| Abstract: | The nature of an inherited platelet disorder was investigated in three siblings affected by severe bleeding. Using whole-exome sequencing, we identified the culprit mutation (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 Rap1 and to perform proper αIIbβ3 integrin inside-out signaling. Expression of CalDAG-GEFI mutant in HEK293T cells abolished Rap1 activation 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 Rac1 GTP-binding. Functional deficiencies were confined to platelets and megakaryocytes with no leukocyte alteration. This contrasts with the phenotype seen in type III leukocyte adhesion deficiency caused 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 megakaryocytes corrected 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.Inherited platelet disorders are rare diseases that give rise to bleeding when platelets fail to fulfill their hemostatic function upon vessel injury. Clinical manifestations include mainly mucocutaneous bleeding, menometrorrhagia and excessive bleeding after surgical intervention or trauma. The study of these diseases allows a better understanding of normal platelet biochemistry and physiology. These inherited disorders include abnormalities of platelet receptors, granules, or signal transduction (Nurden and Nurden, 2011). Signal transduction dysfunction is thought to be the most common cause of platelet inherited disorders; however, only a few have been successfully genotyped.Here, we have identified and characterized the first mutation of RASGRP2 (RAS guanyl-releasing protein-2) in a family suffering severe bleeding. RASGRP2 codes for a major signaling molecule in platelets, calcium-and-DAG-regulated guanine exchange factor-1 (CalDAG-GEFI). It is a guanine nucleotide exchange factor (GEF) that is critical for Ras-like GTPase activation whose target is mainly Rap1 in platelets (Crittenden et al., 2004; Bergmeier et al., 2007; Cifuni et al., 2008; Stefanini et al., 2009). Rap1 is one of the most predominant small GTPases in platelets and constitutes a key signaling element that governs platelet activation by directly regulating integrin-mediated aggregation and granule secretion (Chrzanowska-Wodnicka et al., 2005; Zhang et al., 2011). Mice lacking CalDAG-GEFI not only have major defects in platelet function, with a reduced ability to form thrombi upon vascular injury, but they also have impaired neutrophil functions (Crittenden et al., 2004; Bergmeier et al., 2007; Carbo et al., 2010). To date, no pathological mutation in RASGRP2 has been reported in man and knowledge about the phenotype linked to human CalDAG-GEFI deficiency is lacking. |
