Inverse immunostaining pattern for synthesized versus endocytosed alpha-granule proteins in human bone marrow megakaryocytes

The time of appearance and pattern of expression of several α-granule proteins, von Willebrand factor (VWF), fibrinogen and immunoglobulins (Ig) were examined and compared in human bone marrow megakaryocytes (MK) using an immunocytochemical approach. VWF is synthesized by immature MK, whereas it has been shown that fibrinogen is incorporated from the plasma into α-granules. The present study was undertaken in order to determine whether there are chronological and morphological differences in the expression of VWF and fibrinogen in vivo in human MK. Seven paraffin-embedded biopsies of normal human bone marrow were labelled with specific antibodies for VWF and for fibrinogen, detected by the alkaline phosphatase anti-alkaline phosphatase (APAAP) method, and analysed by immunomorphometry. We found a clear, statistically significant, difference in the labelling pattern of VWF and fibrinogen. The expression of other endocytosed α-granule proteins, immunoglobulins G and A, was therefore studied in bone marrow MK from two patients with multiple myeloma, one with monoclonal IgG and one with monoclonal IgA. The immunostaining pattern was similar to that of fibrinogen and different from VWF, and characteristic of endocytosed α-granule proteins. This study demonstrates that: (i) immunohistochemical staining of MK α-granules proteins distinguishes the peripheral cockade distribution pattern of endocytosed protein from the perinuclear pattern of endogenously synthesized proteins; (ii) VWF is present in human bone marrow MK when fibrinogen is not yet detectable; (iii) VWF synthesis ceases while fibrinogen is still being incorporated; (iv) immunoglobulins can be detected in MK cytoplasm, with a staining pattern resembling that of fibrinogen.