Intrinsic Impairment of Platelet Production in Persistent/Chronic ITP
Authors: Riviere Etienne , Viallard Jean-François , Dias Juliana Vieira , Pons Anne Cecile , Couffinhal Thierry , Pellegrin Jean-Luc , James Chloe 
Published: October 2019

Abstract
Abstract 624
Persistent/chronic immune thrombocytopenias (PC-ITP) are acquired thrombocytopenias characterized by a platelet count less than 100 G/L, lasting respectively more than 3 months/one year. It is a diagnosis of exclusion, and other causes of acquired thrombocytopenia such as neoplasms, infections, autoimmune diseases or drugs must have been ruled out. It is commonly admitted that the pathogenesis of ITP associates the presence of antiplatelet antibodies that bind to mature platelets, leading to their elimination by macrophages through Fc-mediated recognition mechanisms mainly in the spleen or liver, or both. These antibodies are also thought to bind to megakaryocytes impairing platelet production. Newly available TPO receptor agonists are highly effective in chronic ITP patients, with a dramatic increase in platelet count, which suggests a crucial role of altered megakaryopoiesis in this disorder. Because antiplatelet antibodies are only found in 20% of PC-ITP, we hypothesized that some of these patients might have an intrinsic defect in megakaryopoiesis rather than an immune-mediated dysmegakaryopoiesis. We thus analyzed in vitro megakaryocyte differentiation and proplatelet formation in 9 PC-ITP patients, 4 acute ITP patients (A-ITP), and 9 healthy controls (CTRL).
All PC-ITP patients had ITP criteria regarding last international consensus (Neunert C et al, Blood 2011), with a slowly progressing thrombocytopenia (76 G/L, range: 29–97) lasting more than 3 months (median: 17 months; range: 7–36 months), no anti-platelet antibodies and normal medullar density and number of megakaryocytes. All A-ITP patients also matched ITP criteria. All samples were taken at the time of diagnosis, before any treatment was administered. They were compared with 9 controls including patients undergoing valvular replacement or healthy bone marrow donors with normal blood count. To analyze whether there was a defect of megakaryopoiesis that was cell-intrinsic, we isolated CD34 positive cells from the bone marrow and analyzed in vitro megakaryocytic differentiation with TPO-mimetic romiplostim. Proliferation was measured at days 3, 6 and 10 and compared in the 3 groups by a proliferation coefficient. Membrane maturation was assessed at day 6 and 10 by flow cytometry (FC) after CD41-FITC and CD42-PE staining. Megakaryocytic ploidy was measured at day 10 by FC after propidium iodure and CD41 staining. At day 8, large mature megakaryocytes were isolated after discontinuous HSA density gradient and proplatelets forming megakaryocytes were counted from between days 9 and 13. Late mature megakayocytes (day 12–13) were observed with a confocal microscope to qualitatively analyze proplatelet formation.
We did not observe any difference between A-ITP or PC-ITP patients and controls in term of proliferation, ploidy, or expression of surface differentiation markers (CD41, CD42). In contrast, PC-ITP-derived megakaryocytes showed a defect in proplatelet formation, as only 12% of large, mature megakaryocytes were able to form proplatelets in liquid culture at day 11 vs 37% in CTRL (p=0,046) and 39% in A-ITP (p=0,03), 11% at day 12 vs 43,2% in CTRL (p=0,024) and 46% in A-ITP (p=0,0002), and 10% at day 13 vs 44,5% in CTRL (p=0,015) and 46% in A-ITP (p=0,03). Besides, we observed that proplatelet-forming megakaryocytes from ITP patients had less proplatelets per megakaryocyte and less bifurcation per proplatelet.
In conclusion, our study shows that megakaryocytes from patients with persistent/chronic ITP have an intrinsic defect in megakaryocyte development that is independent from the medullar environment. This defect affects proplatelet formation and further investigations are now needed to better describe mechanisms underlying proplatelet alteration in this disease.

Disclosures:
Viallard: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.