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J. Biol. Chem., Vol. 279, Issue 43, 44250-44257, October 22, 2004

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

Felix A. Ruiz, Christopher R. Lea¶, Eric Oldfield¶, and Roberto Docampo||

From the Laboratory of Molecular Parasitology, Department of Pathobiology and Center for Zoonoses Research, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802 and the ^¶Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Inorganic polyphosphate (polyP) has been identified and measured in human platelets. Millimolar levels (in terms of P_i residues) of short chain polyP were found. The presence of polyP of 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.

Received for publication, June 4, 2004 , and in revised form, July 14, 2004.

^* This work was supported in part by the National Institutes of Health Grants AI23259 (to R. D.) and GM50694 (to E. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Unidad de Investigacion, Hospital Universitario "Puerta del Mar," Ave. Ana de Viya 21, 11009 Cadiz, Spain.

^|| To whom correspondence should be addressed at: Dept. of Pathobiology and Center for Zoonoses Research, Laboratory of Molecular Parasitology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 2001 S. Lincoln Avenue, Urbana, IL 61802. Tel.: 217-333-3845; Fax: 217-244-7421; E-mail: rodoc{at}uiuc.edu.

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