blister and Thrombocytopenia

Mutations in the MYH9 gene cause autosomal dominant MYH9-related diseases (MYH9-RD) that associate macrothrombocytopenia with various other clinical conditions. The mechanisms giving rise to giant platelets remain poorly understood.. To study the proplatelet formation (PPF) derived from megakaryocytes (MKs) generated in vitro from 11 patients with MYH9-RD with different mutations, compared with controls.. Proplatelet formation from cultured patients' MKs was evaluated with or without blebbistatin or the ROCK inhibitor Y27632. Myosin IIA and actin distribution were studied in spreading MKs on different surfaces by immunoconfocal analysis. Kinetic studies of contractility were performed on spreading MKs and the impact of blebbistatin on the maturation of the patients' MKs was evaluated by electron microscopy.. We show that in vitro MKs of 11 patients formed significantly fewer proplatelets than controls. MKs from MYH9-RD displayed an abnormal spreading on polylysine, fibronectin and collagen, with a disorganized actin network and a marked increase in stress fiber formation. Traction force microscopy studies demonstrated an elevated level of contractile forces in adherent mutated MKs. The myosin II inhibitor blebbistatin and the ROCK inhibitor Y27632 both rescued the proplatelet formation defect and normalized the ultrastructural characteristics of MYH9-RD MKs. Altogether, our results show that in MYH9-RD, mutations modify the overall MYH9 function and provoke a proplatelet defect through an excess of actomyosin contractility in spreading MKs. These results may promote new therapeutic strategies aimed at reducing this actomyosin contractility.

Topics: Actomyosin; Blood Platelets; Cells, Cultured; Heterocyclic Compounds, 4 or More Rings; Humans; Molecular Motor Proteins; Mutation; Myosin Heavy Chains; Nonmuscle Myosin Type IIA; Thrombocytopenia

 Inactivation of the mouse Myh9 gene (Myh9Δ) or its mutation in MYH9-related diseases leads to macrothrombocytopenia. Paradoxically, previous studies using in vitro differentiated megakaryocytes showed an increased capacity for proplatelet formation when myosin was absent or inhibited..  To explore the origin of the thrombocytopenia induced by myosin deficiency, we studied proplatelet formation using bone marrow explants of wild-type (WT) and Myh9Δ mouse where megakaryocytes have matured in their native environment..  A dramatic decrease in the number and complexity of proplatelets was observed in megakaryocytes from Myh9Δ mice, while inhibition of myosin activity by blebbistatin increased proplatelet formation from WT mature megakaryocytes. Moreover, Myh9Δ megakaryocytes had a smaller size than the WT cells. These data indicate that myosin deficiency acts negatively on proplatelet formation, probably by impairing in situ megakaryocyte maturation, while myosin activity is dispensable at the latest stage of proplatelet formation. In addition, ultrastructural examination of Myh9Δ bone marrow revealed an increased proportion of megakaryocytes exhibiting signs of non-apoptotic cell death as compared with the WT mice..  These data indicate that thrombocytopenia in Myh9Δ mice results from defective development of megakaryocyte size, impaired proplatelet formation and increased cell death.

Topics: Animals; Blood Platelets; Bone Marrow; Caspase 3; Cell Death; Cell Lineage; Cell Survival; Female; Heterocyclic Compounds, 4 or More Rings; In Situ Nick-End Labeling; Male; Megakaryocytes; Mice; Microscopy, Electron, Transmission; Mutation; Myosin Heavy Chains; Nonmuscle Myosin Type IIA; Thrombocytopenia