Human CalDAG-GEFI gene (RASGRP2) mutation affects platelet function and causes severe bleeding.

The nature of an inherited platelet disorderwas investigated in three siblings affected by severe bleeding. Using whole- exome sequencing, we identified the culpritmutation (cG742T) in the RAS guanyl-releasing protein-2 (RASGRP2) gene coding for calcium- and DAG-regulated guanine exchange factor-1 (CalDAG-GEFI). Platelets from individuals carrying the mutation present a reduced ability to activate Rap1and to perform proper αIIbβ3 integrin inside-out signaling. Expression of CalDAG-GEFI mutant in HEK293T cells abolished Rap1activation upon stimulation. Nevertheless, the PKC- and ADP-dependent pathways allow residual platelet activation in the absence of functional CalDAG-GEFI. The mutation impairs the platelet’s ability to form thrombi under flow and spread normally as a consequence of reduced Rac1GTP-binding. Functional deficiencies were confined to platelets and megakaryocyteswith no leukocyte alteration. This contrasts with the phenotype seen in type III leukocyte adhesion deficiencycaused by the absence of kindlin-3. Heterozygous did not suffer from bleeding and have normal platelet aggregation; however, their platelets mimicked homozygous ones by failing to undergo normal adhesion under flow and spreading. Rescue experiments on cultured patient megakaryocytescorrected the functional deficiency after transfection with wild-type RASGRP2. Remarkably, the presence of a single normal allele is sufficient to prevent bleeding, making CalDAG-GEFI a novel and potentially safe therapeutic target to prevent thrombosis.