OC 55.1 - Decreased platelet RABGAP1L (RAB GTPase Activating Protein 1 Like) expression in RUNX1 haplodeficiency - RUNX1 regulates RABGAP1L

Background: RABGAPs are GTP-hydrolysis activating proteins (GAPs) that negatively regulate RABs, proteins linked to vesicle trafficking. RABGAP1L is expressed in erythroid and megakaryocyte (MK) lineages. Little is known about RABGAP1L role in platelets/MK. Patients with germline RUNX1 haplodeficiency (RHD) have thrombocytopenia, platelet dysfunction and a predisposition to myeloid malignancies. RHD platelets and MK have abnormalities including impaired responses to activation, granule deficiencies, and defective endocytosis/protein trafficking. Platelet expression profiling of our RHD patient using Affymetrix gene chips showed that RABGAP1L was decreased compared to healthy controls.

Aims: To understand the role of RABGAP1l in platelets and MK.

Methods: We studied RABGAP1L levels in platelets from RHD patients, its regulation by RUNX1 in PMA-treated megakaryocytic HEL cells, and effect of siRNA RABGAP1L knockdown on uptake of albumin in HEL cells.

Results: On immunoblotting platelet RABGAP1L was decreased in 3 RHD patients compared to healthy individuals (Figure 1). In silico analyses of 2000 bp of RABGAP1L promoter region from ATG showed 3 RUNX1 consensus binding sites: -803/-798, -1611/-1606 and -1630/-1626. Chromatin immunoprecipitation (ChIP) performed using HEL cells and anti-RUNX1 antibody showed enrichment by PCR of promoter regions encompassing 3 RUNX1 sites. EMSA showed RUNX1 binding to site 2. RUNX1 overexpression increased and siRNA downregulation decreased RABGAP1L on immunoblotting indicating RUNX1 regulates RABGAP1L. In prior studies endocytosis of albumin and fibrinogen (not synthesized by human MK), was altered in RHD platelets and MK. We examined uptake and retention over 24 hours of Alexa fluor-488 albumin in HEL cells. In studies in progress, albumin uptake was increased at 30 and 120 minutes on siRNA KD of RABGAP1L compared to control cells (Figure 2).

Conclusion(s): These studies provide first evidence that RABGAP1L is a RUNX1 target and downregulated in RHD platelets/MK. They suggest it regulates albumin endocytosis in megakaryocytic cells. Supported by NHLBI grants HL-137376 and HL-109568.