Marked bleeding diathesis in patients with platelet dysfunction due to a novel mutation in RASGRP2, encoding CalDAG-GEFI (p.Gly305Asp)

Abstract

Congenital platelet function disorders are often the result of defects in critical signal transduction pathways required for platelet adhesion and clot formation. Mutations affecting RASGRP2, the gene encoding the Rap GTPase activator, CalDAG-GEFI, give rise to a novel, and rare, group of platelet signal transduction abnormalities. We here report platelet function studies for two brothers (P1 and P2) expressing a novel variant of RASGRP2, CalDAG-GEFI(p.Gly305Asp). P1 and P2 have a lifelong history of bleeding with severe epistaxis successfully treated with platelet transfusions or rFVIIa. Other bleedings include extended hemorrhage from minor wounds. Platelet counts and plasma coagulation were normal, as was αIIbβ3 and GPIb expression on the platelet surface. Aggregation of patients' platelets was significantly impaired in response to select agonists including ADP, epinephrine, collagen, and calcium ionophore A23187. Integrin αIIbβ3 activation and granule release were also impaired. CalDAG-GEFI protein expression was markedly reduced but not absent. Homology modeling places the Gly305Asp substitution at the GEF-Rap1 interface, suggesting that the mutant protein has very limited catalytic activity. In summary, we here describe a novel mutation in RASGRP2 that affects both expression and function of CalDAG-GEFI and that causes impaired platelet adhesive function and significant bleeding in humans.

Keywords: Bleeding; RASGRP2; dysfunction; platelets; signaling.

Figure 1:. Altered platelet function in patients with novel mutation in RASGRP2.

(A,B) Impaired aggregation, integrin activation and granule secretion in platelets from patients (P1, P2) with lifelong bleeding diathesis. (A) Aggregation response of control and patients’ platelets activated with ADP (10μM), epinephrine (Epi, 50μM), collagen (Col, 8μg/mL), TRAP-6 (20μM), thrombin (0.1 U/mL, washed platelets), arachidonic acid (AA, 0.5mM), ristocetin (1.2mg/mL), A23187 (2.5μM), or PMA (4μM). (B) Flow cytometric analysis of fibrinogen binding to αIIbβ3 (left panel), surface expression of P-selectin (CD62P, middle panel) and surface expression of LAMP-3 (CD63, right panel) for PRP activated with ADP (10μM) or PMA (4μM) followed by labeling with fibrinogen-FITC or the indicated antibodies. Results are shown as mean fluorescence intensity (MFI) after subtracting the MFI measured in unstimulated platelets. (C) Family pedigree (top panel) and localization of the c.914G>A transition and G305D aa substitution within RASGRP2 (bottom panel). Filled and partially filled black symbols indicate subjects homozygous and heterozygous for the mutation. The father was not genotyped. (D) Upper panel: Immunoblot analysis for CalDAG-GEFI in platelet lysates from a healthy human (control), P1, P2, a control mouse (WT) and a Rasgrp1−/− mouse (KO). White arrow indicates the band for CalDAG-GEFI. Bottom panel: densitometric quantification of band intensities. The CalDAG-GEFI to β-actin band intensity ratio is shown. Ratios determined in P1/P2 and KO samples were normalized towards human control and WT mouse samples, respectively. (E) A homology model of the Cdc25/REM domain of CalDAG-GEFI (dark grey, cartoon representation), interacting with Rap1B (light grey, lines representation). This model was generated in pymol and is based on the crystal structure of Epac2 in complex with Rap1B[7,13]. Arrows point to the homologous positions of CalDAG-GEFI G305D and G248W, shown in sphere representation (white).