Ultrastructural analysis of the distribution of the vitronectin receptor (α(v)β3) in human platelets and megakaryocytes reveals an intracellular pool and labelling of the α-granule membrane

Abstract

The vitronectin receptor (VnR or α(v)β3) belongs to the cytoadhesin subclass of the integrin family. This subclass consists of two receptors which have the β3 subunit in common: GP IIb-IIIa complexes (or α(IIb)β3) and VnR. We report the subcellular distribution of VnR within human platelets as determined by immunogold staining of ultrathin frozen sections and transmission electron microscopy. Monoclonal antibodies directed against: (i) the α(v) subunit (LM142, AMF7, CLB-706), or (ii) an epitope specific to the complex (LM609) were used. Although VnR is present on platelets, it is a minor component. We therefore first compared several different staining procedures to detect this integrin. Optimal localization of VnR was obtained using a multistep procedure in which biotinylated anti-mouse IgG and a monoclonal anti-biotin antibody provided staining enhancement. Results showed that although present on the surface, α(v)β3 was mostly detected in internal membrane systems including those of α-granules. Occasionally, platelet sections showed special vesicular structures covered by gold particles. These were often localized at the edge or immediately under the plasma membrane and their origin remains unclear. An internal pool of α(v)β3 was confirmed by flow cytometry and by using platelets from a patient with type I Glanzmann's thrombasthenia arising from a GP IIb gene defect. We also investigated the presence of VnR in megakaryocytes (MR) obtained from normal human bone marrow. A fluorescence study showed VnR in small MK with unilobulated nuclei, suggesting that synthesis of this integrin occurs early during megakaryocytopoiesis. In mature cells, VnR expression had decreased relative to GP IIb-IIIa, although intracellular staining was present in EM and α-granules were again labelled.