Discriminating young platelets on human leukocyte antigen-I expression highlights their extremely high reactivity potential

Figures

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  Figure 1. Human leukocyte antigen I (HLA-I) expression level is a sensitive marker of platelets according to their age. A, (a), Representative fluorescence-activated cell sorting plots showing the 3 arbitrarily designed subsets of platelets based on their HLA-I surface expression. 10% lowest HLA signal defined as old platelets (HLA-I low), 80% intermediate HLA signal defined as midle-aged platelets (HLA-I dim) and 10% highest HLA signal defined as young platelets (HLA-I high) (b), Representative histograms of the platelet size as measured by the FSC parameter of a LSRFortessa cell analyzer. (c) Representative histograms of the expression level of HLA-I molecules assessed by calculating the ratio mean fluorescence intensity (MFI) HLA-I/forward scatter (FSC) among the 3 platelet subsets (± SEM, n = 25). B, Left panel, Representative fluorescence-activated cell sorting plots of ribosomal P-protein (Pribo+) expression among GPIbβ+ platelets among the 3 platelet subsets. Right panel, Percentage of platelets expressing ribosomal P-protein according to their HLA-I expression level (% ± SEM, n = 4). C, Expression level of the main glycoproteins was assessed by calculating the ratio GP MFI/FSC…

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  Figure 2. Cell-sorted human leukocyte antigen I (HLA-I)high platelets reveal the features of young platelets. A, Gating strategy for the sorting and re-analysis of the HLA-I low, dim and high platelet subpopulations representing, respectively, 10% of the lowest, 20% of the intermediate and 10% of the highest HLA-I signal. B, Transmission electron micrographs of sorted platelets for size and platelet activation-related shape changes.

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  Figure 3. The young human leukocyte antigen I (HLA-I)high platelets are more reactive than old ones in response to soluble agonists. In response to different soluble agonists, the proportion of platelets positive for (A) P-selectin or (B) fibrinogen was evaluated by flow cytometry on the whole platelet population (left panels) and on the 3 platelet subsets according to their HLA-I surface expression (right panels). Graphs are representative of the lowest concentration of each agonist inducing maximal activation without saturation (adenosine di-phosphate (ADP), 5 μM; collagen-related peptide (CRP), 1 μg/mL; thrombin receptor-activating peptide (TRAP), 100 μM (T); convulxin, 6 nM; U46619, 1 μM) (% ± SEM ; not significant; P > .05, ∗∗P < .01, ∗∗∗P < .001; 1-way analysis of variance n = 3–9). Dose-response to different agonists for (C) P-selectin or (D) fibrinogen was evaluated using flow cytometry on the 3 platelet subsets according to their HLA-I surface expression after ADP (left panels) or CRP (right panels) activation. Exposure of platelet activation markers is indicated as percentage of positive platelets. The dots indicate the mean value (n = 3) and SEM and the dotted lines show the…

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  Figure 4. Human leukocyte antigen-Ihigh platelets are more prone to become COllagen And Thrombin activation platelets. Dual labelling of platelets with annexin-V and procaspase-activating compound 1 (A) or von Willebrand factor (B) after simultaneous incubation with 100μM thrombin receptor-activating peptide (TRAP) and 1-μg/mL collagen-related peptide (CRP) (T + C) was evaluated using flow cytometry on the 3 different platelet subsets according to their human leukocyte antigen-I surface expression (% ± SEM; not significant; P > .05, ∗∗P < .01, ∗∗∗P < .001; 2-way analysis of variance n = 3).