Critical function for the Ras-GTPase activating protein RASA3 in vertebrate erythropoiesis and megakaryopoiesis

Abstract

Phenotype-driven approaches to gene discovery using inbred mice have been instrumental in identifying genetic determinants of inherited blood dyscrasias. The recessive mutant scat (severe combined anemia and thrombocytopenia) alternates between crisis and remission episodes, indicating an aberrant regulatory feedback mechanism common to erythrocyte and platelet formation. Here, we identify a missense mutation (G125V) in the scat Rasa3 gene, encoding a Ras GTPase activating protein (RasGAP), and elucidate the mechanism producing crisis episodes. The mutation causes mislocalization of RASA3 to the cytosol in scat red cells where it is inactive, leading to increased GTP-bound Ras. Erythropoiesis is severely blocked in scat crisis mice, and ∼94% succumb during the second crisis (∼30 d of age) from catastrophic hematopoietic failure in the spleen and bone marrow. Megakaryopoiesis is also defective during crisis. Notably, the scat phenotype is recapitulated in zebrafish when rasa3 is silenced. These results highlight a critical, conserved, and nonredundant role for RASA3 in vertebrate hematopoiesis.

RASA3 is a critical inhibitor of RAP1-dependent platelet activation

“…The same studies identified the dual specificity GAP, RASA3, as the most abundant RAP-GAP expressed in platelets, with protein expression levels comparable to that of CalDAG-GEFI. An important confirmation that RASA3 may be a critical regulator of platelet function came from our findings that a G125V mutation in Rasa3 (Rasa3 scat ) is associated with severe thrombocytopenia in mice (33) and from recent studies by Molina-Ortiz et al, who described increased RAP1 activation in megakaryocytes from mice expressing a catalytically inactive mutant of RASA3 (34).…”

“…Studies on the role of RASA3 in the activation response of human platelets are not possible currently, as specific inhibitors do not exist and patients with mutations in the gene have not been identified. Recent studies in mice demonstrated that expression of RASA3 mutants leads to high embryonic lethality and severe thrombocytopenia (33,34). To test whether the embryonic lethality of Rasa3 mutant mice is caused by defective platelet function, we deleted Rasa3 both systemically (Rasa3 Figure 1D), including cutaneous and jugular lymphatics and the thoracic duct (Supplemental Figure 2), in which platelets and the lymphovenous valve are required to prevent backflow of blood into the lymphatic vasculature (36).…”

“…It will be interesting to see whether mutations in RASA3 affect peripheral lymphocyte counts by modulating the activity of RAS, RAP, or both. While Rasa3 scat/scat mice also exhibit a profound anemia (33), the defect in rbc is very mild in Rasa3 hlb/hlb mice (L.L. Peters, unpublished observations).…”

Emerging EPO and EPO receptor regulators and signal transducers

“…One is the Ras-GTPase activating protein Rasa3, which when mutated in Scat mice leads to anemia and thrombocytopenia. 56 A second is neurofibromin (Nf1), for which mutations have been associated with juvenile myelomonocytic leukemia, including anemia due to limited EPC differentiation. 57 Regulation of RAS-modulated targets is also important for EPOdependent erythropoiesis.…”

“…47 Important downstream EPOR signal transducers are also being discovered ( Figure 1C). Within a central RAS/MEK module, these include MASL1, 63 Rasa3, 56 and neurofibrin, 57 that act to balance ERK1/2 signaling and EPC production. For EPC cytoprotection, an EPO-induced Spi2A serpin and small molecule inhibitors of leached lysosomal cathepsins have emerged that lessen ROSassociated damage.…”