Fig 1.
Effect of simultaneous stimulation with collagen and CXCL12 in low doses on platelet aggregation.
PRP was simultaneously stimulated by 0.05–0.2 μg/ml of collagen and 1–10 ng/ml of CXCL12 for 5 min. 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 (isolated platelets recorded as 0%, and platelet-poor plasma recorded as 100%). The blue line indicates small aggregates (9–25 μm); green line, medium aggregates (25–50 μm); red line, large aggregates (50–70 μm). The lower panel presents the distribution (%) of aggregated particle size as measured by laser-scattering. The effects of simultaneous stimulation with 10 ng/ml of CXCL12 and 0.2 μg/ml or less of collagen (A), and simultaneous stimulation with 10 ng/ml or less of CXCL12 and 0.2 μg/ml of collagen (B) are shown.
Fig 2.
Effect of simultaneous stimulation with TRAP or ADP, and CXCL12 in low doses on platelet aggregation.
PRP was simultaneously stimulated by 7 μM of TRAP (A) or 0.5 μM of ADP (B) and 10 ng/ml of CXCL12 for 5 min. 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 (isolated platelets recorded as 0%, and platelet-poor plasma recorded as 100%). The blue line indicates small aggregates (9–25 μm); green line, medium aggregates (25–50 μm); red line, large aggregates (50–70 μm). The lower panel presents the distribution (%) of aggregated particle size as measured by laser-scattering. The representative results are shown.
Fig 3.
Effect of simultaneous stimulation with convulxin and CXCL12 in low doses on platelet aggregation.
PRP was simultaneously stimulated by 20 ng/ml of convulxin and 10 ng/ml of CXCL12 for 5 min. 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 (isolated platelets recorded as 0%, and platelet-poor plasma recorded as 100%). The blue line indicates small aggregates (9–25 μm); green line, medium aggregates (25–50 μm); red line, large aggregates (50–70 μm). The lower panel presents the distribution (%) of aggregated particle size as measured by laser-scattering.
Fig 4.
Effect of anti-CXCR4 or anti-CXCR7 monoclonal antibody on the platelet aggregation induced by simultaneous stimulation with collagen and CXCL12 in low doses.
PRP was pretreated with 10 μg/ml of control IgG, anti-CXCR4 monoclonal antibody (A), or anti-CXCR7 monoclonal antibody (B) for 3 min and then simultaneously stimulated by 0.2 μg/ml of collagen and 10 ng/ml of CXCL12 for 5 min. The reaction was terminated by the addition of ice-cold EDTA solution. The black line indicates the percentage of transmittance of each sample (isolated platelets recorded as 0%, and platelet-poor plasma recorded as 100%). The blue line indicates small aggregates (9–25 μm); green line, medium aggregates (25–50 μm); red line, large aggregates (50–70 μm). The lower panel presents the distribution (%) of aggregated particle size as measured by laser-scattering.
Fig 5.
Effect of simultaneous stimulation with collagen and CXCL12 in low doses on the secretion of PDGF-AB from human platelets.
PRP was simultaneously stimulated by the vehicles, 0.05–0.3 μg/ml of collagen and the vehicle, 10 ng/ml of CXCL12 and the vehicle, or 0.05–0.3 μg/ml of collagen and 10 ng/ml of CXCL12 for the indicated time. The dose of collagen achieving a % transmittance of 10%-30% was adjusted individually. The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. The conditioned mixture was centrifuged at 10,000 × g at 4°C for 2 min, and the supernatant was then subjected to ELISA for PDGF-AB. The results obtained from 5 individuals are shown. Each value represents the mean ± SEM. *p<0.05, compared to the value of control. NS: no significant differences between the indicated pairs.
Fig 6.
Effect of simultaneous stimulation with collagen and CXCL12 in low doses on the release of sCD40L from human platelets.
PRP was simultaneously stimulated by 0.15–0.3 μg/ml of collagen and 10 ng/ml of CXCL12 for 15 min. The dose of collagen achieving a % transmittance of 10%-30% recorded was adjusted individually. The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. The conditioned mixture was centrifuged at 10,000 × g at 4°C for 2 min, and the supernatant was then subjected to ELISA for sCD40L. The results obtained from 5 independent individuals are shown. Each value represents the mean ± SEM. *p<0.05, compared to the value of control. **p<0.05, compared to the value of collagen alone. ***p<0.05, compared to the value of CXCL12 alone. NS: no significant differences between the indicated pairs.
Fig 7.
Effect of simultaneous stimulation with collagen and CXCL12 in low doses on the phosphorylation of p44/p42 MAPK in human platelets.
PRP was simultaneously stimulated by 0.4 μg/ml of collagen and 10 ng/ml of CXCL12 for the indicated time. The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. The lysed platelets were subjected to Western blot analysis using antibodies against phospho-specific p44/p42 MAPK or GAPDH. The histogram shows a quantitative representation of the collagen and CXCL12-induced levels obtained from a densitometric analysis. The phosphorylation is expressed as the fold increase compared to the levels of platelets without stimulation. The representative results obtained from 5 independent individuals are shown.
Fig 8.
Effect of simultaneous stimulation with collagen and CXCL12 in low doses on the phosphorylation of p38 MAPK in human platelets.
