Size-Controlled Synthesis of Granular Polyphosphate Nanoparticles at Physiologic Salt Concentrations for Blood Clotting

Abstract

Size-controlled granular polyphosphate (PolyP) nanoparticles were synthesized by precipitation in aqueous solutions containing physiological concentrations of calcium and magnesium. We demonstrate using dynamic light scattering (DLS) that the solubility is correlated inversely with PolyP chain length, with very long chain PolyP (PolyP1000+, more than 1000 repeating units) normally found in prokaryotes precipitating much more robustly than shorter chains like those found in human platelet dense granules (PolyP80, range 76–84 repeating units). It is believed that the precipitation of PolyP is a reversible process involving calcium coordination to phosphate monomers in the polymer chain. The particles are stable in aqueous buffer and albumin suspensions on time scales roughly equivalent to catastrophic bleeding events. Transmission electron microscopy images demonstrate that the PolyP nanoparticles are spherical and uniformly electron dense, with a particle diameter of 200–250 nm, closely resembling the content of acidocalcisomes. X-ray elemental analysis further reveals that the P/Ca ratio is 67:32. The granular nanoparticles also manifest promising procoagulant effects, as measured by in vitro clotting tests assaying contact pathway activity.

Degradation and hemostatic properties of polyphosphate coacervates

“…Either some of the precipitated polyphosphate dissolves away and induces its effect in a soluble form, or proteins from plasma directly bind to the precipitated polyphosphate coacervate and activate. In the time frame of the clotting assay used here the latter postulation is more likely, as degradation studies showed that these coacervates are highly stable for 48 h. In agreement with these results, it has recently been shown that calcium polyphosphate nanoparticles accelerate fibrin formation in citrated plasma [18].…”

“…This observation might be an artifact caused by an interaction between the polyphosphate coacervate and phospholipids in the aPTT agent used for these experiments. Prothrombinase complex consisting of Factor Va and Factor Xa assembles on negatively charged phospholipids, and in their absence thrombin formation would be significantly hindered [18]. In vivo and in the whole blood clotting assay, phospholipids are present in large number on platelets and other blood cells, eliminating such an artifact.…”

“…In vivo and in the whole blood clotting assay, phospholipids are present in large number on platelets and other blood cells, eliminating such an artifact. There is also a possibility for some factors in the contact pathway to bind with the polyphosphate coacervate surface, which is presumably negatively charged [18], affecting their potency. Further studies are indeed required to identify the reason for the observed significant delay in aPTT.…”

“…This approach could be debated since, in vivo, platelets release polyphosphates and calcium simultaneously resulting in the formation of polyphosphate precipitates, coacervates or particles; it is therefore essential to determine if calcium polyphosphate precipitates/coacervates are equally as important as their precursor, soluble NaPP, in hemostasis. Recently, Donovan et al considered this postulation using calcium polyphosphate nanoparticles and showed that these nanoparticles are also a potent activator of the contact pathway [18].…”

Clotting Activity of Polyphosphate‐Functionalized Silica Nanoparticles

“…[9] Delivery of a polyP payload on a nanoparticle carrier could potentially be optimized to target the site of injury while minimizing the impact on the systemic circulation. Effective targeting and control would in theory minimize the risk of thrombotic complications that limit procoagulant therapy.…”