Optimal enhancement of fibrin clot turbidity requires relatively long polyP polymers. Fibrin clots were prepared by adding thrombin to fibrinogen that had been preincubated with Ca²⁺ and polyP, and clot turbidity (A₄₀₅) was quantified 60 minutes after thrombin addition. (A) Clot turbidity as a function of polyP polymer size (all at 150μM phosphate). The value for x = 0 is in the absence of added polyP, while “Bact” indicates S typhimurium–derived polyP. The inset focuses on polyP preparations shorter than 180mers. (B) Clot turbidity as a function of polyP concentration, using the following polyP polymer lengths: 65mers (▵), 158mers (■), 700mers (○), polyP type 65 (●), polyP type 75⁺ (□), and polyP₄₀₀₊ (▾). (C) PP_i abrogates the ability of polyP to enhance clot turbidity. Fibrinogen was preincubated with calcium ions, 150μM polyP₄₀₀₊, and the indicated concentrations of phosphate-containing substances, after which thrombin was added. The substances tested were: monophosphate (▿), PP_i (▴), triphosphate (□), ADP (○), and ATP (●). The dotted line represents clot turbidity in the absence of polyP. (D) PP_i, but not other polyP, polymers abrogates the ability of polyP₄₀₀₊ to enhance clot turbidity. Phosphate polymers of the indicated lengths (all at 150μM phosphate) were included in fibrin-clotting reactions as performed in panel C. The open bar with the asterisk represents clot turbidity in the absence of polyP, while the open bar with “0” represents clot turbidity with polyP₄₀₀₊, but no added small phosphate polymer.