Novel Polyphosphate-Binding Proteins in Human Plasma and Platelet Releasates

Inorganic polyphosphates (polyP) are negatively charged, linear phosphate polymers found throughout biology. PolyP is secreted in substantial amounts by activated human platelets and it also accumulates in many infectious microorganisms. We have shown that polyP is a potent hemostatic regulator, acting at multiple points in the clotting cascade; in particular, polyP triggers the contact pathway, enhances FXI activation by thrombin, accelerates factor V activation, and modulates fibrin clot structure. Since there may be other, as-yet undiscovered (patho)physiologic roles for polyP, we undertook a study to identify the major polyP-binding proteins in human plasma and in releasates from activated human platelets.

PolyP was end-labeled by biotinylation, then bound to streptavidin coated onto magnetic beads (Dynabeads), polystyrene microtiter plates, or surface plasmon resonance sensorchips. PolyP-Dynabeads were exposed to plasma or to releasates from platelets activated by TRAP; the beads were then washed repeatedly, and polyP-binding proteins were eluted with 1 M LiCl. Eluted proteins were separated by 2-D gel electrophoresis and were identified by mass spectrometry. Some additional proteins known or suspected to bind to polyP were also analyzed in parallel. Binding affinities for selected proteins were quantified by microtiter plate assays or by surface plasmon resonance approaches.

Candidate poly-P binding proteins identified in plasma pull-downs included parvulin, factor H, complement C1q, complement C1q tumor necrosis factor-related protein, C4 binding protein, and apolipoprotein E. Candidate poly-P binding proteins identified in pull-downs of platelet-releasate included platelet factor 4, multimerin, thrombospondin, and cyclophilin. High affinity binding to polyP was also quantified for thrombin, FXI, FXIa, kallikrien, histones and the isolated polyphosphate-binding domain of E. coli exopolyphosphatase.

PolyP binds with high affinity to certain proteins that participate in the coagulation cascade as well as several additional proteins. Some of these are involved in reactions known to be modulated by polyP, but several have yet to be evaluated. Further investigation is indicated into potential roles for polyP in modulation of the complement cascade, prolyl isomerase activity, angiogenesis, and platelet responses.

Smith:University of Illinois: Patents & Royalties. Choi:University of Illinois: Patents & Royalties. Morrissey:University of Illinois: Patents & Royalties.