Inorganic polyphosphate is an abundant component of acidocalcisomes of bacteria and unicellular eukaryotes. Human platelet dense granules strongly resemble acidocalcisomes, and we recently showed that they contain substantial amounts of polyphosphate, which is secreted upon platelet activation. We now report that polyphosphate is a potent hemostatic regulator, accelerating blood clotting by activating the contact pathway and promoting the activation of factor V, which in turn results in abrogation of the function of the natural anticoagulant protein, tissue factor pathway inhibitor. Polyphosphate was also found to delay clot lysis by enhancing a natural antifibrinolytic agent, thrombin-activatable fibrinolysis inhibitor. Polyphosphate is unstable in blood or plasma, owing to the presence of phosphatases. We propose that polyphosphate released from platelets or microorganisms initially promotes clot formation and stability; subsequent degradation of polyphosphate by blood phosphatases fosters inhibition of clotting and activation of fibrinolysis during wound healing.factor V ͉ platelets ͉ tissue factor pathway inhibitor ͉ acidocalcisomes ͉ dense granules P olyphosphate (polyP) is widely distributed in biology, being found in bacteria, fungi, plants, and animals (1). The biologic functions of polyP have been studied most extensively in prokaryotes and unicellular eukaryotes, in which high levels of polyP accumulate in acidic organelles known as acidocalcisomes. PolyP in these organisms can reach chain lengths of several hundred phosphate units. In unicellular organisms, polyP has been shown to play essential roles in stress responses and virulence (1, 2), although its function in higher eukaryotes, including man, has not been extensively investigated. Recently, we reported that dense granules of human platelets strongly resemble acidocalcisomes and contain millimolar levels of polyP (with chain lengths of Ϸ70-75 phosphate units), making acidocalcisomes the only known class of organelle conserved during evolution from bacteria to humans (3). Human platelets each have three to eight dense granules (also called ␦ granules), a type of secretory granule that contains serotonin, ADP, ATP, and PP i in addition to polyP. Patients with dense granule defects exhibit bleeding diatheses, underscoring the role of these secretory granules in hemostasis (4). Platelets contain Ϸ0.74 nmol polyP per 10 8 platelets, which is secreted after stimulation by platelet agonists such as thrombin (3). Therefore, released polyP could readily attain a concentration of 3 M in whole blood, and this could be far higher in platelet-rich thrombi. (Concentrations of polyP are expressed in this paper as phosphate monomer.)The importance of platelets in hemostasis suggested to us that polyP may play an important role in the blood clotting system. In this study, we found that polyP of the size released from activated platelets has a strongly net procoagulant effect, which is exerted at several levels in the clotting͞fibrinolysis system: PolyP activate...

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 ϳ70 -75 phosphate units was identified by 31 P 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 A 1 -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 Ca and 2) the effect of ionomycin, which could not take Ca 2؉ out of acidic organelles and was more effective after alkalinization of this compartment by the previous addition of nigericin, monensin, or NH 4 Cl. 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.

Recent work has shown that acidocalcisomes, which are electron-dense acidic organelles rich in calcium and polyphosphate, are the only organelles that have been conserved during evolution from prokaryotes to eukaryotes. Acidocalcisomes were first described in trypanosomatids and have been characterized in most detail in these species. Acidocalcisomes have been linked with several functions, including storage of cations and phosphorus, polyphosphate metabolism, calcium homeostasis, maintenance of intracellular pH homeostasis and osmoregulation. Here, we review acidocalcisome ultrastructure, composition and function in different trypanosomatids and other organisms.

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