PRP was simultaneously stimulated by 0.4 μg/ml of collagen and 10 ng/ml of CXCL12 for the indicated time. The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. The lysed platelets were subjected to Western blot analysis using antibodies against phospho-specific p38 MAPK or GAPDH. The histogram shows a quantitative representation of the collagen and CXCL12-induced levels obtained from a densitometric analysis. The phosphorylation is expressed as the fold increase compared to the levels of platelets without stimulation. The representative results obtained from 5 independent individuals are shown.
Fig 9.
Effect of SB203580 on the platelet aggregation induced by simultaneous stimulation with collagen and CXCL12 in low doses.
PRP was pretreated with 30 μM of SB203580 or vehicle for 3 min and then simultaneously stimulated by 0.3 μg/ml of collagen and 10 ng/ml of CXCL12 for 5 min. The reaction was terminated by the addition of ice-cold EDTA solution. The black line indicates the percentage of transmittance of each sample (isolated platelets recorded as 0%, and platelet-poor plasma recorded as 100%). The blue line indicates small aggregates (9–25 μm); green line, medium aggregates (25–50 μm); red line, large aggregates (50–70 μm). The lower panel presents the distribution (%) of aggregated particle size as measured by laser-scattering. The representative results obtained from 5 healthy donors are shown.
Table 1.
Effect of SB203580 on platelet aggregation by low doses of collagen and CXCL12.
Fig 10.
Effect of SB203580 on the phosphorylation of p38 MAPK in human platelets induced by simultaneous stimulation with collagen and CXCL12 in low doses.
PRP was pretreated with 30 μM of SB203580 or vehicle for 3 min and then simultaneously stimulated by 0.1–0.2 μg/ml of collagen and 10 ng/ml of CXCL12 for 5 min. The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. The lysed platelets were subjected to Western blot analysis using antibodies against phospho-specific p38 MAPK, total p38 MAPK, or GAPDH. The histogram shows a quantitative representation of the co-stimulation with collagen and CXCL12-induced levels obtained from a densitometric analysis. The phosphorylation is expressed as the fold increase compared to the levels of collagen alone, presented as lane 1. Each value represents the mean ± SEM of 5 times independent experiments. *p<0.05, compared to the value of CXCL12 alone. **p<0.05, compared to the value of collagen alone. ***p<0.05, compared to the value of simultaneous stimulation of collagen and CXCL12.
Fig 11.
Effects of SB203580 on the secretion of PDGF-AB and the release of sCD40L from human platelets induced by simultaneous stimulation with collagen and CXCL12 in low doses.
PRP was pretreated with 30 μM of SB203580 or vehicle for 3 min and then simultaneously stimulated by 0.1–0.2 μg/ml of collagen and 10 ng/ml of CXCL12 for 5 min (A) or 15 min (B). The dose of collagen achieving a % transmittance of 10%-30% recorded in an aggregometer was adjusted individually. The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. The conditioned mixture was centrifuged at 10,000 × g at 4°C for 2 min, and the supernatant was then subjected to ELISA for PDGF-AB (A) or sCD40L (B). The results from of 5 independent individuals are shown. Each value of PDGF-AB (A) or sCD40L (B) represents the mean ± SEM. *p<0.05, compared to the value of CXCL12 alone. **p<0.05, compared to the value of collagen alone. ***p<0.05, compared to the value of simultaneous stimulation of collagen and CXCL12.
Fig 12.
Effects of simultaneous stimulation with collagen and CXCL12 in low doses on the phosphorylation of HSP27 and release of phosphorylated-HSP27 in human platelets.
PRP was simultaneously stimulated by 0.4 μg/ml of collagen and 10 ng/ml of CXCL12 for the indicated time. The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. The lysed platelets were subjected to Western blot analysis using antibodies against phospho-specific HSP27 (Ser-78) or GAPDH. The histogram shows a quantitative representation of the collagen and CXCL12-induced levels obtained from a densitometric analysis. The phosphorylation is expressed as the fold increase compared to the levels of platelets without stimulation. The representative result is shown (A). PRP was pretreated with 30 μM of SB203580 or vehicle for 3 min and then simultaneously stimulated by 0.1–0.2 μg/ml of collagen and 10 ng/ml of CXCL12 for 5 min. The reaction was terminated by addition of ice-cold EDTA (10 mM) solution. The extracts of platelets were then subjected to SDS-PAGE with a subsequent Western blot analysis using antibodies against phospho-specific HSP27 (Ser-78), total HSP27, or GAPDH. The histogram shows a quantitative representation of the collagen and CXCL12-induced levels obtained from a densitometric analysis. The phosphorylation is expressed as the fold increase compared to the levels of collagen alone, presented as lane 1 (B). PRP was pretreated with 30 μM of SB203580 or vehicle for 3 min and then stimulated by 0.1–0.2 μg/ml of collagen and 10 ng/ml of CXCL12 for 15 min. The reaction was terminated by the addition of ice-cold EDTA solution. The mixture was centrifuged at 10,000 × g at 4°C for 2 min, and the supernatant was then subjected to ELISA for phosphorylated-HSP27 (Ser-78) (C). Each value represents the mean ± SEM of the 5 times independent experiments. *p<0.05 compared to the value of CXCL12 alone. **p<0.05 compared to the value of collagen alone. ***p<0.05 compared to the value of collagen and CXCL12 (B, C).