Abstract Number: LPB0127
Meeting: ISTH 2021 Congress
Background: Heterozygous germline RUNX1 mutations lead to thrombocytopenia and impaired platelet function and granule contents. Our previous studies in a patient with RUNX1 mutation showed that platelet albumin was decreased (Sun et al, Blood 103: 948-54, 2004). Human platelet α-granules contain numerous proteins, some synthesized (PF4 and VWF) and others incorporated by endocytosis (fibrinogen, albumin and IgG) by megakaryocytes (MK).
Aims: To understand the mechanisms leading to decreased platelet albumin and granule defects in RUNX1 haplodeficiency (RHD).
Methods: We studied endocytosis of fluorescent-labeled albumin, fibrinogen and IgG in platelet suspensions (0-90 min) and in PMA-treated megakaryocytic HEL cells (up to 24 hours) using flow cytometry and immunofluorescence microscopy. We studied alterations in caveolin-1.
Results: In platelets, protein uptake was time- and concentration-dependent. Uptake of albumin, fibrinogen and IgG was decreased in two patients (father and daughter) with RHD (c.352-1 G>T) (mean fluorescent intensity <50% of normal). In HEL cells, uptake of albumin and fibrinogen was time- and concentration-dependent. On siRNA RUNX1 knockdown, uptake/retention of albumin and fibrinogen over 24 hrs was higher than in control siRNA cells. Caveolin-1 and flotillin-1, two caveolae-associated membrane proteins linked to endocytosis, were increased on immunoblotting. Caveolin-1 downregulation (siRNA) in RUNX1-deficient HEL cells abrogated the increase in uptake of albumin, but not fibrinogen. On immunofluorescence microscopy staining of immobilized HEL cells albumin (but not fibrinogen) colocalized with CAV-1 in wild type cells at 30 min; this colocalization was disrupted on RUNX1 knockdown.
Conclusions: Platelets with RUNX1 haplodeficiency have defective endocytosis of albumin, fibrinogen and IgG. With RUNX1 knockdown, in MK cells there is caveolin-1 upregulation, increased albumin uptake and defective caveolin-dependent trafficking of albumin. These studies provide insights into diverse mechanisms leading to α-granule abnormalities and handling of proteins by platelets and megakaryocytes in RHD. (Supported by Grants HL-109568, HL-137376, HL137207).
To cite this abstract in AMA style:Del Carpio-Cano F, Mao G, Alam M, Wurtzel J, Goldfinger L, Rao AK. Defective Platelet Endocytosis of Albumin in RUNX1 Haplodeficiency Associated with Altered Caveolin-dependent Albumin Trafficking in Megakaryocytic Cells [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/defective-platelet-endocytosis-of-albumin-in-runx1-haplodeficiency-associated-with-altered-caveolin-dependent-albumin-trafficking-in-megakaryocytic-cells/. Accessed August 16, 2022.
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