Time-dependent degradation and tissue factor (TF) addition blunt platelet polyP procoagulant activity. (A) Analysis of polyP stored for various times: 60 ng/lane of polyP was ran on a 2% agarose gel and visualized by negative DAPI staining. Lane 1 indicates freshly isolated platelet polyP; Lane 2, polyP stored for 3 months; Lane 3, 3.5-year-old platelet polyP (as provided to Faxälv et al); Lane 4, buffer; and Lane 5, long chain polyP. A molecular-weight DNA standard is given to the left. (B) Time-dependent loss of polyP procoagulant activity: real-time thrombin formation with fluorogenic substrate in normal human platelet-poor plasma triggered by the addition of 10 µg/mL of freshly prepared platelet polyP, platelet polyP stored for 3 months, 3.5-year-old polyP, and vehicle control (buffer). Thrombin formation triggered by fresh platelet polyP in FXII plasma is blotted for control (FXII def.). (C) Platelet polyP-triggered (1, 10 µg/mL) and TF-triggered (0.6 pM) thrombin formation in platelet-poor plasma. Agents were applied separately. (D) Thrombin generation by TF and a combined application of TF-platelet polyP. Graphs are representative of n = 5 experiments.