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

Abstract

Polyphosphate (polyP), a biopolymer of inorganic phosphate, is widely distributed in living organisms. In platelets, polyP is released upon activation and plays important roles in coagulation and tissue regeneration. However, the lack of a specific quantification method has delayed the in-depth study of polyP. The fluorescent dye 4′,6-diamidine-2-phenylindole dihydrochloride (DAPI) has recently received attention as a promising probe for the visualization and quantification of cellular polyP levels. In this study, we further optimized quantification conditions and applied this protocol in quantification of platelet polyP levels in a Japanese population. Blood samples were collected from non-smoking, healthy Japanese subjects (23 males, 23 females). Washed platelets were fixed and probed with DAPI for fluorometric determination. PolyP levels per platelet count were significantly higher in women than that in men. A moderate negative correlation between age and polyP levels was found in women. Responsiveness to CaCl2 stimulation was also significantly higher in women than that in men. Overall, our optimized protocol requires neither purification nor degradation steps, reducing both the time and bias for reproducible quantification. Thus, we suggest that despite its low specificity, this DAPI-based protocol would be useful in routine laboratory testing to quantify platelet polyP levels efficiently and economically.

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“…It is important to evaluate the expression of platelet activation markers, such as CD62P, to confirm the release of polyP by activated platelets. However, to stabilize polyP levels, it is necessary to incubate blood samples at least overnight at 22-25˚C ( 30 ). Furthermore, it was difficult to normalize the venipuncture skill, which can easily influence platelet conditions; thus, platelet activation was not evaluated in the present study.…”

“…Prior to centrifugation, 0.5 ml ACD-A (Terumo) was added to each blood sample, then the samples were incubated for 10 min to prevent unexpected coagulation, which is often observed in female blood samples ( 30 ). Platelets were collected using the dual-spin protocol of pure-platelet rich plasma (PRP) preparation (1st soft spin: 472 x g for 10 min; 2nd hard spin: 578 x g for 5 min, at 22-25˚C).…”

“…After storage at 4˚C for 24 h, the fixed platelets were centrifuged at 578 x g for 5 min at 22-25˚C, after which the supernatants were carefully aspirated. The resulting platelet pellets were gently suspended in Milli-Q water, as previous observations found that Milli-Q water does not increase background of fluorescence signal quantification, and enables DAPI to invade the cytoplasm without perforation ( 30 ). Platelets were counted using an automated hematology analyzer and then adjusted to the appropriate range (<4.5x10 7 /ml) for polyP quantification.…”

“…DAPI (Dojindo Molecular Technologies, Inc.) was added to each tube at a concentration of 4 µg/ml and incubated for 30 min at 22-25˚C. Fluorescence intensity was measured using a fluorometer (FC-1; Tokai Optical Co., Ltd.) with excitation and emission wavelengths of 425 and 525 nm, respectively ( 30 ).…”

“…Due to the lack of specific probes, it is difficult to accurately quantify platelet polyP levels in an efficient manner. Therefore, our methods are modeled using previous studies ( 30 , 32 , 33 ) showing that the right shift of excitation and emission wavelengths (350 and 470 nm) from those wavelengths used for DNA examination increases the specificity of DAPI against polyP, with some modifications. Specifically, quantification and visualization of platelet polyP using DAPI was optimized through the determination of the optimum light wavelengths for fluorescence, as well as for sample fixation and choice of the aqueous solution.…”