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

Stereo view of 3D structure of the SVMP (Bothropasin) showing molecular interaction with the compound 5d.

<p>The interaction between the compound and active site residues are shown using a line representation colored by parent atoms except for carbon (green color). The ligand is presented in the ball and stick form with their parent color except for carbon (white color). Hydrogen bonds are presented as white dotted lines.</p

Effect of compound 5d on EC venom-induced fibrino(geno)lytic activity.

<p>EC venom (0.2 µg) was pre-incubated separately with different doses of compound 5d (1∶0; 1∶1; 1∶5; 1∶10; 1∶25 and 1∶50; venom: compound; w/w; Lane 2–7 respectively, Lane-1 represents substrate alone and Lane-8 represents substrate with 10 µg of compound 5d alone) for 10 min at 37°C. The reaction was initiated by adding respective substrates (A) fibrin and (B) fibrinogen and incubated for 30 min. Samples were electrophoresed on 10% SDS-PAGE and the gels were processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106364#s2" target="_blank">materials and methods</a> section.</p

UV-VIS spectral studies of compound 5d in presence of CaCl₂ and ZnCl₂.

<p>The mixture of compound 5d (1 mM) and different concentrations of (A) CaCl₂ and (B) ZnCl₂ (0–2.0 mM) in a final volume of 1 mL PBS. The samples were monitored by spectroscopic scanning with the wavelength range of 200 to 300 nm.</p

Schematic representation for the synthesis of apigenin structural analogues.

<p>Schematic representation for the synthesis of apigenin structural analogues.</p

Effect of compound 5d on EC venom induced myotoxicity.

<p>EC venom (5 µg) was pre-incubated separately with varied doses of compound 5d (1∶0; 1∶5; 1∶10 and 1∶25; venom: compound; w/w) for 10 min at 37°C. The samples were injected intramuscularly into group of mice (n = 5) in a total volume of 50 µL saline. Mice were anaesthetized and sacrificed after 3 h and the assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106364#s2" target="_blank">materials and methods</a> section. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106364#s3" target="_blank">Results</a> are expressed as mean ± SEM of three independent experiments. <b>***</b><i>p</i><0.001, <b>**</b><i>p</i><0.01, a - significant compared to saline control group and b - significant compared to venom alone group.</p

Effect of compound 5d on EC venom-induced serum LDH and CPK levels in experimental animals in an independent injection experiment.

<p>EC venom (5 µg) was intramuscularly injected to the group of mice (n = 5) followed by compound 5d (250 µg) injection at various time intervals (0, 2.5, 5, 7.5 and 10 min) to the same site where venom had been injected, and then the respective assay was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106364#s2" target="_blank">materials and methods</a> section. Data are presented as the mean ± SEM, <b>***</b><i>p</i><0.001, <b>*</b><i>p</i><0.05, a - significant compared to saline control group and b - significant compared to venom alone group.</p><p>Effect of compound 5d on EC venom-induced serum LDH and CPK levels in experimental animals in an independent injection experiment.</p

Neutralization of hemorrhagic activity of EC venom by compound 5d.

<p>EC venom (2 µg) was pre-incubated separately with various doses of compound 5d (1∶0 to 1∶10; venom: compound; w/w) in a total volume of 50 µL PBS for 10 min at 37°C. The samples were then injected intradermally into experimental animals (n = 5) and the assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106364#s2" target="_blank">materials and methods</a> section. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106364#s3" target="_blank">Results</a> are expressed as repetitive pictures of three independent experiments.</p

Effect of compound 5d on caseinolytic and gelatinolytic activities of EC venom.

<p>EC venom (3 µg) was pre-incubated separately with varied doses of compound 5d (1∶0; 1∶1; 1∶2; 1∶5; 1∶10; 1∶25 and 1∶50; venom: compound; w/w; Lane 1–7 respectively, Lane-8 represents 100 µg of compound 5d alone) for 10 min at 37°C. Samples were electrophoresed on gels impregnated separately with (A) casein (0.2%) and (B) gelatin (1%) as substrate. The gels were then processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106364#s2" target="_blank">materials and methods</a> section.</p

Effect of compound 5d on procoagulant activity of EC venom.

<p>EC venom (0.6 µg) was pre-incubated with compound 5d at various doses and the procoagulant assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106364#s2" target="_blank">materials and methods</a> section. Data are presented as the mean ± SEM, <b>***</b><i>p</i><0.001, a - significant compared to saline control group and ‡ - non-significant compared to venom alone group.</p><p>Effect of compound 5d on procoagulant activity of EC venom.</p