Non-activating ITGB3 mutation in ß3 cytoplasmic region causes macrothrombocytopenia with impaired aIIbß3/RhoA pathway

Almost all mutations of ITGA2B or ITGB3 identified in congenital macrothrombocytopenia induce constitutive activation of aIIbß3. However, whether concomitant aIIbß3 activation is essential for macrothrombocytopenia development remains unknown. Recently, we identified the ß3(R734C) mutation that does not induce aIIbß3 activation in macrothrombocytopenia. Here, we generated ß3(R734C) knock-in (KI) mice and investigated the abnormalities in the platelet/megakaryocyte biology of KI mice and a family with ß3(R734C)-related macrothrombocytopenia. Macrothrombocytopenia was observed in heterozygous ß3(R734C) subjects with reduced expression of aIIbß3. Compared to those in the wild-type (WT) mice, platelet counts decreased to 76 and 40% in heterozygous (Hetero) and homozygous (Homo) KI mice, respectively. Platelet aIIbß3 expression was decreased in KI mice, and aIIbß3 activation was not detected on non-stimulated KI platelets. Platelet aggregation, agonist-induced JON/A binding and P-selectin expression were impaired in KI mice. Platelet spreading on fibrinogen was also impaired in KI mice with ADP or thrombin stimulation. Additionally, filopodia and lamellipodia formation was impaired in fibrinogen-adhered megakaryocytes of KI mice. Moreover, RhoA activation was significantly impaired in fibrinogen-adhered megakaryocytes of KI mice. Proplatelet formation in KI mice was impaired with abnormal morphology. Platelet lifespan was shortened in Homo mice. Our data newly revealed that constitutive aIIbß3 activation is not essential for the development of macrothrombocytopenia. We also showed that ß3(R734C) mutation impairs the inside-out and outside-in signaling of aIIbß3, and abnormal actin rearrangement and impaired RhoA activation may play major roles in leading to macrothrombocytopenia. Overall design: To gain further understanding of ß3(R734C) mutation, we performed a gene expression analysis using RNA-sequence (RNA-seq), using WT and ß3(R734C) KI murine platelets. Putative differentially expressed genes generated by RNA-seq were identified (2 fold change cut-off).