Abstract
Abstract 1138
Inorganic polyphosphates (polyP) are negatively charged, linear phosphate polymers that are abundant in platelet dense granules and secreted upon platelet activation. We recently reported that polyP may be the long-sought (patho)physiologic activator of the contact pathway with important roles in inflammation and thrombosis. We also reported that polyP opposes the action of many anticoagulant drugs and thus has potential as a general procoagulant agent to treat bleeding. Studies of the role of polyP in blood clotting would be facilitated by being able to covalently attach probes – including fluorophores and biotin – to the ends of polyP. For therapeutic applications, it would also be advantageous to covalently immobilize polyP onto solid supports like collagen sponges and wound dressings. We now report that a wide variety of primary amine-containing compounds can be covalently attached to the terminal phosphates of polyP via phosphoramidate linkages. This allows essentially the full armamentarium of protein chemistry to be employed in modifying polyP.
We have developed and optimized reaction conditions under which EDAC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide) efficiently promotes the covalent coupling of compounds with primary amines to polyP via the formation of stable phosphoramidate linkages with the terminal phosphate groups (see figure).
Using 31P NMR, we have confirmed that EDAC-mediated reaction between primary amines and polyP results in stable phosphoramidate linkages with the terminal phosphate groups. We have used this chemistry to efficiently float polyP onto amine-derivatized microtiter plates and chromatography beads, and have used this presentation of polyP to quantify the binding affinities of thrombin, kallikrein, and factor XIa for polyP. We have also successfully attached fluorescent probes to the termini of polyP and thereby visualized the incorporation of polyP into fibrin clots. We have also demonstrated that polyP covalently attached to solid supports via phosphoramidate linkages retains potent procoagulant activity. And finally, we have found that attaching small organic molecules to the terminal phosphates of polyP protects polyP from degradation by exopolyphosphatases such as alkaline phosphatase, which should prolong its in vivo half-life considerably. These findings facilitate more extensive studies of the biological role(s) of polyP, as well as development of enhanced polyP-based treatments for bleeding.
Smith:University of Illinois: Patents & Royalties. Morrissey:University of Illinois: Patents & Royalties.
