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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; Argentina Fil: 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; Argentina Fil: 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; Argentina Fil: 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; Argentina Fil: 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; Argentina Fil: Pierdominici, Marta S.. Departamento de Hematología, Hospital Ramos Mejía, Buenos Aires, Argentina; Argentina Fil: 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; Argentina Fil: Riveros, Dardo Alberto. Centro de Educación Médica e Investigaciones Clínicas “Norberto Quirno”; Argentina Fil: García, Alejandro Jorge. Centro de Educación Médica e Investigaciones Clínicas “Norberto Quirno”; Argentina Fil: 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; Argentina Fil: 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; Argentina Fil: 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; Argentina

Similarly to nucleated cells, platelet life span is also controlled by an intrinsic apoptotic program that triggers collapse of the mitochondrial inner membrane potential, activation of caspases-3, -8 and -9, phosphatidylserine (PS) externalization and microparticle shedding. The aim of the present study was to investigate platelet apoptosis in adult patients with immune thrombocytopenia (ITP) under different treatment conditions and to search for its relationship with the type of auto-antibody and the platelet-activation status. Twenty-four patients with chronic ITP, age 42 (21-80) years (median and range) diagnosed according to current criteria (Rodeghiero et al, 2009) were included after written informed consent in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee from Instituto de Investigaciones Medicas Alfredo Lanari. Platelet count was 38x109/L (6-85). Platelet apoptosis was evaluated by phosphatidylserine (PS) exposure on the platelet surface using FITC-conjugated Anexin-V, mitochondrial electrochemical potential changes (ΔΨm) using the cell penetrating lipophilic cationic fluorochrome JC-1, and activated caspase-3 (a-casp3) measured by the cell-penetrating carboxyfluorescein-labelled fluoromethyl ketone tetrapeptide (FAM-DEVD-FMK). These parameters were studied in resting platelets and after stimulation with calcium ionophore (A23187). Platelet activation was evaluated by FITC-PAC-1 binding to activated GPIIb-IIIa and GPIb-IX internalization using PE-CD42b, in resting conditions and after stimulation either with ADP or TRAP. Apoptosis and activation parameters were evaluated by flow cytometry. In resting conditions, platelets from ITP patients showed increased PS expression and a- casp3 and abnormal ΔΨm (table 1). TablePSΔΨma-casp3Patients19.6 (1.9-82.0)31.2 (5.8-92.4)11.3 (1.8-40.9)Controls4.7 (1.9-10.8)10.3 (2.2-27.5)4.3 (1.9-8.2)p (Mann-Whitney)0.002 After stimulation with A23187, ITP platelets had similar levels of PS expression (p=0.305, n=20) and ΔmΨ (p=0.383, n=25) compared to normal controls. However, an increased sensitivity to the apoptotic stimulus was evidenced by elevated levels of a-casp3 at low and high A23187 concentrations (1-3 mM, p=0.097; 6-10 mM, p=0.002). Platelet apoptosis was not related to platelet activation, as PAC-1 binding was not increased in ITP platelets (basal p=0.847, ADP-induced p=0.059, TRAP-induced p=0.103, n=16). Besides, internalization of GPIb-IX after ADP and TRAP stimulation was also normal (p=NS, n=9 for both agonists). Platelets from ITP patients bearing two of the most frequently found auto-antibodies (5 with anti-GPIIb-IIIa and 1 with anti-GPIb-IX) had similar levels of PS expression and ΔmΨ at resting conditions than those who were negative for these auto-antibodies (n=14) (p=0.265 and 0.148, respectively). There were no differences in apoptosis markers either in resting platelets or after stimulation when comparing untreated patients (n=9) vs patients under any kind of treatment (n=15) (resting conditions, PS p=0.737; ΔmΨ p=0.270; stimulated, PS p=0.966; ΔmΨ p=0.987), although platelet count was similar in both groups. Normal platelets incubated during 1 hour with plasma from ITP had higher a-casp3 than those incubated with normal plasma (n=12 and 9, respectively, p=0.027), suggesting a plasmatic component could be responsible for the apoptotic stimulus. In conclusion, increased platelet apoptosis in ITP patients could be induced by a plasmatic factor, contributing to thrombocytopenia in this entity. Disclosures Riveros: Roche: Speakers Bureau.