ROS release induced by LPS promotes potentiation of aggregation.

<p>Washed platelets (4 x 10⁸/ml) were pre-incubated with 10μM DCFH-DA in the presence or absence of vehicle, LPS from <i>E</i>. <i>coli</i> O111:B4 (0.5 or 1μg/mL) or LPS RS (1μg/mL), superoxide dismutase- SOD (30U/mL) or catalase (300U/mL) before being activated with U46619 (0.25μM). Samples were analysed by flow cytometry and the levels of ROS released were detected and expressed as % increase above levels detected in unstimulated platelets (A and B). Human-washed platelet aggregation was performed by optical aggregometry following stimulation with U46619 (0.25μM) in the presence or absence of LPS from <i>E</i>. <i>coli</i> (1μg/mL) after 3 min of incubation with superoxide dismutase- SOD (30U/mL) or catalase (300U/mL) (C). Cumulative data represent mean values ± SEM (n = 4). (Anova-Bonferroni test, * P≤ 0.05; Test t student ^# P≤ 0.01; ^{# #} P≤ 0.01).</p

LPS stimulates ERK1/2 and PLA₂- phosphorylation via TLR4 and Akt.

<p>Washed human platelets were incubated for 3 minutes with LPS RS (1μg/mL) or Akt inhibitor IV (5μM) before activation with U46619 (0.25μM) in the presence or absence of vehicle or LPS from <i>E</i>. <i>coli</i> (1μg/mL) and were analysed by immunoblotting using antiphospho- ERK1/2 (A and B) and antiphospho-PLA₂ (A and C) antibodies. Total levels of 14-3-3ζ were measured on each sample as a loading control. Cumulative data represent mean values ± SEM (n = 4). (Anova-Bonferroni test, * P≤ 0.05; ** P≤ 0.01).</p

Exposure of platelets to LPS potentiates PI3K/Akt signalling.

<p>Washed human platelets with or without treatment with U46619 (0.25μM) in the presence or absence of LPS from <i>E</i>. <i>coli</i> O111:B4 (1μg/mL) were analysed by immunoblotting using antiphospho-Akt (Ser473) antibody. Total levels of 14-3-3 ζ were measured on each sample as a loading control (A). Human-washed platelet aggregation was performed by optical aggregometry activated with U46619 (0.25μM) in the presence or absence of LPS from <i>E</i>. <i>coli</i> O111:B4 (1μg/mL) after 3 min of incubation with LY294002 (20μM), Cal (60μM) or Akt inhibitor IV (5μM) (B). The effect of U46619 and LPS- induced fibrinogen binding and P-selectin exposure after incubation with LY294002 (20μM), Cal (60μM) or Akt inhibitor IV (5μM) were measure in PRP by flow cytometry (C and D). Washed platelets (4 x 10⁸/ml) were pre-incubated with 10μM DCFH-DA in the presence or absence of LY294002 (20μM), Cal (60μM) or Akt inhibitor IV (5μM) before being activated with U46619 (0.25μM) in the presence or absence of LPS from <i>E</i>. <i>coli</i> O111:B4 (1μg/mL) and ROS levels were analysed by flow cytometry (E). Cumulative data represent mean values ± SEM (n = 4). (Anova-Bonferroni test, * P≤ 0.05; ** P≤ 0.01; *** P≤ 0.001; Test t student ^{# #} P≤ 0.01).</p

The structures of quercetin aglycone and plasma metabolites.

<p>Quercetin is part of the flavonol subclass of flavonoids. Metabolites of quercetin include methylated (4′-<i>O</i>-methyl quercetin: tamarixetin), sulphated (quercetin-3′-sulphate) and glucuronidated (quercetin-3-glucuronide) counterparts.</p

The role of ERK1/2 in LPS-mediated platelet potentiation.

<p>Washed human platelets stimulated or not with U46619 (0.25μM) in the presence or absence of LPS from <i>E</i>. <i>coli</i> O111:B4 (1μg/mL) were analysed by immunoblotting using antiphospho- ERK1/2 antibody. Total levels of 14-3-3 ζ were measured on each sample as a loading control (A). Human-washed platelet aggregation was performed by optical aggregometry following stimulation with U46619 (0.25μM) in the presence or absence of LPS from <i>E</i>. <i>coli</i> O111:B4 (1μg/mL) after 3 min of incubation with Cobimetinib (100μM) before activation with U46619 (0.25μM) (B). The effect of U46619 (0.25μM) and LPS- induced fibrinogen binding and P-selectin exposure after incubation with Cobimetinib (100μM) were measured in PRP by flow cytometry (C and D). Washed platelets (4 x 10⁸/mL) were pre-incubated with 10μM DCFH-DA in the presence or absence of Cobimetinib (100μM) before being activated with U46619 (0.25μM) in the presence or absence of LPS from <i>E</i>. <i>coli</i> O111:B4 (1μg/mL) and ROS levels were analysed by flow cytometry (E). Cumulative data represent mean values ± SEM (n = 4). (Anova-Bonferroni test, * P≤ 0.05; ** P≤ 0.01; *** P≤ 0.001; Test t student ^# P≤ 0.05; ^{# #}P≤ 0.01).</p

Purification of rhinocerase from rotofor separated venom fractions.

