RASA3 is a RAS and RAP1 GTPase-activating protein that moves between the cytosol and the plasma membrane, and when localized to the platelet plasma membrane it inhibits platelet activation in homeostatic balance against RASGRP2. RASA3 negatively regulates RAS, which transmits cytokine signaling for erythropoiesis and megakaryopoiesis. This spontaneous point mutation in the GTPas-activating protein RASA3 causes a conservative G125V substitution in a conserved glycine that is positioned between the two C2 domains. This has been shown to result in mislocalization to the reticulocyte cytosol rather than the plasma membrane and the loss of GAP activity. Although a Rasa3 null allele causes homozygous embryonic lethality with edema and hemorrhaging in the head and trunk, this severe combined anemia and thrombocytopenia (scat) mutation instead causes cycles of catastrophic bone marrow failure. A less severe phenotype is caused by the ENU-induced H794L point mutation of Rasa3hlb381 (Stock No. 005343), which causes thrombocytopenia at 6 weeks of age, but Rasa3hlb381 homozygotes are reported to be viable, fertile, and normal in size.
The scat mutation arose spontaneously in the BALB/cBy inbred strain at The Jackson Laboratory. Heterozygous intercrosses yield approximately 15% liveborn homozygotes, rather than the expected 25%, indicative of embryonic lethality. At birth homozygotes are small and pale with bruising and petechiae. Nearly one quarter were found to develop intracranial bleeding. Homozygotes display severe anemia and thrombocytopenia. They also have leukopenia, with decreased levels of both B and T cells, although ablation of lymphocytes through the co-expression of the Prkdcscid or Rag1tm1Mom allele does not prevent the disease phenotype. Splenectomy of homozygotes in remission does, however, prevent a return of the disease phenotype, and the disease phenotype can be transferred to wildtype hosts from homozygotes by transfer of hematopoietic stem cells. The initial report of Peters et al. found that 61.2% of homozygotes died by 25 days of age, with splenomegaly, gastrointestinal and rectal bleeding found at necropsy. Those homozygotes that survive the initial crisis phase, which begins in utero and ends at approximately 9 days of age, enter a remission phase with no anemia or thrombocytopenia and reduced leukopenia. The average length of this remission is 11 days, then a second crisis phase occurs in which the majority of homozygotes again die, but a few again enter a remission. This cyclic crisis and remission has not been reported for other alleles of RASA3. Peripheral blood smears and hematological assessment from homozygotes in the second crisis phase show severe poikilocytosis, anisocytosis, and polychromatophilia, a cell hemoglobin concentration mean of only 4.6, mean hematocrit of only 14.8, platelet count less than 20% of normal, percent reticulocytes more than 4 times normal and other abnormalities. Bone marrow shows decreased marrow cellularity during bone marrow failure and there are increased numbers of megakaryocytes in the bone marrow and spleen, with a highly disorganized demarcation membrane system, no platelet-forming areas, few granules, and frequent hypersegmented nuclei and excess rough ER. Hartman et al. found a delay at the G1 checkpoint of the basophilic and polychromatophilic stages of erythroid differentiation, an increase in peroxide levels at the proerythroblastic stage, with a trend for increased reactive oxygen species throughout erythroid differentiation, in reticulocytes, and in the bone marrow, which resolves to normal in mature cells. They also found decreased apoptosis in the proerythroblastic, polychromatic, and orthochromatic stages in the bone marrow and spleen, and lower levels of galectin 1 during crisis compared with wildtype. Peters et al. found that a platelet-specific antibody is generated in homozygotes and also in heterozygous females late in pregnancy, and that wildtype hosts transplanted with splenocytes from homozygotes also develop this platelet-specific antibody. Curiously, splenectomized homozygous females bred to heterozygous males failed to produce affected offspring, although sibling intercrossing those unaffected offspring generated more than 10% of affected offspring, showing that at least one wildtype allele of Rasa3 in the mother is required to generate the phenotype in the offspring independent of the genotype of the embryo.
