Abstract
Polyphosphate (polyP) is released from activated platelets and activates the intrinsic coagulation pathway. However, polyP may also be involved in various pathophysiological functions related to platelets. To clarify these functions, we established a cytochemical method to reproducibly visualize polyP in platelets. Platelets obtained from healthy non-smoking donors were suspended in phosphate-buffered saline and quickly immobilized on glass slides using a Cytospin. After fixation and membrane permeabilization, platelets were treated with 4′,6- diamidino-2-phenylindole (DAPI) and examined using a fluorescence microscope with a blue-violet excitation filter block (BV-2A). Fixed platelets were also subjected to immunocytochemical examination to visualize serotonin distribution. Under the optimized conditions for polyP visualization, immobilized platelets were fixed with 10% neutral-buffered formalin for 4 h or longer and treated with DAPI at a concentration of 10 µg/mL in 0.02% saponin- or 0.1% Tween-20-containing Hanks balanced salt solution as a permeabilization buffer for 30 min at room temperature (22–25 °C). Based on the results obtained by using activated platelets, treatment with alkaline phosphatases, and serotonin release, the DAPI+ targets were identified as polyP. Therefore, this cytochemical method is useful for determining the amount and distribution of polyP in platelets.
Highlights
Regenerative therapy using platelet-rich plasma (PRP) is inspired by the process of wound healing [1,2,3]
Platelets suspended in phosphate-buffered saline (PBS) were immobilized on glass slides and fixed in 10% neutral-buffered formalin for 1, 2, 4, or 18 h
Our results indicated that Triton X-100 at the concentration used (0.1%) may be too strong for surfactant action to retain some important compounds involved in DAPI visualization
Summary
Regenerative therapy using platelet-rich plasma (PRP) is inspired by the process of wound healing [1,2,3] This therapy appears to be primarily dependent on biomolecules, including growth factors, released from platelets [4]. These biomolecules are released upon activation and are immediately entrapped by newly formed fibrin fibers surrounding the platelets and do not diffuse away. Newly formed fibrin fibers activate platelets to further grow a blood clot via a positive feedback loop Taken together, these results indicate that the function of platelets in PRP therapy includes, but is not limited to, their supply of growth factors. Platelets take the initiative to control their growth factor retention and release
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