Initiation of clotting via the contact pathway is most efficient with very long polyP polymers. (A) Clotting was initiated by preincubating 5μM polyP with citrated plasma for 3 minutes at 37°C, after which calcium chloride was added and the time to clot formation recorded. PolyP specific activities were calculated by comparing polyP-initiated clotting times to a standard curve in which clotting was initiated by varying kaolin concentrations (see supplemental Figure 1C), yielding “kaolin equivalents” on a mass basis. Activities of sized-fractionated polyP preparations (▾) are compared with platelet-derived polyP (▿; also indicated with arrows), bacterial-derived polyP (“Bact”), polyP type 65, and PolyP₁₀₀₀₊. The inset focuses on polymers shorter than 200mers. The point for platelet polyP was plotted at its mean polymer length (80mers). (B) Inhibition of contact-pathway–initiated clotting by an excess of small polyP polymers. Plasmas were preincubated for 3 minutes 37°C with a combination of 10μM polyP₁₀₀₀₊ and the indicated concentrations of shorter phosphate/polyP preparations (identified as “inhibitor” on the x-axis), after which calcium chloride was added to allow clotting. The short phosphates were: monophosphate (▿), PP_i (▴), and triphosphate (□) (none of which had any effect); and 11mers polyP (▾), 16mers polyP (○), 35mers polyP (■), 53mers polyP (▵), and 83mers (●) (which prolonged the clotting times in a concentration-dependent manner). The dotted line represents the clotting time in the absence of polyP. Data in all panels are mean ± SE (n = 3).