Table 1.
Demographic characteristics and cardiovascular risk factors.
Figure 1.
Platelet activation in MI patients.
Panel A: Left. Flow cytometric analysis of platelet CD62p surface expression in patients with MI, sCAD and healthy donors. Cells were stained with specific mAb anti P-selectin, as described in Materials and Methods. Ten patients were analyzed for each group. Right. Quantification of CD62p expression on the surface of platelets from MI, sCAD and HD. Absolute numbers of surface antigens expressed/cell (MESF). (Anova and Bonferroni t-test; * P<0.05 MI vs HD; no significant differences were found between stableCAD and HD). Panel B: Platelet surface expression of CD62p in MI (•), sCAD (▴) and HD (○) patients in the presence of increasing doses of ADP. Left panel shows the percentage of positive cells. Right panel shows the absolute numbers of surface antigens expressed/cell (MESF). Data from 7 patients of each group are expressed as means ± S.D. (Anova and Bonferroni t-test; * P<0.05 MI vs HD; no significant differences were found between stable CAD and HD).
Figure 2.
Platelets from MI patients contain mRNA for PKCε.
Panel A: Representative RT-PCR analysis of CD41 and CD45 expression in MI, sCAD and healthy donor platelets. Equal amounts of total cDNA were amplified by PCR to detect the indicated mRNA expression. CD45 is not expressed in isolated platelets, indicating the absence of nucleated cell contaminants. Panel B: Representative RT-PCR analysis of PKCε expression in MI, sCAD and healthy donor platelets. Equal amounts of total cDNA were amplified by PCR to detect PKCε mRNA. Platelets from MI patients express PKCε. PC: positive control; NC: negative control. Panel C: Analysis of PKCε RNA expression in MI, sCAD and HD platelets. The samples positive to PKCε mRNA expression in MI, sCAD and HD were respectively 21, 8, 3 out of 72 (24 each group) (Chi-square test: p = 0.0001 MI vs HD; p = 0.0001 MI vs sCAD; no significant differences were found between sCAD and HD). Panel D: Quantitative analysis of PKCε mRNA expression by real-time PCR. Equal amounts of total cDNA were amplified by PCR. The expression level of PKCε mRNA in each patient was compared with the mean expression in the HD group. Data from 10 patients of each group are shown (Mann Whitney test: p = 0.001 MI vs HD; p = 0.009 MI vs sCAD; no significant differences were found between sCAD and HD). Panel E: PKCε sequencing in MI patients. The presence of PKCε was further confirmed in MI patients by bi-directional sequencing. Representative fragment of the cDNA sequences (reverse strand) of PKCε encompassing the exons 1 and 2 (from nucleotide 11 to nucleotide 397). Patients #1, #3 and #7 belonging to the MI group showed the presence (in heterozygosity) of the rs12615152 (c.294C>T, p.Cys98Cys).
Figure 3.
Platelets from MI patients express PKCε protein.
Panel A: A representative Western blot assay for the detection of total PKCε protein expression in platelets from PKCε mRNA-positive MI, sCAD and HD. β-Actin was assayed for protein loading. Panel B: Densitometric analysis of PKCε protein expression normalized against β-actin in PKCε mRNA-positive MI, sCAD and HD platelets. Data are expressed as means ± S.D. (Anova and Bonferroni t-test; * P<0.05 MI vs HD; # P<0.05 MI vs sCAD; no significant differences were found between sCAD and HD). Panel C: PKCε protein expression in mature and immature platelets from healty donors (HD) and MI patients. Cells were simultaneously labelled with Thyazole Orange (TO) -to visualize the immature platelet fraction (IPF) - and anti-PKCε mAb, and analyzed by flow cytometry. Three populations were identified within the CD41+ cell subset: PKCε negative reticulated platelets (TO+EPS−); PKCε positive reticulated platelets (TO+EPS+); PKCε positive mature platelets (TO−EPS+); (Data from 3 patients/group, expressed as means ± S.D. t-Test ** P<0.001; * P<0.05).
Figure 4.
PKCε protein expression in platelets correlates with their activation levels.
Panel A: Flow cytometric analysis of platelet CD62p surface expression in patients with MI, sCAD, healthy donor and in all the sample (ALL), on the basis of PKCε expression. Cells were stained with specific mAb anti P-selectin (CD62p). Seven patients were analyzed for each group (MI: 2 PKCε negative and 5 PKCε positive samples; sCAD: 4 PKCε negative and 3 PKCε positive samples; HD: 4 PKCε negative and 3 PKCε positive samples). Data is expressed as mean ± S.D (Anova and Bonferroni t-test). Panel B: Flow cytometric analysis of CD62p surface expression in PKCε negative and positive platelets. Cells were treated with ADP and compared with untreated platelets (resting). Ten patients were analyzed for each group. Data is expressed as mean ± S.D (Anova and Bonferroni t-test).
Figure 5.
PKCε protein transfection in normal platelets induces hyper-responsiveness to ADP-mediated activation.
Panel A. Western blot detection of total PKCε protein expression in transfected platelets. Healthy donor platelets were incubated with ProteoJuice medium in the presence or absence (negative control) of recombinant PKCε (rhPKCε). K562 cells were used as positive control. β-Actin was assayed for protein loading. Panel B: The expression of CD62p on the surface of activated platelets was compared to the expression of CD62p on resting platelets. rhPKCε-transfected platelets were significantly more reactive than activated control platelets. Left panel shows the percentage of positive cells. Right panel shows the absolute numbers of surface antigens expressed/cell (MESF). Data from 5 independent experiments (each symbol is related to one experiment) are expressed as means ± S.D. (Anova and Bonferroni t-test; * P<0.05 activated platelets vs resting platelets – II vs I and IV vs III; # P<0.05 rhPKCε-transfected platelets vs activated control platelets – IV vs II).
Figure 6.
PKCε enhances platelet adhesion to fibrillar type I collagen under flow conditions.
rhPKCε-transfected and control platelets were reconstituted in whole blood, previously deprived from PRP, and tested for their adhesion capacity under flow. Mepacrine-loaded platelets (5–7×108/ml) and washed erythrocytes (hematocrit 42–45%) suspended in plasma, were perfused for 3 minutes over immobilized fibrillar type I collagen. Surface coverage was measured on an area of 0.07 mm2 after 3 minutes of perfusion at 600 s−1 or 1500 s−1 and is shown as mean ± 95% confidence intervals of at least 3 separate experiments. Results are shown relative to the values observed in untreated blood cell suspensions (control) (Anova and Bonferroni t-test; * p<0.01). Representative single-frame images of each surface are also shown.