<p><i>A</i>. Superdex 75 gel filtration chromatogram obtained during the elution of proteins from rotofor separated venom fractions 3 and 4. The peaks were numbered at the particular fractions which were used for analysis by SDS-PAGE and for analysis of the serine protease activity. <i>B</i>. 100 µl of the selected fractions shown in figure <i>A</i> were used to measure serine protease activity using Arg-AMC fluorescent substrate. Each bar shows the mean ± S.D. (<i>n</i> = 3). The hydrolytic activity measured for P2 was taken as 100%. <i>C</i>. 100 µl of the selected fractions indicated in figure <i>A</i> were analysed by SDS-PAGE (10%) and silver stained.</p

Glycosylation detection on rhinocerase.

<p>Deglycosylation was performed by mixing 100 µg of rhinocerase with 5 units of N glycosidase F in 50 µl of 0.02M Tris-HCl pH 7.4. 20 µg of glycosylated (lane1) and deglycosylated (lane2) rhinocerase were run in two separate SDS-PAGE (10%) gels and transferred to two PVDF membranes. <i>A</i>. One PVDF membrane was treated with rhinocerase-specific antibody. <i>B</i>. The second PVDF membrane was used to detect glycosylation using the ECL glycosylation detection module. Data are representative of three separate experiments.</p

Quercetin is metabolised by platelets.

<p>Platelets (8×10⁸ cells.mL⁻¹) pretreated with quercetin (50 µM) and tamarixetin (50 µM) for 5, 40, 60 or 120 min were lysed with 50% methanol and 0.1% formic acid. Extracts from lysed platelets were separated by HPLC utilising UV detection (λ: 210 nm) over a period of 20 min, before identification of the protonated masses of compounds using mass spectrometry. Mass spectrums show the ([M+H⁺]⁺) of quercetin (molecular mass: 303 Da), the platelet metabolite (molecular mass: 317 Da) and the external control spike, myricetin (molecular mass: 319 Da) at 5 min (A: with plasma control inset) and 60 min (B). EIC and mass spectrums of standards show tamarixetin (EIC: C.i-RT: 7.5 min; mass spectrum: C.ii-molecular mass: 317 Da), quercetin (EIC: (D.i)-RT: 7 min; mass spectrum: (D.ii)-molecular mass: 303 Da) with inset MS spectrum showing the absence of tamarixetin (ii) and myricetin (EIC: (E.i)-RT: 6.5 min; mass spectrum: (E.ii)-molecular mass: 319 Da). Data represent 3 individual experiments using platelets isolated from 3 different blood donors.</p

Quercetin and tamarixetin associate with platelets.

<p>Quercetin (50 µM) and tamarixetin (50 µM) were incubated with platelets (8×10⁸ cells.mL⁻¹) for 5, 40, 60 or 120 min prior to lysis with 50% (v/v) methanol and 0.1% (v/v) HCl. Compounds within extracts obtained from platelet lysates spiked with myricetin (external control compound of similar structure) were separated over 60 min using HPLC analysis with photodiode array detection. Chromatograms show quercetin ((A)-RT: 46.1 min) and tamarixetin ((B)-RT: 51.6 min) associated with platelets through comparison with standards (quercetin: D; tamarixetin: 3E), untreated platelets (plasma control: C) and the external control, myricetin ((A, B)-RT: 39 min; F: standard). Insets show UV absorbance spectrum (λ: 360 nm) of detected compounds. Data represent 3 individual experiments.</p

Rotofor based separation of <i>B. g. rhinoceros</i> venom proteins.

<p><i>A.</i> SDS-PAGE (10%) was run with 50 µg of <i>B. g. rhinoceros</i> venom and stained with Coomassie brilliant blue. Several proteins with different molecular weights are present in this venom. <i>B</i>. 2 mg of venom were mixed with non-reducing rotofor buffer containing ampholytes with pI 6–8 and separated under non-denaturing conditions. In total 10 fractions (indicated by the numbers at the top of the gel) were collected. 10 µl of each fraction were run in SDS-PAGE (10%) and stained with Coomassie brilliant blue. <i>C</i>. 20 µl of each rotofor fraction were used to measure serine protease activity using Arg-AMC fluorescent substrate. The data represents the mean ± S.D. (<i>n</i> = 3). The hydrolytic activity measured for fraction 5 was taken as 100%.</p