RUNX1 Haplodeficiency Reduces Platelet Endocytosis of Albumin and Fibrinogen and Impairs Megakaryocyte Intracellular Trafficking

Introduction:RUNX1 is a major hematopoietic transcription factor that regulates megakaryopoiesis and platelet formation and function. Germline RUNX1 haplodeficiency (RHD) is characterized by thrombocytopenia, impaired platelet function and granule deficiencies. Our previous studies in a patient with RUNX1 mutation showed that platelet albumin and fibrinogen were decreased (Sun et al, Blood 103: 948-54, 2004) .Human platelet α-granules contain numerous proteins, some synthesized (e.g., PF4 and VWF) and others (e.g., fibrinogen, albumin and IgG) are incorporated by endocytosis, a process poorly understood in platelets/megakaryocytes (MK).

Methods: To understand the mechanisms leading to the decreased platelet albumin and fibrinogen, we performed studies on endocytosis in platelets from RHD patients, megakaryocytic HEL cells and MK differentiated from CD34 ⁺ hematopoietic stem and progenitor cells (HSPCs). 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 hrs) using flow cytometry and immunofluorescence microscopy, and assessed proteins linked to endocytosis.

Results:Platelet uptake of albumin, fibrinogen and IgG was time- and concentration dependent. Uptake of all 3 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 (KD), uptake/retention of albumin and fibrinogen over 24 hrs was increased compared to control siRNA cells. Caveolin-1 and flotillin-1, two caveolae-associated membrane proteins linked to endocytosis, and LAMP2 (linked to lysosomal trafficking) and RAB11 (linked to recycling endosomes) were increased on RUNX1 KD on immunoblotting. Caveolin -1 downregulation (siRNA) in RUNX1-deficient HEL cells abrogated the increase in uptake of albumin, but not fibrinogen. At 30 min, in control cells albumin was strongly colocalized with caveolin-1 on immunofluorescence studies. This colocalization was abolished on CAV-1 KD. With RUNX1 KD there was a decrease in colocalization with caveolin-1, and an increase with flotillin-1 both at 30 (Figure 1a) and 120 min. Thus, RUNX1 KD alters endocytosis and trafficking of albumin from caveolin-1 to flotillin-1 bearing vesicles. In experiments where HEL cells were incubated in medium without albumin for 24 hours, cell albumin decreased over time in both control cells and RUNX1-deficient cells with lower levels in the latter. LAMP2 levels were increased on RUNX1 KD, which may provide a mechanism (lysosomal degradation) for this. With respect to fibrinogen, at 30 or 120 min, there was no colocalization of fibrinogen with caveolin-1 or flotillin-1, indicating fibrinogen uptake is not caveolin-dependent. In control cells, there was low/moderate fibrinogen colocalization with RAB11 (marker for recycling endosomes); this was markedly increased with RUNX1 KD (Figure 1b), along with increased RAB11 level on immunoblotting, suggesting that RUNX1 KD is associated with altered fibrinogen trafficking towards recycling endosomes. Recently, IFITM3 has been implicated in platelet fibrinogen uptake in nonviral sepsis. We found HEL cell IFITM3 was increased on RUNX1 KD; however, IFITM3 siRNA KD did not decrease fibrinogen uptake. We studied endocytosis in in vitro differentiated MKs for 12 days from human CD34 ⁺ HSPCs; upon lentiviral shRNA KD of RUNX1 the uptake of albumin and fibrinogen was increased compared to control cells .

Conclusions: We provide the first evidence that 1) platelet endocytosis of albumin and fibrinogen is impaired in RHD and 2) RUNX1-deficient megakaryocytic cells have enhanced endocytosis but defective trafficking of albumin and fibrinogen, mediated by distinct mechanisms, and associated with increases in caveolin 1, flotillin-1, RAB11, LAMP2 and IFITM3. These studies provide new insights into mechanisms governing endocytosis by platelets and MKs relevant to the α-granule deficiencies in RHD.