Human Platelet Dense Granules Contain Polyphosphate and Are Similar to Acidocalcisomes of Bacteria and Unicellular Eukaryotes

TLDR

Inorganic polyphosphate has been identified in human platelets, and the dense granules’ acidity, density, and polyP content resemble acidocalcisomes of bacteria and unicellular eukaryotes. PolyP of about 70–75 phosphate units was detected by 31P NMR and gel electrophoresis, and its preferential localization to platelet dense granules was confirmed by gradient centrifugation. Platelets contain millimolar amounts of short‑chain polyP that localizes to dense granules, which also store calcium, potassium, and enzymatic activities, and release polyP, pyrophosphate, and serotonin upon thrombin stimulation, indicating evolutionary conservation of acidocalcisome‑like organelles.

Abstract

Inorganic polyphosphate (polyP) has been identified and measured in human platelets. Millimolar levels (in terms of Pi residues) of short chain polyP were found. The presence of polyP of approximately 70-75 phosphate units was identified by 31P NMR and by urea-polyacrylamide gel electrophoresis of platelet extracts. An analysis of human platelet dense granules, purified using metrizamide gradient centrifugation, indicated that polyP was preferentially located in these organelles. This was confirmed by visualization of polyP in the dense granules using 4',6-diamidino-2-phenylindole and by its release together with pyrophosphate and serotonin upon thrombin stimulation of intact platelets. Dense granules were also shown to contain large amounts of calcium and potassium and both bafilomycin A1-sensitive ATPase and pyrophosphatase activities. In agreement with these results, when human platelets were loaded with the fluorescent calcium indicator Fura-2 acetoxymethyl ester to measure their intracellular Ca2+ concentration ([Ca2+]i), they were shown to possess a significant amount of Ca2+ stored in an acidic compartment. This was indicated by the following: 1) the increase in [Ca2+]i induced by nigericin, monensin, or the weak base, NH4Cl, in the nominal absence of extracellular Ca2 and 2) the effect of ionomycin, which could not take Ca2+ out of acidic organelles and was more effective after alkalinization of this compartment by the previous addition of nigericin, monensin, or NH4Cl. All of these characteristics of the platelet dense granules, together with their known acidity and high density (both by weight and by electron microscopy), are similar to those of acidocalcisomes (volutin granules, polyP bodies) of bacteria and unicellular eukaryotes. The results suggest that acidocalcisomes have been conserved during evolution from bacteria to humans.