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 B1, absent in patients with paroxysmal nocturnal hemoglobinuria.Design and Methods: We compared expression of proteinase 3 and neutrophil antigen B1 by flow cytometry and western blotting in normal and paroxysmal nocturnal hemoglobinuria cells and measured cytoplasmic and soluble levels of proteinase 3 by ELISA assay 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: Consequently, we showed that membrane-bound proteinase 3 is deficient in patient 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 but not disease cells. 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 proteases inhibitor enhanced expression of this epitope on protease activated receptor-1 located C-terminal to the thrombin cleavage site on platelets.Conclusions: We demonstrate that glycosylphosphatidyl inositol-anchored proteins deficiency in paroxysmal nocturnal hemoglobinuria results in decreased membrane-bound and soluble proteinase 3 levels. This phenomenon may constitute another mechanism in contributing to a prothrombotic propensity in paroxysmal nocturnal hemoglobinuria.