Localization of Short-Chain Polyphosphate Enhances its Ability to Clot Flowing Blood Plasma

Abstract

Short-chain polyphosphate (polyP) is released from platelets upon platelet activation, but it is not clear if it contributes to thrombosis. PolyP has increased propensity to clot blood with increased polymer length and when localized onto particles, but it is unknown whether spatial localization of short-chain polyP can accelerate clotting of flowing blood. Here, numerical simulations predicted the effect of localization of polyP on clotting under flow, and this was tested in vitro using microfluidics. Synthetic polyP was more effective at triggering clotting of flowing blood plasma when localized on a surface than when solubilized in solution or when localized as nanoparticles, accelerating clotting at 10-200 fold lower concentrations, particularly at low to sub-physiological shear rates typical of where thrombosis occurs in large veins or valves. Thus, sub-micromolar concentrations of short-chain polyP can accelerate clotting of flowing blood plasma under flow at low to sub-physiological shear rates. However, a physiological mechanism for the localization of polyP to platelet or vascular surfaces remains unknown.

Figure 1. Numerical simulations predict localization of polyP accelerates thrombin production at low shear rates.

Two-dimensional numerical simulations of the human blood coagulation cascade, comparing the generation of thrombin by polyP dispersed throughout a cylindrical channel versus polyP immobilized on the channel surface. The channel was 20 mm long with a radius of 2 mm. The overall number of polyP molecules was the same in all simulations (7.54 × 10⁻⁹ moles). (A) Plots show [thrombin], which is the sum of concentrations of thrombin and meizothrombin, for a two-dimensional longitudinal cut of the cylinder at 500 s into the simulation. (B) The fold difference in the maximum [thrombin] generated in the channel when polyP was surface-immobilized (SI-polyP) versus dispersed (D-polyP) at varying shear rates.

Figure 2. PolyP induces clotting of flowing blood plasma when localized on a surface at sub-physiological shear.

(A) Schematic of biotinylated synthetic polyP (cyan) patterned onto the surface of half of a microfluidic channel, which induces production of thrombin and clotting (blue) of flowing blood plasma (grey). (B) Images of fluorescent-labeled agents flowing and patterned along one side of a microfluidic channel. Biotinylated lipids (tagged red) self-assembled on the channel wall. Non-biotinylated lipids (not tagged in these images) were simultaneously flowed and patterned on the other side of the chamber using laminar flow patterning. Then, streptavidin (tagged green) was flowed through and bound to the biotinylated lipids, followed by flowing biotinylated polyP labeled with DAPI (cyan), which bound streptavidin. A substrate (blue) for thrombin was activated, indicating initiation of clotting, selectively on patterned polyP₄₀₀ (300 nmol/m²). Scale bar is 250 μm. (C) Quantifying of the amount of SI-polyP by measuring the fluorescence of DAPI bound to it. Channels with SI-polyP were compared to channels without polyP and to channels treated with polyP diluted with biotinylated PEG. Inset is a standard curve of known concentrations of solubilized D-polyP, which was used to calculate the surface concentration of SI-polyP in coated channels. (D) The clotting times of normal human plasma flowing through channels coated with polyP₄₀₀ at a shear rate of 1 s⁻¹. *p = < 0.01 compared to controls without polyP. Data indicate mean ± SEM, n = 3.

Figure 3. The microfluidic system used to measure clotting over a range of shear rates.

(A) Schematic of the microfluidic system. Box (dashed lines) indicates the region where shear rates were varied and clot times were measured. (B) Fluorescence images showing that clotting was detected by the cessation of flow of tracer beads (pink) and by the cleavage of a substrate for thrombin (blue). Scale bar is 250 μm. (C) Assessing the range of clotting times in this flow system by adding various concentrations of FVIIa to the plasma. Data points indicate mean ± SEM, n = 3–4. Red circles indicate p = <0.05 between the data points, and blue circles indicate p = <0.01 between the data points.

Figure 4. PolyP accelerates clotting best when spatially localized onto surfaces, compared to soluble polyP and nanoparticles of polyP.

(A) Clotting times of plasma by polyP₁₆₀ at varying shear rates, comparing three states of polyP₁₆₀: solubilized, self-assembled nanoparticles, and surface-immobilized. (B) Time-lapse images showing SI-polyP₁₆₀ initiating clotting (detected by non-flowing beads) from the channel wall (dashed lines). Scale bar is 250 μm. (C) Comparing three states of polyP₇₀: solubilized, surface-immobilized onto the microfluidic channels, and immobilized onto silica nanoparticles. Clotting tendencies of plasma containing silica nanoparticles coated with polyP₇₀ (SNP-polyP₇₀) compared to soluble and surface-immobilized polyP₇₀ under shear in the microfluidic device. (D) Comparing two states of long-chain polyP: surface immobilized polyP₄₀₀ and nanoparticles of self-assembled polyP_>1000. (E) Schematic summarizing the relationship between spatial distribution of polyP and the acceleration of clotting in the above experiments. Data points indicate mean ± SEM, *p < 0.001, **p < 0.0001, n = 3–4. Statistical analysis represents comparisons between whole curves.