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

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

The relationship between individual levels of released phosphorylated-HSP27 and the levels of PDGF-AB induced by collagen in type 2 DM patients.

<p>The level of phosphorylated-HSP27 and PDGF-AB 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. The baseline levels in unstimulated samples were subtracted from each of the individual released phosphorylated-HSP27 levels and the PDGF-AB levels. Each data were collected with the platelet counts, and 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

Representative patterns of platelet aggregation induced by various doses of collagen as detected by an aggregometer with laser-scattering system and representative data showing the collagen-induced HSP27 phosphorylation in platelets from type 2 DM patients.

<p>PRP from type 2 DM patients was stimulated by various doses of collagen (0, 0.1, 0.3 and 1.0 μg/ml) in an aggregometer at 37°C for 4 min with a stirring speed of 800 rpm. (A) Time-dependent changes in the platelet aggregation after stimulation by 0 μg/ml (<i>i</i>), 0.1 μg/ml (<i>ii</i>), 0.3 μg/ml (<i>iii</i>), and 1.0 μg/ml (<i>iv</i>) are shown. The black line indicates the percentage of transmittance of each samples (the isolated platelets were recorded as 0%, and platelet free plasma was 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). (B) The reaction was terminated by the addition of an ice-cold EDTA (10 mM) solution. The extracts of platelets were subjected to a Western blot analysis using antibodies against phospho-specific HSP27 (Ser-78 and Ser-82), total HSP27 and P2Y12. The bands were quantified using the ImageJ software program as the counts of pixels. The bands of phospho-HSP27 and the bands of total HSP27 were normalized to the total HSP27 bands and the P2Y12 bands, respectively. The ratio (phospho-HSP27/total HSP27 and total HSP27/P2Y12) is presented for each value. Upper panel indicates the results of a Western blot analysis. In the left-lower panel, the semi-black bars indicate the phosphorylation ratio of HSP27 (Ser-78), and the black bars indicate the phosphorylation ratio of HSP27 (Ser-82). In the right-lower panel, the black bars indicate the ratio of total HSP27/P2Y12.</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

The relationship between individual levels of HSP27 phosphorylation (Ser-78) in the platelets and the released phosphorylated HSP27 levels induced by collagen in 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 phosphorylated HSP27 levels in the conditioned mixture after platelet aggregation stimulated by 0.3 μg/ml of collagen for 30 min were collected with the platelet counts. Each data about the phosphorylation of HSP27 in platelets and the levels of released phosphorylated-HSP27 were determined by a Western blot analysis using the ImageJ software program and ELISA, respectively. These data were plotted and analyzed by linear regression analysis. (a) Whole subjects (n = 27) were plotted. (b) The residual subjects after excluding what concentration of phosphorylated-HSP27 could not be detected (n = 25) were plotted.</p

The effect of exogenous recombinant phosphorylated-HSP27 on the platelet aggregation induced by collagen in healthy subjects.

<p>Representative patterns of platelet aggregation as detected by an aggregometer with laser-scattering system are presented. PRP from healthy subjects was stimulated by 1 μg/ml of collagen or vehicle with 3 μg/ml of recombinant phosphorylated-HSP27 or vehicle in an aggregometer at 37°C for 4 min with a stirring speed of 800 rpm. The black line indicates the percentage of transmittance of each samples (the isolated platelets were recorded as 0%, and platelet free plasma was 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 distribution (%) of aggregated particle size measured by laser-scattering method was presented as a lower panel.</p