizes the cardinal role of starvation and autophagy in the management of diseases and disorders associated with platelet hyperactivity.</p

Effect of compound 4f on A23187 induced (A) Translocation of cytosolic cytochrome C and activation of caspase-3 (B) Caspase-9 and (C) Caspase-3 activities and (D) PS externalization in PRP and washed platelets.

<p>Values are presented as mean ± SEM (n = 5), expressed as percentage increase in (B & C) caspase activity and (D) Annexin V-FITC fluorescence and expressed as percentage increase in apoptotic platelets expressing PS relative to control. ***<i>p</i><0.001; significant compared to control. ^#<i>p</i><0.05, ^##<i>p</i><0.01, ^###<i>p</i><0.001; significant compared to A23187.</p

Effect of compound 4f on (A) Collagen induced protein phosphorylation (B) A23187 induced γ-glutamyltransferase activity (C) MTT cell viability assay (D) LDH release in platelets.

<p>(A) Lane I- resting platelets (untreated). Lane II- platelets treated with Collagen (1 µg/mL). Lanes III, IV and V- pre-loaded platelets with collagen and incubated with 4f in increasing concentration of 25, 50 and 100 µM respectively. Values are presented as mean ± SEM (n = 5). ***<i>p</i><0.001; significant compared to control. ^#<i>p</i><0.05, ^##<i>p</i><0.01, ^###<i>p</i><0.001; significant compared to agonist.</p

Efficacy of Ibuprofen and its derivatives measured in terms of (A) Reduction potential (B) DPPH radical scavenging activity in comparison with Quercetin.

<p>Values are presented as mean ± SEM (n = 5).</p