Evidence that differential packaging of the major platelet granule proteins von Willebrand factor and fibrinogen can support their differential release.

Abstract

von Willebrand factor (VWF) and fibrinogen are major storage proteins of platelet alpha-granules. VWF is synthesized by the megakaryocyte, the cell from which platelets bud, and fibrinogen is delivered to alpha-granules by endocytosis. Considering biosynthetic origins, VWF and fibrinogen might be differentially packaged within platelets. We applied immunofluorescence microscopy to provide whole platelet, global information on the distributions of VWF and fibrinogen. The distribution of VWF and fibrinogen were characterized in both the resting state and handling activated human platelets. Full cell volume image stacks were collected by spinning-disk confocal microscopy, corrected for a small pixel shift between green and red channels, deconvolved, and visualized in a three-dimensional space. In sum, we found that there was little overlap in the distribution of VWF and fibrinogen in resting state platelets. In an important control, the distributions of green and red secondary antibodies overlapped completely when different color secondary antibodies directed against the same first antibody were used. Moreover, the same result was observed using different first antibodies and switching second antibody color to switch the color of VWF and fibrinogen staining. No accumulation of fibrinogen in late endosomes or lysosomes was detected by co-staining with LAMP2, a late endosome/lysosome membrane protein. Significantly, we found that in handling activated platelets there was differential retention of fibrinogen-positive granules relative to VWF positive granules. Our results indicate that VWF and fibrinogen are differentially packaged in human platelets. Moreover, the results suggest that differential packaging could support differential release of alpha-granule proteins.