Detection of PrPc on the surfaces of activated CD55⁻ platelets of PNH patients.

(A) PrPc molecule schematic showing the location of mAb epitopes. Monoclonal antibodies FH11 and 1562 bind to sites on the N-terminal part of PrPc from a hydrophobic putative transmembrane segment (TM1), whereas 6H4 antibody binds to the C-terminal part of PrPc. (B) Two-color flow cytometry analysis of PrPc mAb binding to resting and TRAP-activated platelets of a PNH patient. CD55 antibody detected 2 platelet populations, CD55⁺ normal (11%) and CD55⁻ PNH platelets (89%). Resting CD55⁺platelets displayed low levels of PrPc surface expression, but most resting CD55⁻ platelets (depicted in gray) were negative for PrPc. Activation of platelets led to up-regulation of PrPc not only on CD55⁺ but surprisingly also on CD55⁻platelets. PrPc up-regulation on CD55⁻ platelets, in contrast to CD55⁺ platelets, could not be detected by mAb 6H4, demonstrating that the C-terminal part of the molecule was not accessible for binding. Similar results were obtained in all PNH patients evaluated (n = 8). (C) Western blot analysis of PrPc expression in CD55⁻ platelets of PNH patients. Immunoblots were developed using mAb 6H4 without (A) or in the presence of (B) competing peptide. Lane 1, normal platelets; lanes 2 and 3, platelets of 2 PNH patients with major PNH platelet clone (more than 95% of platelets were CD55⁻). PrPc present in PNH platelets contained the 6H4 epitope and exhibited slightly lower molecular weight than PrPc on normal platelets.