Fluorescent Cytochemical Detection of Polyphosphates Associated with Human Platelets

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.

Fluorometric Quantification of Human Platelet Polyphosphate Using 4′,6-Diamidine-2-phenylindole Dihydrochloride: Applications in the Japanese Population

“…Regarding possible degradation by phosphatases, we did not find that orthovanadate (1 mM) significantly increased polyP values. As a result, the exogenous addition of 0.1% CaCl 2 to the platelets reduced platelet polyP levels in a concentration-dependent manner, and the amount of polyP remaining in the platelets approximately corresponded to the cytochemical visualization data shown in the present and previous studies [17].…”

“…Growth factors and fibrinogen contained in platelets are major factors involved in tissue regeneration. In a previous study [ 17 ], however, we confirmed that activated platelets secrete polyP along with the growth factors [ 26 ]. If polyP has a potent ability in tissue repair and regeneration [ 10 , 27 ], we expect PCs containing significant amounts of polyP to be applicable for regenerative therapy, as shown previously [ 28 ], and hypothesized that polyP may be utilized as a marker of the quality of PCs, i.e., a marker of their predictable clinical outcomes.…”

“…PolyP stored in and released from platelets was visualized using DAPI [ 17 , 52 ]. Briefly, the control and activated platelets were immobilized on glass slides using a Cytospin 4 cytocentrifuge (Thermo Fisher Scientific, Waltham, MA, USA) and fixed with 10% neutral buffered formalin for 24 h. After washing with PBS, the platelets were stained with PBS containing 0.1% Tween-20 (FUJIFILM Wako Pure Chemical, Osaka, Japan) and 10 μg/mL DAPI in the presence of Phalloidin-iFluor 555 (1:200 dilution) (Abcam, Cambridge, UK) for 30 min.…”

“…Therefore, at present, DAPI is superior to other dyes in terms of commercial availability, sensitivity, and specificity. In a previous cytochemical study, we demonstrated that the optimized DAPI-staining protocol can be used to easily visualize the distribution of polyP in platelets and the extracellular spaces in a semi-quantitative manner [ 17 ]. In contrast, due to the need for extraction and purification, techniques for polyP quantification, such as RAMAN microscopy and enzyme assays, have not been accepted as routine laboratory methodologies [ 12 ].…”

Platelet polyphosphate and SARS-Cov-2 mRNA-vaccine-induced inflammatory side effects: a pilot study

“…Due to the lack of specific probes, it is difficult to accurately quantify platelet polyP levels in an efficient manner. In previous studies, 17,18 we optimized the previous methods, especially those related to sample preparation and the parameter of light wavelengths in fluorescent determination, for quantification and visualization of platelet polyP using DAPI. Although not completely excluding the possible count of DAPI-bound, non-polyP substances, this method enables rapid quantification of polyP levels without sacrificing recovery or reproducibility.…”