Ultrastructural demonstration of tubular inclusions coinciding with von Willebrand factor in pig megakaryocytes

Abstract

The appearance of von Willebrand factor (vWF) in bone marrow megakaryocytes was studied by standard electron microscopy (EM) and immuno-EM using an original purification technique. Eighty percent pure megakaryocytes were isolated from porcine rib bone marrow using Percoll gradients followed by counterflow centrifugation. Activation was prevented by prostacyclin and prefixation with low concentrations of glutaraldehyde. In early megakaryoblasts, standard EM revealed the presence of tubular structures in the small vesicles located in the Golgi area, in the small immature alpha-granules and in the rare mature alpha-granules. Immunolabeling for vWF was simultaneously observed in small vesicles and small alpha-granules, mainly in the Golgi zone. In mature megakaryocytes, standard EM showed that tubular structures were numerous, regularly spaced, and aligned in parallel. Immunolabeling for vWF was intense, restricted to the alpha-granules, and distributed in a similar manner to porcine platelets. Gold particles were located eccentrically at one pole of the alpha-granule, labeling only its periphery or outlining one side of an elongated granule. Tubule profiles could be seen underlying the immunolabeling and were usually located at one side of the granule. In conclusion, this study demonstrates the presence of tubular structures in megakaryocyte alpha- granules, their association with vWF, and the appearance of both in the Golgi-associated vesicles.

Von Willebrand Disease: From In Vivo to In Vitro Disease Models

“…5 Platelet-secreted VWF constitutes 20% of the total VWF protein and is enriched in VWF HMWMs. 6,7 When WPBs fuse with the endothelial membrane, the tubulated VWF multimers uncoil, and are released as long strings into the circulation. These ultralarge VWF multimers are proteolyzed by the enzyme ADAMTS13 into smaller subunits and circulate as coiled inactive VWF units, which are activated by vascular damage.…”

“…Moving through the Golgi system, the propeptide is cleaved and multimers will form, before being either secreted constitutively as low molecular weight multimers (LMWMs) or packed as high molecular weight multimers (HMWMs) in the α‐granules in megakaryocytes or in a tubular conformation into the WPBs of endothelial cells 5 . Platelet‐secreted VWF constitutes 20% of the total VWF protein and is enriched in VWF HMWMs 6,7 …”