Identification of two novel mutations in RASGRP2 affecting platelet CalDAG-GEFI expression and function in patients with bleeding diathesis

Abstract

The RASGRP2 gene encodes the Ca2+ and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), which plays a key role in integrin activation in platelets and neutrophils. We here report two new RASGRP2 variants associated with platelet dysfunction and bleeding in patients. The homozygous patients had normal platelet and neutrophil counts and morphology. Platelet phenotyping showed: prolonged PFA-100 closure times; normal expression of major glycoprotein receptors; severely reduced platelet aggregation response to ADP and collagen (both patients); aggregation response to PAR1 and arachidonic acid markedly impaired in one patient; PMA-induced aggregation unaffected; platelet secretion, clot retraction, and spreading minimally affected. Genetic analysis identified two new homozygous variants in RASGRP2: c.706C>T (p.Q236X) and c.887G>A (p.C296Y). In both patients, CalDAG-GEFI protein was not detectable in platelet lysates, and platelet αIIbβ3 activation, as assessed by fibrinogen binding, was greatly impaired in response to all agonists except PMA. Patient neutrophils showed normal integrin expression, but impaired Mn2+-induced fibrinogen binding. In summary, we have identified two new RASGRP2 mutations that can be added to this rapidly growing form of inherited platelet function disorder.

RasGRP2 Structure, Function and Genetic Variants in Platelet Pathophysiology

“…In humans, we reported defective spreading over fibrinogen of platelets from patients expressing an inactive form of RasGRP2 [58]. However, other studies on platelets isolated from individuals lacking the GEF showed only minimal spreading impairment [59,60]. Whereas the effect of RasGRP2 deficiency on platelet spreading relies on consequences of defective integrin "inside-out" activation or on its direct involvement in "outside-out" signaling remains to be fully determined.…”

Subcellular localization of Rap1 GTPase activator CalDAG‐GEFI is orchestrated by interaction of its atypical C1 domain with membrane phosphoinositides

“…6,12,13 In humans, mutations in CDGI also cause impaired platelet function and a moderate to severe bleeding phenotype. 10,[14][15][16][17][18][19] Interestingly, in vitro and in vivo platelet function is markedly impaired in mice and humans expressing a truncated CDGI lacking the atypical C1 regulatory domain. 13,16 The role of this C1 domain in CDGI function, however, is not understood.…”

Platelets at the vascular interface

“…Consistent with these conclusions, complete CalDAG-GEFI deficiency is ill tolerated as it causes moderate to severe bleeding in humans and mice. 51,[53][54][55][58][59][60] At the same time, CalDAG-GEFI/RAP1 signaling has to be restrained by RASA3 in circulating platelets to avoid severe thrombocytopenia and blood-lymphatic mixing in the developing embryo. Together, these specialized mechanisms ensure the tight regulation of platelet adhesiveness, which is crucial for the platelets' ability to efficiently form a stable and self-limiting hemostatic plug at sites of vascular injury.…”

“…result is a near-immediate integration of second messenger generation and integrin activation. Lack of CalDAG-GEFI leads to impaired thrombus formation and bleeding 51,[53][54][55][57][58][59][60]. CalDAG-GEFI-mediated RAP1 activation is antagonized by the GTPase activating protein (GAP), RASA3 61.…”