Microparticles and platelets enhance FXI activation in plasma to cleave chem163S. (A) Purified 6his-chem163S was incubated with FXIa in assay buffer with or without microparticles. Aliquots were removed every 5 minutes for 30 minutes, and the level of 6his-chem163S was measured with the 6his-chem163S ELISA. (B-D) Contact pathway was initiated by kaolin or Polyp in PRP and PPP. (B) Purified 6his-chem163S (10 μM) was incubated in control plasma with or without phospholipids after contact pathway activation. Aliquots of reactions were removed every 5 minutes for 30 minutes, and the levels of 6his-chem163S in each aliquot were analyzed by 6his-chem163S ELISA. (C) After activation by kaolin or Polyp for 30 minutes, the concentration of endogenous chem163S was determined by the chem163S ELISA. In some experiments, hirudin (1.5 U/mL) was added to block thrombin activity. (D) FXIa fluorogenic substrate D-LPR-ANSNH-C3H7•2HCl was incubated in PPP or PRP after contact pathway activation. Fluorescence was monitored every 15 seconds, and the FXIa concentration was calculated by interpolation from a FXIa standard curve. Data from 3 independent experiments were pooled to show mean ± standard error of the mean.
