Representative data showing the effect of rivaroxaban on platelet aggregation induced by collagen.

<p>PRP was pretreated with various doses of rivaroxaban for 15 min, and then stimulated by 1.0 μg/ml collagen or vehicle for 5min. The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. Platelet aggregation was detected by an aggregometer with laser scattering system. The black line indicates the percentage of transmittance of each sample (PRP recorded as 0%, and PPP was recorded as 100%). The blue line indicates small aggregates (9–25 μm); the green line, medium aggregates (25–50 μm); the red line, large aggregates (50–70 μm). The distributions (%) of aggregated particle size were measured by AUC of each particle size. Representative results obtained from ten healthy donors are presented.</p

Representative data showing the effect of collagen on platelet aggregation and HSP27 phosphorylation (Ser-78) in platelets in patients after rivaroxaban administration.

<p>PRP was stimulated by various doses of collagen for 5 min, and the reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. The black line indicates the percentage of transmittance of each sample (PRP recorded as 0%, and PPP was recorded as 100%). The blue line indicates small aggregates (9–25 μm); the green line, medium aggregates (25–50 μm); the red line, large aggregates (50–70 μm). The distributions (%) of aggregated particle size were measured by AUC of each particle size. The lysates of platelets were subjected to Western blot analysis using antibodies against HSP27, or phospho-specific HSP27 (Ser-78). Representative results obtained from five patients before rivaroxaban administration (A) and after the administration for 2 days (B). In the lower panel, the histogram shows quantitative representation of the collagen-induced phosphorylation levels obtained from laser densitometric analysis. The levels are expressed as the fold increase to the basal levels presented as lane 1. Each value represents the mean ± SEM. *p<0.05, compared to the value of the vehicle alone.</p

Effects of rivaroxaban and edoxaban on the collagen-induced phosphorylation of HSP27 in human platelets.

<p>PRP was pretreated with various doses of rivaroxaban (A) or edoxaban (B) for 15 min, and then stimulated by 1.0 μg/ml collagen or vehicle for 5 min. The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. The lysate of platelets was subjected to Western blot analysis using antibodies against phospho-specific HSP27 (Ser-78 and Ser-82), total HSP27 or GAPDH. Representative results of rivaroxaban from ten healthy donors (A) and results of edoxaban from five healthy donors (B) are presented. The histogram shows quantitative representation of the collagen-induced levels obtained from laser densitometric analysis. The phosphorylation levels are expressed as the fold increase to the basal levels presented as lane 1. Each value represents the mean ± SEM. *p<0.05, compared to the value of the vehicle alone, and **p<0.05, compared to the value of collagen alone.</p

Effects of rivaroxaban and edoxaban on the collagen-induced phosphorylation of p44/p42 MAP kinase in human platelets.

<p>PRP was pretreated with various doses of rivaroxaban (A) or edoxaban (B) for 15 min, and then stimulated by 1.0 μg/ml collagen or vehicle for 5 min. The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. The lysate of platelets was subjected to Western blot analysis using antibodies against phospho-specific p44/p42 MAP kinase, p44/p42 MAP kinase or GAPDH. Representative results of rivaroxaban from ten healthy donors (A) and results of edoxaban from five healthy donors (B) are presented. The histogram shows quantitative representation of the collagen-induced levels obtained from laser densitometric analysis. The phosphorylation levels are expressed as the fold increase to the basal levels presented as lane 1. Each value represents the mean ± SEM. *p<0.05, compared to the value of the vehicle alone, and **p<0.05, compared to the value of collagen alone.</p

Effect of rivaroxaban on the collagen-stimulated PDGF-AB secretion from human platelets.

<p>PRP was pretreated with 1000 ng/ml of rivaroxaban or vehicle for 15 min, and then stimulated by 1.0 μg/ml collagen or vehicle for 5 min. The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. The conditional mixture was centrifuged at 10,000 x g at 4°C and the supernatants were then subjected to an ELISA for PDGF-AB. Values for unstimulated platelets have been subtracted from each data point. Results from ten healthy donors are shown. Each value represents the mean ± SEM. N.S. designates no significant difference between the indicated pairs.</p

Effect of rivaroxaban on the collagen-induced phosphorylation of p38 MAP kinase in human platelets.

<p>PRP was pretreated with various doses of rivaroxaban for 15 min, and then stimulated by 1.0 μg/ml collagen for 5 min. The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. The lysate of platelets was subjected to Western blot analysis using antibodies against phospho-specific p38 MAP kinase, p38 MAP kinase or GAPDH. Representative results obtained from ten healthy donors are presented. The histogram shows quantitative representation of the collagen-induced levels obtained from laser densitometric analysis. The phosphorylation levels are expressed as the fold increase to the basal levels presented as lane 1. Each value represents the mean ± SEM. N.S. designates no significant difference between the indicated pairs.</p

Effect of rivaroxaban on the collagen-stimulated HSP27 release from human platelets.

<p>PRP was pretreated with 1000 ng/ml of rivaroxaban or vehicle for 15 min, and then stimulated by 1.0 μg/ml collagen or vehicle for 5 min. The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. The conditional mixture was centrifuged at 10,000 x g at 4°C and the supernatants were then subjected to an ELISA for phosphorylated HSP27. Values for unstimulated platelets have been subtracted from each data point. Results from ten healthy donors are shown. Each value represents the mean ± SEM. *p<0.05, compared to the value of the collagen alone.</p