A Novel View of Paroxysmal Nocturnal Hemoglobinuria (PNH) Pathogenesis: Do Pathologic PNH Hematopoietic Stem/Progenitor Cells (HSPCs) Displace Normal HSPCs From Their Niches in Bone Marrow Because They Are More Motile Due to Defective Adhesion and Enhanced Migratory Properties?
Authors: Ratajczak Janina , Liu Rui , Natarajan Nagendra , Maciejewski Jaroslaw P. , Sharma Vivek R. , Ratajczak Mariusz Z 
Published: October 2019

Abstract
Abstract 732

Background
. Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon acquired hemolytic anemia that results from the expansion of hematopoietic stem cells with a mutation in one of the enzymes (PIG-A) responsible for glycosylphosphatidylinositol (GPI anchor) biosynthesis, which is a post-translation modification of proteins associated with lipid rafts on the cell membrane surface. Some of these proteins are involved in the resistance of erythrocytes to lysis by the final product of complement cascade (CC) activation, C5b-C9, also known as the membrane attack complex (MAC). As we reported, the CXCR4 receptor, which binds a-chemokine stromal derived factor-1 (SDF-1) in regulating the trafficking of hematopoietic stem/progenitor cells (HSPCs), is also associated with lipid rafts (Blood 2005;105:40-8). In addition, we recently demonstrated that the bioactive lipid sphingosine-1-phosphate (S1P), which is a major chemoattractant directing egress of HSPCs from bone marrow (BM) into peripheral blood (PB) during mobilization, is released from erythrocytes by C5b-C9/MAC (Leukemia 2010;24:976-85). Hypothesis. Based on this finding, we hypothesized that HSPCs are continuously mobilized from the BM of PNH patients due to the susceptibility of PIG-A-deficient erythrocytes to CC activation, which elevates the free S1P level in plasma, as well as to their defective adhesion in the BM microenvironment due to impaired lipid raft formation. Experimental strategies. To address this hypothesis, peripheral blood mononuclear cells (PBMNC) were isolated from 6 PNH patients and stained with the fluorescent variant of aerolysin (FLAER), which binds GPI anchor and thus identifies normal, but not PNH, cells in FACS analysis. PNH patient-derived cells were tested for i) the level of CD34 antigen expression, ii) chemotaxis in response to SDF-1 and S1P, and iii) adhesion to fibronectin and bone marrow stromal cells. Results. We observed in PNH patients ∼3-fold higher expression of CD34 antigen on FLAER– cells circulating in PB than FLAER+ cells, which suggests that PNH-mutated HSPCs are preferentially released/mobilized into PB. Next, in Transwell chemotaxis assays followed by in vitro clonogenic assays with cells collected from the lower Transwell chambers, we observed that FLAER– cells responding to SDF-1 are ∼20 times more enriched in migrating clonogenic BFU-E and CFU-GM progenitors than their normal FLAER+ counterparts. Moreover, in parallel experiments, FLAER– CFU-GM that were plated over BM-derived fibroblasts or fibronectin in the presence of SDF-1 and S1P (known activators of VLA-4–VCAM-1-mediated cell adhesion) exhibited impaired adhesion in comparison to normal FLAER+ CFU-GM cells. Conclusions. Based on these observations, we propose a novel view of the pathogenesis of PNH and the expansion of PNH-affected cells in BM. Accordingly, the lack of PIG-A protein, which plays an important role in lipid raft formation, confers an advantage to PNH-affected HSPCs, which become more mobile. These cells are preferentially mobilized into PB in response to S1P released from C5b-C9/MAC-lysed erythrocytes. Thus, PNH-mutated HSPCs over time may outcompete normal HSPCs for their niches in BM, due to their increased motility, and contribute to the PNH type of hematopoiesis.

Disclosures:
No relevant conflicts of interest to declare.