An improved method for the isolation of dense storage granules from human platelets

Area of Science:

• Biochemistry
• Cell Biology
• Hematology

Background:

• Platelets play a crucial role in hemostasis and thrombosis.
• Dense granules within platelets store key signaling molecules like serotonin and nucleotides.
• Understanding the composition and metabolic state of dense granule contents is vital for platelet function research.

Purpose of the Study:

• To isolate and characterize the dense storage granules from human platelets.
• To quantify the concentrations of serotonin, adenosine triphosphate (ATP), and adenosine diphosphate (ADP) within these granules.
• To determine the metabolic activity and origin of nucleotides stored in dense granules.

Main Methods:

• Human platelets were pretreated with metabolic inhibitors (rotenone, 2-deoxyglucose).
• French press homogenization was used to isolate dense storage granules.
• Concentrations and specific radioactivities of serotonin, ATP, and ADP were measured in isolated granules and platelet homogenates.

Main Results:

• Dense granules were isolated with approximately 20% yield.
• Serotonin showed 40-60 fold enrichment in dense granules compared to homogenate.
• ATP and ADP were 40-fold enriched, with an ATP to ADP ratio of 0.68-0.70, matching secreted nucleotides.
• Specific radioactivity of granule ATP and ADP was negligible, indicating they are not newly synthesized.

Conclusions:

• The isolated dense granules contain significantly enriched levels of serotonin, ATP, and ADP.
• The ATP and ADP stored in dense granules are metabolically inactive and share the same ratio as secreted nucleotides.
• These findings support the conclusion that dense granule nucleotides are pre-synthesized and not derived from the metabolically active intracellular pool.