FXa-initiated clotting is accelerated by shorter polyP polymers than are required for initiating clotting via the contact pathway. PolyP (5μM) was added simultaneously to normal plasma together with 333 pM FXa and calcium chloride, and time to clot formation was measured. (A) PolyP specific activities were calculated by comparing polyP clot times to a standard curve in the absence of polyP, but in which varying FVa concentrations were added to plasma (not shown). The FVa concentration (in nM FVa) that yielded the same clotting time as a given polyP preparation was then divided by the polyP concentration (5000 nM). Sized-fractionated polyP preparations (▾) are compared with bacterial-derived polyP (“Bact”) and polyP type 65. The inset focuses on polymers shorter than 200mers. (B) Shortening of the FXa clotting time by polyP is independent of FXII. PolyP preparations of the indicated polymer sizes (all at 20μM phosphate) were added to FXa-initiated clotting reactions conducted with either pooled normal plasma (white bars) or FXII-deficient plasma (black bars). Clotting times with polyP were normalized to the clotting time of the respective plasma without polyP. Data in all panels are mean ± SE (n = 3).