A Novel Case of Familial Thrombocytopenia with Evidence for a Critical Role of Proteins Involved in Ca²⁺ Signaling in Megakaryocytopoiesis

Abstract number: P2133

Nurden¹ P, Debili² N, Vainchenker² W, Hillman³ A, Bobe⁴ R, Pasquet¹ JM, Bredoux⁴ R, Corvazier⁴ E, Nurden¹ A, Enouf⁴ J

¹¹Laboratoire Hématologie, IFR4/FR21, Pessac, France ²²U362 Inserm, Villejuif, France ³³RCSI, Dublin, Ireland ⁴⁴U348 Inserm, Hôpital Lariboisière, Paris, and the French Network for Inherited Platelet Disorders.

Inherited thrombocytopenia are known to have multiple origins. Here we describe a novel profile with autosomal dominant inheritance affecting a brother, his sister and their father. Bleeding was severe during childhood, their platelet count remains less than 20 G/L and agglutinates were present in blood taken on all anticoagulants. Platelet size is mostly normal with occasional enlarged forms. Electron microscopy revealed platelet agglutinates composed of adjacent discoid platelets with areas of the plasma membranes in very close contact. Increased amounts of VWF and sometimes also Fg are seen at the platelet surface but not P-selectin. FACs analysis revealed the normal presence of GPIIb-IIIa but GPIb-X was variable depending on the antibody. Exon 28 of the VWF gene was normal as was the gene encoding GPIbalpha ruling out type 2B and platelet-type VWD. Basal levels of Ca²⁺ were increased in the platelets and there were signs of filamin degradation. We investigated the role of proteins involved in Ca²⁺ signaling, focusing on platelet Sarco/Endoplasmic Reticulum Ca²⁺ATPases (SERCAs)-type 2b, 3a and 3b and plasma membrane Ca²⁺ATPases (PMCAs)-type 1b and 4b, a possible target of caspase-3 in early phase apoptosis. Inositol 1, 4, 5 trisphosphate receptors (InsP₃-Rs) types 1–3 were also analyzed. In comparison with normal platelets, those from the patients showed strikingly higher expressions of the PMCA4b isoform and InsP₃-R3 protein. In addition, the unusual presence of cleaved polyadenosine diphosphate (ADP)-ribose polymerase protein in platelets of the patients, showed a caspase-3 activity that contrasted with normal platelets. In vitro, megakaryocyte (MK) culture from blood CD34⁺ cells in the presence of SCF and TPO showed impaired MK maturation and confirmed an abnormal platelet production. Taken together, the results suggest that the regulation of proteins involved in Ca²⁺ signaling during megakaryocytopoiesis is necessary for a normal platelet production and that abnormalities in Ca²⁺ metabolism are a potential cause of inherited thrombocytopenias.