A nonsynonymous SNP in the ITGB3 gene disrupts the conserved membrane-proximal cytoplasmic salt bridge in the αIIbβ3 integrin and cosegregates dominantly with abnormal proplatelet formation and macrothrombocytopenia

We report a 3-generation pedigree with 5 individuals affected with a dominantly inherited macrothrombocytopenia. All 5 carry 2 nonsynonymous mutations resulting in a D723H mutation in the 133 integrin and a P53L mutation in glycoprotein (GP)Ib alpha. We show that GPIb alpha-L53 is phenotypically silent, being also present in 3 unaffected pedigree members and in 7 of 1639 healthy controls. The beta(3)-H723 causes constitutive, albeit partial, activation of the alpha(IIb)beta(3) complex by disruption of the highly conserved cyto-plasmic salt bridge with arginine 995 in the alpha(IIb) integrin as evidenced by increased PAC-1 but not fibrinogen binding to the patients' resting platelets. This was confirmed in CHO alpha(IIb)beta(3)-H723 transfectants, which also exhibited increased PAC-1 binding, increased adhesion to von Willebrand factor (VWF) in static conditions and to fibrinogen under shear stress. Crucially, we show that in the presence of fibrinogen, alpha(IIb)beta(3)-H723, but not wild-type alpha(IIb)beta(3), generates a signal that leads to the formation of proplatelet-like protru-sions in transfected CHO cells. Abnormal proplatelet formation was confirmed in the propositus's CD34(+) stem cell-derived megakaryocytes. We conclude that the constitutive activation of the alpha(IIb)beta(3)-H723 receptor causes abnormal proplatelet formation, leading to incorrect sizing of platelets and the thrombocytopenia observed in the pedigree.