Apoptosis markers in ITP platelets.

<p>A) Unstimulated platelets from ITP patients were washed and incubated either with FITC-anexin-V to detect phosphatidylserine (PS) exposure (n = 21), JC-1 to evaluate loss of ΔΨm (n = 24) or FAM-DEVD-FMK to measure active caspase 3 (aCasp3) (n = 12) as described in Materials and methods. Samples were analyzed by flow cytometry within 1 hour of processed. Box plot represent percentage of platelets displaying apoptotic markers in ITP patients (P) and controls (C). Wilcoxon signed rank test *p<0.05, **p<0.01, ***p<0.001. Representative examples of FITC-anexin-V binding (B), active caspase 3 detection (C, D) and loss of ΔΨm measurement (E, F) in control and ITP platelets are shown. G) Platelet apoptosis was induced by the addition of 1–3 μmol/L A23187 and evaluated by PS exposure (p = NS, n = 20) and ΔΨm measurement (p = NS, n = 23). H) Active caspase 3 was evaluated after apoptosis induction with 1–3 and 6–10 μmol/L A23187 (p = NS and **p<0.01, respectively).</p

Apoptosis in ITP platelets according to clinical and laboratory data.

<p>A) ITP patients were grouped according to the presence/absence of auto-antibodies and increased or normal PS exposure on autologous platelets. The incidence of platelet apoptosis tended to be higher in patients with auto-antibodies than in those with no-detectable auto-antibodies (Fisher exact test, p = 0.063, OR: 10.8, 95% CI = 0.99–117). B) The incidence of platelet apoptosis was similar in patients without treatment and under any kind of treatment (Fisher exact test, p = 1.00, OR: 1.429, 95% CI = 0.271–7.521). C) PS exposure and loss of ΔΨm before (B) and during eltrombopag treatment (TRA). Data represents mean+SD from three ITP patients (Paired t test, PS, p = NS; loss of ΔΨm *p<0.05). D) ITP patients were grouped according to their Bleeding scale (ITP Bleeding Scale-IBLS) and the number of ITP patients with increased platelet apoptosis was plotted in each group (Chi squared test, p = 0.732).</p

Apoptosis markers in normal platelets incubated with ITP plasma.

<p>Unstimulated normal platelets were washed and incubated either with ITP or control plasma during 1 hour. Then, platelets were washed and incubated with FITC-anexin-V, JC-1 or FAM-DEVD-FMK as described in Materials and Methods, and analysed by flow cytometry within 1hour of processed. Box plot represent percentage of apoptotic platelets measured as A) PS exposure (Mann-Whitney test p = NS, n = 10), B) loss of ΔΨm (*p<0.05, n = 12) and C) aCasp3, (p = NS, n = 18).</p

Induction of normal platelet apoptosis by ITP samples in the presence of normal autologous CD3⁺ lymphocytes.

<p>Unstimulated normal platelets and autologous CD3⁺ lymphocytes were separately purified and incubated either in ITP or control samples during 1 hour. Then, samples were washed, incubated with FITC-Anexin-V and analysed by flow cytometry within 1 hour of processed to detect PS exposure. A) Percentage of PS expression on normal platelets incubated with ITP plasma (n = 14) and control plasma (n = 13) (Mann Whitney test, ****p<0.0001) in the presence of autologous CD3⁺ lymphocytes. B) A representative example showing percentage of FITC-Anexin-V staining on platelets incubated with ITP plasma and normal plasma is shown. C) Comparison between fold increase in PS exposure on normal platelets incubated with individual ITP recalcified plasma (ITP plasma) and its corresponding platelet-adsorbed plasma (PA plasma), both in the presence of autologous normal CD3+ lymphocytes (Paired t test, p<0.01). Dotted line represents PS exposure of control samples. D) Percentage of PS expression on normal platelets incubated with autologous CD3⁺ lymphocytes in the presence of purified IgG from ITP plasma (n = 8) and control plasma (n = 6) (Mann Whitney test, **p<0.01) E) PS exposure induced by ITP plasma samples in the presence (+) or absence (-) of 20 μmol/L leupeptin (n = 10, paired t test ***p<0.001).</p