Non-activating ITGB3 mutation in β3 cytoplasmic region causes macrothrombocytopenia with impaired αIIbβ3/RhoA pathway

Almost all mutations of ITGA2B or ITGB3 identified in congenital macrothrombocytopenia induce constitutive activation of αIIbβ3. However, whether concomitant αIIbβ3 activation is essential for macrothrombocytopenia development remains unknown. Recently, we identified the β3(R734C) mutation that does not induce αIIbβ3 activation in macrothrombocytopenia. Here, we generated β3(R734C) knock-in (KI) mice and investigated the abnormalities in the platelet/megakaryocyte biology of KI mice and a family with β3(R734C)-related macrothrombocytopenia. Macrothrombocytopenia was observed in heterozygous β3(R734C) subjects with reduced expression of αIIbβ3. 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 αIIbβ3 expression was decreased in KI mice, and αIIbβ3 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 αIIbβ3 activation is not essential for the development of macrothrombocytopenia. We also showed that β3(R734C) mutation impairs the inside-out and outside-in signaling of αIIbβ3, and abnormal actin rearrangement and impaired RhoA activation may play major roles in leading to macrothrombocytopenia.