Advances in the diagnosis and therapy of paroxysmal nocturnal hemoglobinuria

Abstract

PNH is an uncommon acquired hemolytic anemia that often manifests with hemoglobinuria, abdominal pain, smooth muscle dystonias, fatigue, and thrombosis. The disease results from the expansion of hematopoietic stem cells harboring a mutation in a gene, PIG-A, that is required for the biosynthesis of a lipid moiety, glycosylphosphatidylinositol (GPI), that attaches dozens of different proteins to the cell surface. Thus, PNH cells are deficient in cell surface GPI anchored proteins; this deficiency on erythrocytes leads to intravascular hemolysis since certain GPI anchored proteins normally function as complement regulators. Free hemoglobin released from intravascular hemolysis leads to circulating nitric oxide depletion and is responsible for many of the clinical manifestations of PNH, including fatigue, erectile dysfunction, esophageal spasm, and thrombosis. Interestingly, rare PIG-A mutations can be found in virtually all healthy control subjects leading to speculation that PIG-A mutations in hematopoietic stem cells are common benign events. However, recent data reveals that most of these mutations in healthy controls are not derived from stem cells. The recently FDA approved complement inhibitor eculizumab has been shown to decrease hemolysis, decrease erythrocyte transfusion requirements, decrease the risk for thrombosis and improve quality of life for PNH patients.

Figure 1. Biosynthesis of GPI-anchored proteins

GPI-anchored biosynthesis takes place in the endoplasmic reticulum. PIG-A is one of 7 genes required for the first step (see Table 1), the transfer of N-acetylglucosamine (GlcNAc) from uridine 5′-diphospho-N-acetylglucosamide (UDP-GlcNAc:PI ) to phosphatidylinositol (PI) to yield GlcNAc-PI. After synthesis of the mature GPI precursor, the cognate protein is attached and then transported to the plasma membrane where the GPI-anchored protein resides in membrane rafts. PIG-A mutations lead to a defect in the first step in GPI anchor biosynthesis resulting in intracellular degradation of the cognate protein and a lack of cell surface GPI anchored proteins.

Figure 2. Nitric oxide scavenging in PNH

Under normal conditions (A) Nitric oxide synthase (NOS) combines with oxygen (O₂) and arginine to form nitric oxide (NO) and citrulline. (B) Intravascular hemolysis releases free hemoglobin into the plasma. Oxygen bound Fe²⁺ from free hemoglobin enters the plasma and converts NO to inert nitrite and oxidizes hemoglobin to methemoglobin. In addition, intravascular hemolysis releases erythrocyte arginase, which depletes arginine, the substrate for NOS. Depletion of NO at the tissue level leads to many of the symptoms of PNH including smooth muscle dystonias.

Figure 3. Flow cytometric diagnosis of PNH. (A)

Cell surface GPI anchored proteins can be detected using individual monoclonal antibodies (e.g, anti-CD55, anti-CD59, anti-CD14 etc.) or FLAER. Monoclonal antibodies bind to the protein portion of the cell which may vary based on the cell type and level of differentiation. FLAER binds to the glycan portion of the GPI anchor and binds to virtually all GPI anchored proteins. (B) Flow cytometric analysis of granulocytes from a PNH patient showing GPI anchor deficient cells (left peak) and small population of normal GPI anchor replete cells (right peak).

Figure 4. Blockade of terminal complement activation

Classical, alternative and lectin pathways converge at the point of C3 activation. The lytic pathway is initiated with the formation of C5 convertase and leads to the assembly of the C5b, C6, C7, C8, (n) C9 membrane attack complex. Eculizumab binds to C5 and prevents the formation of the membrane attack complex by reducing cleavage of C5 into C5a and C5b.