calcimycin and Hemoglobinuria--Paroxysmal

Complement-induced procoagulant alteration of red blood cell (RBC) membranes in paroxysmal nocturnal haemoglobinuria (PNH) was examined. Microvesicles, deficient in acetylcholinesterase, were generated and released from PNH RBC upon complement activation. The microvesicles generated from complement-activated PNH RBC accelerated factor Xa-dependent plasma coagulation more than those generated from RBC by the treatment with ionophore A23187. When assessed by factor Xa-catalysed prothrombin activation, complement activation enhanced procoagulant properties of both normal and PNH RBC similarly, although PNH RBC were lysed but normal RBC were not. This enhancement of factor Xa-dependent prothrombinase activity of complement-activated RBC was inhibited by the treatment of the RBC with annexin V, a protein with binding affinity for anionic phospholipids especially for phosphatidylserine (PS). Neither the enhanced procoagulant properties of RBC nor apparent RBC population with annexin V-binding affinity were demonstrated before complement activation in any of the four PNH patients studied. PS-externalized PNH RBC and microvesicles may contribute to the removal of PNH RBC from the circulation. We conclude that although PNH RBC do not constantly exhibit enhanced procoagulant properties in vivo, complement activation induces a procoagulant alteration of RBC membranes with microvesicle formation, potentially contributing to the thrombogenesis in PNH.

Topics: Annexin A5; Blood Coagulation; Calcimycin; CD59 Antigens; Complement System Proteins; Erythrocyte Membrane; Factor Xa; Hemoglobinuria, Paroxysmal; Hemolysis; Humans

Erythrocytes shed membrane vesicles in response to many stimuli. It has been previously demonstrated that glycan phosphatidylinositol-linked (GPI-linked) proteins such as decay accelerating factor and acetylcholinesterase are concentrated in these vesicles relative to the erythrocyte membrane. We have examined the requirement for GPI-linked proteins for the process of vesiculation. Erythrocytes that do not express GPI-linked proteins, obtained from patients with paroxysmal nocturnal hemoglobinuria (PNH), release between 10% and 50% of the quantity of vesicles as normal cells in response to the Ca2+ ionophore A23187. Platelets from the same patients produced 10% to 20% of the amount of vesicles as normal platelets. In addition, a mutant B-lymphoblastoid cell line that lacks GPI-linked molecules produces about half of the number of vesicles as compared with the wild-type cell line in response to the Ca2+ ionophore. Prior findings indicate that vesiculation is one of the mechanisms that the cell uses to remodel the plasma membrane, as well as protect itself from membrane-damaging agents such as the terminal complement components C5b-9. On the basis of the present results, we conclude that GPI-linked proteins play an important role in membrane vesiculation.

Topics: Acetylcholinesterase; Antibodies, Monoclonal; Antigens, CD; Blood Platelets; Blood Proteins; Calcimycin; Calcium; CD55 Antigens; CD59 Antigens; Cell Death; Cell Line; Erythrocyte Membrane; Erythrocytes; Glycosylphosphatidylinositols; Hemoglobinuria, Paroxysmal; Humans; Membrane Glycoproteins; Membrane Proteins; Reference Values

Glycoinositol phospholipid (GPI) anchor structures derive from sequentially glycosylated inositol phospholipid precursors assembled in the endoplasmic reticulum. To characterize GPI biosynthesis in nontransformed human lymphocytes and to define the GPI synthetic defect underlying deficient expression of GPI-anchored proteins by paroxysmal nocturnal hemoglobinuria (PNH) cells, putative intracellular GPI intermediates were analyzed following [3H]Man labeling of normal and affected lymphocytes. In unstimulated normal peripheral blood lymphocytes, [3H]Man incorporation into GPIs was minimally detectable but after phytohemagglutinin (PHA), allogeneic cell, or anti-CD3 stimulation, assembly of [3H]Man-labeled GPIs markedly increased. Expression of GPIs by prestimulated quiescent PHA blasts could be efficiently induced by phorbol 12-myristate 13-acetate (PMA) and increased by the Ca2+ ionophore A23187 independently of new protein synthesis. Utilizing allogeneically stimulated cells in conjunction with PMA induction, products deriving from [3H]Man labeling of affected CD48- T and natural killer lymphocyte cell lines from five PNH patients were compared to those deriving from unaffected CD48+ cell lines from the same patients or controls. In contrast to unaffected paired control cells, affected cells of all of the patients exhibited a common abnormality in which they assembled dolichol-phosphoryl-Man but failed to express [3H]Man-containing GPIs. These data indicate that 1) significant GPI production in lymphocytes is dependent on prior stimulation of the cells, 2) exposure of lymphocytes to agents which activate protein kinase C induces GPI synthesis, and 3) in five PNH patients affected lymphocytes are uniformly defective in an early GPI biosynthetic step which undermines expression of GPI mannolipids.

Topics: Animals; Calcimycin; Carbohydrate Sequence; Cell Line; Glycosylphosphatidylinositols; Hemoglobinuria, Paroxysmal; Humans; Killer Cells, Natural; Lymphocyte Activation; Mice; Molecular Sequence Data; T-Lymphocytes; Tetradecanoylphorbol Acetate