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

Autor: Taisuke Watanabe, Yutaka Kitamura, Hachidai Aizawa, Hideo Masuki, Tetsuhiro Tsujino, Atsushi Sato, Hideo Kawabata, Kazushige Isobe, Koh Nakata, Tomoyuki Kawase
Jazyk: angličtina
Rok vydání: 2021
Předmět:
Zdroj: International Journal of Molecular Sciences, Vol 22, Iss 14, p 7257 (2021)
Druh dokumentu: article
ISSN: 1422-0067
1661-6596
38872129
DOI: 10.3390/ijms22147257
Popis: 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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