Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Oct 22;3(20):3092-3098.
doi: 10.1182/bloodadvances.2018020834.

Megakaryocytes Package Contents Into Separate α-Granules That Are Differentially Distributed in Platelets

Affiliations
Free PMC article

Megakaryocytes Package Contents Into Separate α-Granules That Are Differentially Distributed in Platelets

Elisabeth M Battinelli et al. Blood Adv. .
Free PMC article

Abstract

In addition to their primary roles in hemostasis and thrombosis, platelets participate in many other physiological and pathological processes, including, but not limited to inflammation, wound healing, tumor metastasis, and angiogenesis. Among their most interesting properties is the large number of bioactive proteins stored in their α-granules, the major storage granule of platelets. We previously showed that platelets differentially package pro- and antiangiogenic proteins in distinct α-granules that undergo differential release upon platelet activation. Nevertheless, how megakaryocytes achieve differential packaging is not fully understood. In this study, we use a mouse megakaryocyte culture system and endocytosis assay to establish when and where differential packaging occurs during platelet production. Live cell microscopy of primary mouse megakaryocytes incubated with fluorescently conjugated fibrinogen and endostatin showed differential endocytosis and packaging of the labeled proteins into distinct α-granule subpopulations. Super-resolution microscopy of mouse proplatelets and human whole-blood platelet α-granules simultaneously probed for 2 different membrane proteins (VAMP-3 and VAMP-8), and multiple granular content proteins (bFGF, ENDO, TSP, VEGF) confirmed differential packaging of protein contents into α-granules. These data suggest that megakaryocytes differentially sort and package α-granule contents, which are preserved as α-granule subpopulations during proplatelet extension and platelet production.

Conflict of interest statement

Conflict-of-interest disclosure: J.N.T. and J.E.I. have financial interest in and are founders of Platelet BioGenesis, a company that aims to produce donor-independent human platelets from human-induced pluripotent stem cells at scale. They are both inventors on this patent. The interests of J.N.T. and J.E.I. were reviewed and are managed by the Brigham and Women’s Hospital and Partners HealthCare in accordance with their conflict-of-interest policies. R.F. has a private equity interest in Platelet Diagnostics, and he is also a founder and consultant for that company. The interests of R.F. are reviewed and managed by the Beth Israel Deaconess Medical Center Office of Compliance and Business Conduct. The remaining authors declare no competing financial interests.

Figures

None
Figure 1.
Figure 1.
Live cell fluorescence and super-resolution structured illumination microscopy reveal distinct subpopulations of α-granules and VAMPs, in mature mouse MKs. Mouse fetal liver derived MKs were cultured overnight with Alexa 488-fibrinogen (FG488) and Alexa 568-endostatin (ENDO568) or Alexa 488-fibrinogen and Alexa 546-fibrinogen (FG546). (A) Structured illumination microscopy of MKs containing fluorescently tagged ENDO and fibrinogen. The yellow boxes, and their corresponding magnified insets, highlight separation of FG488/ENDO568 in the upper panels and colocalization of FG488/FG546 in the lower panels. Images are maximum intensity projections. For FG488/ENDO568, scale bar is 5 µm for full images, and 2 µm for the inset. For FG488/FG546, scale bar is 10 µm for full images, and 5 µm for inset. (B) Time lapse imaging of a proplatelet containing FG488 and ENDO568. White arrowhead denotes fibrinogen-containing granule; open arrowhead denotes ENDO-containing granule. Images were taken every 5 minutes for 1 hour. Scale bar is 5 µm. (C) Structured illumination microscopy of VAMP-3 (green) and VAMP-8 (red). Note that the proplatelet shown in the insets are circular in structure. Images are maximum intensity projections. Scale bar is 25 µm for full images, and 8 µm for insets. For VAMP-3 and VAMP-8 costaining, the Pearson’s R value is 0.21 ± 0.02. Three proplatelet producing MKs were analyzed.
Figure 2.
Figure 2.
Super-resolution structured illumination and immunogold electron microscopy reveals distinct α-granule subpopulations in mouse proplatelets and human platelets. Mouse fetal liver derived MKs were isolated with a BSA gradient. Released mouse proplatelets were stained with antibodies against bFGF, ENDO, TSP, and VEGF and imaged with structured illumination microscopy. (A) A single slice of a composite stack of a released proplatelet. Scale bar is 10 µm. Magnified images show proplatelet shaft (1) and tip (2). (B) Distribution of α-granule cargo proteins were determined by overlaying different staining combinations. Scale bar is 1 µm. (C) Full image of proplatelet tip (left). White boxes highlight granules of distinct protein cargo, magnified on right. Scale bar is 1 µm. (D) Human platelets were probed with antibodies against bFGF, ENDO, TSP, and VEGF and imaged with structured illumination microscopy. Images are maximum intensity projections. Scale bar is 2 µm. (E) Double immunogold labeling of human platelet sections using anti-ENDO and antifibrinogen primary antibodies followed by staining with protein A–gold conjugated to 10 nm (fibrinogen; red arrows) or 15 nm (ENDO; white arrows). Scale bar is 100 nm.

Similar articles

See all similar articles

Publication types

Feedback