Abnormal Proplatelet Formation and Myosin Activation Status in Patients with MYH9-Related Syndrome
Authors: Chen Yolande , Boukour Siham , Nurden Paquita , Seye Ababacar , Bluteau Olivier , Palazzo Alberta , Chang Yunhua , Lordier Larissa , Favier Remi , Schlegel Nicole , Galy Anne , Nurden Alan T. , Raslova Hana , Vainchenker William , Debili Najet 
Published: October 2019

Abstract
Abstract 4826
MYH9-related disorders (MYH9-RD) are characterized by an autosomal-dominant macrothrombocytopenia associated or not with glomerular impairment, hearing loss and/or cataracts. It is caused by mutations of the MYH9 gene (encoding for the nonmuscle myosin heavy chain IIA), present at the heterozygous state in patients.
In knockout animal models, proplatelet formation (PPF) was found to be increased in certain conditions, but decreased in others (Chen et al, Blood 2007, Eckly et al, Blood 2007 and Thrombosis and Haemostasis 2010) and samples with heterozygosity did not show differences with the wild type. More recently, in a mouse model with a mutated MYH9 gene, mice with the mutation showed a phenotype similar to the one found in MYH9-RD, affecting multiple organs and, interestingly, mice heterozygous for the mutation showed an abnormal phenotype as well, although attenuated (Anderson et al, ASH Meeting 2010, Abstract 2527). However, opposite to the initial reports in mouse models with a knockout, proplatelet formation was found to be impaired in 4 patients (Pecci et al, Thrombosis and Haemostasis 2009). Therefore the question for the exact mechanism for thrombocytopenia still stands, so does the question whether the mutated protein causes disease through a dominant negative effect versus a functional haploinsufficiency. To address these questions, we performed studies on patient samples on one hand and seeked to knock down the gene expression in human megakaryocytes (MKs) on the other hand.
To study PPF in these patients, we analyzed MK differentiation in CD34+ cells from 9 patients with mutations in exons encoding for the motor domain (2 patients) or for the coiled coil domain of myosin IIA (7 patients). Compared with cultured MKs derived from healthy donors, a 2 to 5 –fold decrease in PPF was observed in the patients (4.9 ± 1.2% for patients vs. 15.1 ± 1.8% for control) and proplatelet area was decreased in patients as well (threefold decrease, 6156 μm2 for patients versus 18064 μm2 for control). In confocal microscopy with immunofluorescence staining, we observed disorganization of the granules, abnormal spreading and stress fibers, meaning that actin-myosin network is unstable.
We next used a shRNA strategy to knock down MYH9 expression during normal MK differentiation and compared shRNA-treated MKs (with 50% residual MYH9 expression) with those derived from patient CD34+ cells. Treatment with shRNA decreased in vitro PPF, as previously observed for cells from patients. Moreover, shRNA-treated MKs exhibited the same ultrastructural abnormalities as MKs from patients. Normal platelet production is dependent on the formation of branched long proplatelets. Surprisingly the defect in PPF formation seen in patients was rescued by blebbistatin, an inhibitor of class II myosin. In its presence the proplatelets increased by 2 to 5 fold, suggesting that the remaining myosin IIA might be hyperactivated and inhibiting PPF. Ultrastructural studies by electron microscopy performed on cells with blebbistatin treatment corrected abnormalities seen in the patients. Parallel to blebbistatin, ROCK (Y27632) or MLCK inhibitors (P18 or ML7) led to a threefold increase in proplatelet formation. We therefore compared by western blot the status of pMLC2 in patients and control MKs. In 3 out of 6 patients we found an increased pMLC2. In addition, we also found an increased pMLC2 in MKs treated with shRNA with 50% residual myosin expression. Altogether our results show that reducing the protein expression by half was sufficient to recreate features characteristic of the disease in megakaryocytes, while the increased PPF, relative to control, in response to rho/ROCK inhibitors and to myosin inhibitors, suggest that the defective PPF in patients may be related to an activated rho/ROCK pathway and/or increase of the contractile force.

Disclosures:
No relevant conflicts of interest to declare.