Loss of expression of neutrophil proteinase-3: a factor contributing to thrombotic risk in paroxysmal nocturnal hemoglobinuria

Background A deficiency of specific glycosylphosphatidyl inositol-anchored proteins in paroxysmal nocturnal hemoglobinuria may be responsible for most of the clinical features of this disease, but some functional consequences may be indirect. For example, the absence of certain glycosylphosphatidyl inositol-anchored proteins in paroxysmal nocturnal hemoglobinuria cells may influence expression of other membrane proteins. Membrane-bound proteinase 3 co-localizes with glycosylphosphatidyl inositol-linked neutrophil antigen 2a, which is absent in patients with paroxysmal nocturnal hemoglobinuria. Design and Methods We compared expression of proteinase 3 and neutrophil antigen 2a by flow cytometry and western blotting in normal and paroxysmal nocturnal hemoglobinuria cells and measured cytoplasmic and soluble levels of proteinase 3 by enzyme-linked immunosorbent assays in controls and patients with paroxysmal nocturnal hemoglobinuria. Finally, we studied the effects of proteinase 3 on platelet activation using an in vitro aggregometry assay and flow cytometry. Results We showed that membrane-bound proteinase 3 is deficient in patients' cells, but invariantly present in the cytoplasm regardless of disease phenotype. When we isolated lipid rafts from patients, both molecules were detected only in the rafts from normal cells, but not diseased ones. Membrane-bound proteinase 3 was associated with a decrease in plasma proteinase 3 levels, clone size and history of thrombosis. In addition, we found that treating platelets ex vivo with proteinase 3, but not other agonists, decreased the exposure of an epitope on protease activated receptor-1 needed for thrombin activation. Conversely, treatment of whole blood with serine protease inhibitor enhanced expression of this epitope on protease activated receptor-1 located C-terminal to the thrombin cleavage site on platelets. Conclusions We demonstrated that deficiency of glycosylphosphatidyl inositol-anchored proteins in paroxysmal nocturnal hemoglobinuria results in decreased membrane-bound and soluble proteinase 3 levels. This phenomenon may constitute another mechanism contributing to a prothrombotic propensity in patients with paroxysmal nocturnal hemoglobinuria.