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

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 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

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

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

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

The relationship between individual levels of released phosphorylated-HSP27 and the area under the curve (AUC) of platelet aggregation induced by collagen in type 2 DM patients.

<p>The levels of released phosphorylated-HSP27 in the supernatant of the conditioned mixture after platelet aggregation stimulated by 0.3 μg of collagen for 30 min was determined using specific ELISA kits and data were collected with the platelet counts. AUC of platelet aggregation stimulated by 0.3 μg/ml of collagen for 4 min were determined by an aggregometer using LS system recorded individually by the size of aggregates, (A) AUC of small aggregates, (B) AUC of medium aggregates, (C) AUC of large aggregates, (D) AUC of total transmission. Each data were plotted and analyzed by linear regression analysis. (a) Whole subjects (n = 35) were plotted. (b) The residual subjects after excluding what concentration of phosphorylated-HSP27 could not be detected (n = 30) were plotted.</p

The relationship between individual levels of HSP27 phosphorylation (Ser-78) and the change of intracellular HSP27 protein levels induced by collagen in platelets from type 2 DM patients.

<p>The baseline levels in unstimulated samples were subtracted from each of the individual HSP27 phosphorylation ratio (phosphorylated-HSP27/total HSP27) and intracellular HSP27 protein levels stimulated by 0.3 μg/ml of collagen for 4 min, and the net changes are presented as levels of phosphorylated-HSP27 (Ser-78) and changes of HSP27, respectively. Each data were determined by a Western blot analysis using the ImageJ software program and were plotted and analyzed by linear regression analysis.</p

Characteristics of the study subjects.

<p>F indicates female; M, male; BMI, body mass index; sBP, systolic blood pressure; dBP, diastolic blood pressure; HbA_1c, hemoglobin A_1c; Glu, plasma glucose; TC, total cholesterol; TG, triglyceride; HDL, high-density lipoprotein; Plt, platelet counts. The data are presented as the means ± SD.</p><p>Characteristics of the study subjects.</p