Platelet Apoptosis in Adult Immune Thrombocytopenia: Insights into the Mechanism of Damage Triggered by Auto-antibodies

Mechanisms leading to decreased platelet count in immune thrombocytopenia (ITP) are heterogeneous. This study describes increased platelet apoptosis involving loss of mitochondrial membrane potential (ΔΨm), caspase 3 activation (aCasp3) and phosphatidylserine (PS) externalization in a cohort of adult ITP patients. Apoptosis was not related to platelet activation, as PAC-1 binding, P-selectin exposure and GPIb-IX internalization were not increased. Besides, ITP platelets were more sensitive to apoptotic stimulus in terms of aCasp3. Incubation of normal platelets with ITP plasma induced loss of ΔΨm, while PS exposure and aCasp3 remained unaltered. The increase in PS exposure observed in ITP platelets could be reproduced in normal platelets incubated with ITP plasma by adding normal CD3+ lymphocytes to the system as effector cells. Addition of leupeptin -a cathepsin B inhibitor- to this system protected platelets from apoptosis. Increased PS exposure was also observed when normal platelets and CD3+ lymphocytes were incubated with purified IgG from ITP patients and was absent when ITP plasma was depleted of auto-antibodies, pointing to the latter as responsible for platelet damage. Apoptosis was present in platelets from all patients carrying anti-GPIIb-IIIa and anti-GPIb auto-antibodies but was absent in the patient with anti-GPIa-IIa auto-antibodies. Platelet damage inversely correlated with platelet count and decreased during treatment with a thrombopoietin receptor agonist. These results point to a key role for auto-antibodies in platelet apoptosis and suggest that antibody-dependent cell cytotoxicity is the mechanism underlying this phenomenon.Fil: Goette, Nora Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Glembotsky, Ana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Lev, Paola Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Grodzielski, Matías. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Contrufo, Geraldine. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Pierdominici, Marta S.. Departamento de Hematología, Hospital Ramos Mejía, Buenos Aires, Argentina; ArgentinaFil: Espasandin, Yesica Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Riveros, Dardo Alberto. Centro de Educación Médica e Investigaciones Clínicas “Norberto Quirno”; ArgentinaFil: García, Alejandro Jorge. Centro de Educación Médica e Investigaciones Clínicas “Norberto Quirno”; ArgentinaFil: Molinas, Felisa Concepción. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Heller, Paula Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Marta, Rosana Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentin

Galectins: major signaling modulators inside and outside the cell

Galectins control cell behavior by acting on different signaling pathways. Most of the biological activities ascribed to these molecules rely upon recognition of extracellular glycoconjugates and establishment of multivalente interactions, which trigger adaptive biological responses. However, galectins are also detected within the cell in different compartments, where their regulatory functions still remain poorly understood. A deeper understanding of the entire galectin signalosome and its impact in cell behavior is therefore essential in order to delineate new strategies to specifically manipulate both galectin expression and function. This review summarizes our current knowledge of the signaling pathways activated by galectins, their glycan dependence and the cellular compartment where they become activated and are biologically relevant.Fil: Compagno, Daniel Georges. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Jaworski, Felipe Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Gentilini, Lucas Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Contrufo, Geraldine. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Gonzalez Perez, Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Elola, Maria Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; ArgentinaFil: Pregi, Nicolás. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; ArgentinaFil: Rabinovich, Gabriel Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Laderach, Diego Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin

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

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

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