Phosphoramidate End Labeling of Inorganic Polyphosphates: Facile Manipulation of Polyphosphate for Investigating and Modulating Its Biological Activities

Abstract

Polyphosphates, linear polymers of inorganic phosphates linked by phosphoanhydride bonds, are widely present among organisms and play diverse roles in biology, including functioning as potent natural modulators of the human blood clotting system. However, studies of proteinpolyphosphate interactions are hampered by a dearth of methods for derivatizing polyphosphate or immobilizing it onto solid supports. We now report that EDAC (1-ethyl-3-[3-dimetyhlaminopropyl]carbodiimide) efficiently promotes the covalent attachment of a variety of primary aminecontaining labels and probes to the terminal phosphates of polyphosphates via stable phosphoramidate linkages. Using 31 P NMR, we confirmed that EDAC-mediated reactions between primary amines and polyphosphate results in phosphoramidate linkages with the terminal phosphate groups. We show that polyphosphate can be biotinylated, labeled with fluorophores and immobilized onto solid supports; that immobilized polyphosphate can be readily used to quantify protein binding affinities; that covalently derivatized or immobilized polyphosphate retains its ability to trigger blood clotting; and that derivatizing the ends of polyphosphate with spermidine protects it from exopolyphosphatase degradation. Our findings open up essentially the entire armamentarium of protein chemistry to modifying polyphosphate, which should greatly facilitate studies of its biological roles.Inorganic polyphosphate (polyP), a linear polymer of orthophosphate residues linked via phosphoanhydride bonds, is widely distributed in biology and plays important and diverse roles in nature (1,2). We recently showed that polyP is a potent modulator of the blood clotting cascade (3)(4)(5), and an expanding body of research is investigating its roles in other biological systems (6-11). Many technical obstacles remain, for investigating the biological roles of polyP and there is a real need for improved microscale methods for analyzing polyP. In particular, there is a dearth of approaches for covalently modifying polyP or attaching it to solid supports. One of the few published methods for immobilizing polyP onto surfaces is via Lewis acid/base interactions between polyP and zirconia beads (12). Although we have successfully used this method (13), it suffers from relatively high nonspecific binding of proteins to zirconia. Furthermore, this chemistry is not readily adaptable for attaching labels to polyP, or to immobilizing polyP onto the sorts of solid supports routinely used in analyses † This work was supported by grant R01 HL47014 from the National Heart, Lung and Blood institute to J.H.M., grant R01 GM75937 from the National Institute of General Medical Sciences to C.M.R., and postdoctoral fellowship grant 0920045G from the American Heart Association to R.L. Immobilization of polyP onto Polystyrene Microplate Wells and Coagulometer CuvettesA variety of reaction conditions were tested in order to optimize EDAC-mediated covalent coupling of polyP HMW to primary amines displayed on Amine Surface stripwe...

Polyphosphate: an ancient molecule that links platelets, coagulation, and inflammation

“…PolyP is also a highly anionic polymer, and we have shown that it binds tightly to the proteins responsible for initiating the contact pathway. 5,27,71 Furthermore, activation of clotting via polyP has a bell-shaped concentration dependence, consistent with the idea that polyP serves as a template for assembling multiple contact factors. 5,27 The ability of polyP to trigger the contact pathway exhibits a profound dependence on polymer length, with optimal specific activities requiring very long polyP polymers ( Figure 3A).…”

“…Recently, however, we identified reaction conditions under which the terminal phosphates of polyP can be made to enter into stable phosphoramidate linkages with almost any primary amine-containing compound, using the zero-length coupling reagent, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide. 71 We have successfully used this coupling chemistry to covalently attach probes, such as biotin, fluorophores, and polyamines, to the terminal phosphates of polyP, and we have successfully coupled polyP to a variety of solid supports. 71 This opens up a number of possibilities, including: identifying polyP-binding proteins in biologic samples, developing high-throughput screens for polyP inhibitors, using biotin-or fluorophore-tagged polyP to visualize polyP in vitro and in vivo (including flow cytometry), and developing therapeutics based on coupling polyP to solid supports (such as wound dressings) or to targeting molecules or nanoparticles.…”

“…29 PolyP binds to both thrombin and factor XI, and factor XI activation by thrombin exhibits a bell-shaped concentration dependence on polyP, consistent with a template-based mechanism. 5,26,29,71 These studies therefore show that platelet polyP is a potent, natural cofactor for factor XI activation by thrombin, explaining how factor XI functions in normal hemostasis independent of factor XII. …”

Polyphosphate colocalizes with factor XII on platelet-bound fibrin and augments its plasminogen activator activity

“…PolyP 70 was bound to Sepabeads as described previously 36 and incubated with 5 mg of FXII, aFXIIa, bFXIIa, or plasminogen. Flow-through material and subsequent low-salt (50 mM NaCl) and high-salt (1 M NaCl) washes were collected, and fractions were analyzed by western blotting as described previously 32 using horseradish peroxidase-conjugated goat anti-human FXII or horseradish peroxidase-conjugated goat anti-human plasminogen (both Enzyme Research Laboratories).…”

“…This is indicative of an electrostatic interaction with the polymer, as previously shown for other proteins. 30,34,36 Plasminogen circulates in 2 forms: the predominant Glu-plasminogen is described as the "closed" conformation, whereas the truncated Lys-plasminogen, cleaved at the C-terminus by plasmin, is in an "open" conformation and exhibits a shorter half-life. Enhanced binding of Lys-plasminogen to fibrin 38 and the activators tPA and uPA results in more rapid plasmin generation.…”