MEGAKARYOCYTES FROM THE MARROW OF A PATIENT WITH BERNARD-SOULIER SYNDROME LACKED GP Ib AND WERE DEFICIENT IN GP IX

Abstract

Although it is recognized that glycoprotein (GP) Ib/IX complexes are deficient in the platelets of patients with the Bernard-Soulier syndrome (BSS), the nature of the genetic defect remains unknown. We have looked for these GPs in permeabilized megakaryocytes (MK) of a BSS patient employing immunofluorescence (IF) or immunocytochemical procedures combined with electron microscopy. The study involved the use of monoclonal antibodies AP-1 (anti-GP Ib), AP-2 (anti-GP IIb-IIIa) and FMC 25 (anti-GP IX), gifts from Drs. T. Kunicki and M. Berndt respectively. Bound IgG were revealed by biotinylated anti-mouse IgG followed by Texas Red-streptavidin and MK identified in IF by a double-staining procedure using a polyclonal antibody to fibrinogen (Fg). Platelet morphology was typical of BSS with a high percentage of "giant" platelets. Flow cytometry confirmed that platelets of all sizes were negative for AP-1 and FMC 25 but normally bound AP-2. MK from a marrow aspirate obtained by sternal puncture were concentrated on a Percoll gradient. Electron microscopy showed the MK to be of normal size with a normal granule distribution. However, a striking feature was an irregular distribution of the demarcation membranes which often had a vacuolar appearance. Whereas all permeabilized MK from normal individuals were strongly fluorescent with AP-2, AP-1 and FMC 25, those from the BSS patient were negative for AP-1, weakly positive for FMC 25 and normal for AP-2. Incubation of intact cells with AP-1, with bound antibody located by anti-IgG bound to gold particles, confirmed the absence of GP lb from the surface membranes of BSS MK. Our results show that the platelet membrane GP defect in BSS results from an abnormal synthesis and/or stability of both GP lb and GP IX in the MK.