Mechanism of platelet α-granule biogenesis: study of cargo transport and the VPS33B-VPS16B complex in a model system

Platelet alpha-granules play important roles in platelet function. They contain hundreds of proteins that are synthesized by the megakaryocyte or taken up by endocytosis. The trafficking pathways that mediate platelet alpha-granule biogenesis are incompletely understood, especially with regard to cargo synthesized by the megakaryocyte. Vacuolarprotein sorting 33B (VPS33B) and VPS16B are essential proteins for alpha-granule biogenesis, but they are largely uncharacterized. Here, we adapted a powerful method to directly map the pathway followed by newly synthesized cargo proteins to reach alpha-granules. Using this method, we revealed the recycling endosome as a key intermediate compartment in alpha-granule biogenesis. We then used CRISPR/Cas9 gene editing to knock out VPS33B in pluripotent stem cell-derived immortalized megakaryocyte cells (imMKCLs). Consistent with the observations in platelets from patients with VPS33B mutation, VPS33B-knockout (KO) imMKCLs have drastically reduced levels of alpha-granule proteins platelet factor 4, von Willebrand factor, and P-selectin. VPS33B and VPS16B form a distinct and small complex in imMKCLs with the same hydrodynamic radius as the recombinant VPS33B-VPS16B heterodimer purified from bacteria. Mechanistically, the VPS33B-VPS16B complex ensures the correct trafficking of alpha-granule proteins. VPS33B deficiency results in alpha-granule cargo degradation in lysosomes. VPS16B steady-state levels are significantly lower in VPS33B-KO imMKCLs, suggesting that VPS16B is destabilized in the absence of its partner. Exogenous expression of green fluorescent protein-VPS33B in VPS33B-KO imMKCLs reconstitutes the complex, which localizes to the recycling endosome, further defining this compartment as a key intermediate in alpha-granule biogenesis. These results advance our understanding of platelet alpha-granule biogenesis and open new avenues for the study of these organelles.