Non-enzymatic quantification of polyphosphate levels in platelet lysates and releasates

Abstract

Inorganic polyphosphate has been shown to be shed upon platelet activation inducing prothrombotic stimuli on the coagulation system. Several methods have been published to detect and quantify polyphosphate in various cells and tissues, but evaluation of platelet content has only been achieved by indirect detection of orthophosphate after enzymatic digestion, thus, relying heavily on specificity of an exopolyphosphatase that is not commercially available.We present a non-enzymatic method for quantification of platelet-derived polyphosphate featuring optimized extraction on silica spin-columns, followed by specific fluorescence detection using DAPI. This allowed us to quantify polyphosphate in platelet lysates, but also in releasates of TRAP-activated platelets for the first time.Extraction of exogenous polyphosphate from buffer and sample matrices resulted in quantitative yields while removing matrix effects observed with direct fluorescence detection. Treatment of eluted fractions with phosphatase completely abrogated polyphosphate-specific fluorescence arguing for no additional compounds influencing the fluorescence detection. This was confirmed by no change in fluorescence intensity in samples previously treated with DNase and RNase.Taken together, we developed a robust and easily standardizable method to quantify polyphosphate in platelet lysates and releasates that will facilitate polyphosphate related investigations of platelet physiology and coagulation.