Platelet dense granules begin to selectively accumulate mepacrine during proplatelet formation

Area of Science:

• Hematology
• Cell Biology
• Platelet Biology

Background:

• Platelet dense granules (DGs) store essential molecules like calcium, ADP, and serotonin, crucial for hemostasis.
• DGs are traditionally believed to mature fully within megakaryocytes (MKs) before platelet release.

Purpose of the Study:

• To investigate the maturation process of platelet dense granules (DGs) in megakaryocytes (MKs).
• To challenge the existing notion of DG maturation occurring entirely within MKs prior to platelet formation.

Main Methods:

• Utilized vital fluorescent dyes, including mepacrine and LysoTracker, for microscopy-based discrimination of cellular compartments.
• Examined DG labeling in isolated primary mouse platelets, murine fetal liver-derived MKs, human CD34+ cell-derived MKs, and megakaryocytoid cell lines (MEG-01, G1ME2).
• Assessed mepacrine labeling sensitivity to proton ATPase inhibitors in both MKs and platelets.

Main Results:

• In platelets, mepacrine-labeled DGs were distinct from highly acidic compartments (lysosomes) and endolysosomes/alpha granules.
• In MKs, mepacrine labeling significantly overlapped with LysoTracker and DQ™ BSA labeling, indicating accumulation in endolysosomes.
• Mepacrine labeling in MKs was sensitive to proton ATPase inhibitors, unlike in platelets, suggesting different retention mechanisms.

Conclusions:

• Platelet dense granules (DGs) do not fully mature within megakaryocytes (MKs); a distinct maturation step occurs later.
• Mepacrine accumulates in endolysosomes in MKs, but is likely pumped into or retained in separate DGs in platelets.
• Evidence suggests DG maturation initiates in proplatelets during the final stages of MK differentiation.