Summary The pathophysiological mechanisms contributing to the decreased platelet count in immune thrombocytopenia (ITP) are not entirely understood. Here, we investigated the key step of proplatelet formation (PPF) by studying the effect of ITP plasma in thrombopoiesis. Normal cord blood‐derived mature megakaryocytes were cultured in the presence of recalcified plasma from ITP patients, and PPF was evaluated by microscopic analysis. Patient samples induced a dose‐dependent inhibition in PPF, as well as decreased complexity of proplatelet architecture. Although slightly increased, plasma‐induced megakaryocyte apoptosis was not related to PPF impairment. Purified IgG reproduced the inhibitory effect, while platelet‐adsorbed plasma induced its reversion, suggesting the involvement of auto‐antibodies in the inhibition of thrombopoiesis. Impaired PPF, induced by ITP plasmas bearing anti‐GPIIb‐IIIa antibodies, was related to their ability to interfere with the normal function of this integrin, as assessed by megakaryocyte PAC‐1 binding and β3 integrin phosphorylation while the presence of anti‐glycoprotein Ia‐IIa auto‐antibodies was associated with loss of normal inhibition of PPF induced by type I collagen. In conclusion, abnormal thrombopoiesis comprising decreased PPF and morphological changes in proplatelet structure are induced by patient samples, unveiling new mechanisms contributing to decreased platelet count in ITP.

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Mechanisms leading to low platelet count in immune thrombocytopenia (ITP) involves both decreased production and increased destruction of platelet. However, the contribution of these pathologic mechanisms to clinical outcome of individual patients is uncertain. Here we evaluated different pathogenic mechanisms including in vitro megakaryopoiesis, platelet/megakaryocyte (MK) desialylation and MK apoptosis, and compared these effects with thrombopoyesis and platelet apoptosis in the same cohort of ITP patients. Normal umbilical cord blood-CD34+ cells, mature MK derived cells or platelets were incubated with plasma from ITP patients. Despite inhibition of thrombopoiesis previously observed, megakaryopoiesis was normal or even increased. Plasma from ITP patients affected the sialylation pattern of control platelets and this effect occurred concomitantly with apoptosis in 35% ITP samples. However, none of these abnormalities were observed in control MKs incubated with ITP plasma. Addition of mononuclear cells as immune effectors did not lead to phosphatidylserine exposure in MK, ruling out an antibody-mediated cytotoxic effect. These results suggest that both desialylation and apoptosis may be relevant mechanisms leading to platelet destruction although, they do not interfere with MK function. Analysis of these thrombocytopenic factors in individual patients showed no specific distribution pattern. However, the presence of circulating antiplatelet autoantibodies was associated with higher incidence of abnormalities. In conclusion, the causes of thrombocytopenia are multifactorial and may occur together, providing a rational basis for the use of combination therapies targeting concomitant ITP mechanisms in patients with refractory disease.

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