Figure 3.

Binding of FXII to apoptotic cells mediates its cleavage and activation. (A) As indicated, 95 nM FXII was incubated with viable cells (via) or apoptotic cells (ap) at density of 2 × 10⁵ in the presence or absence of 30 nM prekallikrein (PK) and 30 nM high molecular weight kininogen (HK) at 37°C for 30 min. After centrifugation at 2,500 rpm for 5 min, the supernatant was collected and analyzed by western blot with an anti-FXII Ab (i). The levels of FXII, PK, and HK before incubation with cells are shown by western blotting (ii). In three independent experiments, the density of bands was measured by NIH Image J software, and the cleavage of FXII was defined as the ratio of [cleaved FXII chain (48 kDa)]/[uncleaved FXII (80 kDa) plus cleaved FXII chain (48 kDa)] shown as relative band density (iii). **p < 0.01; ***p < 0.001. (B) In a cell-free system, 95 nM FXII was mixed with 30 nM PK and 30 nM HK followed by addition of 50 nM phosphatidylserine (PS) or phosphatidylcholine (PC) liposomes, one half of the mixture was immediately centrifuged (0 min), and the other half was incubated at 37°C for 60 min (60 min). After centrifugation at 55,000 rpm for 5 h, the supernatant was collected for analysis by western blotting using anti-FXII Ab (i, upper panel). The levels of FXII before incubation with liposomes are shown by western blotting (i, lower panel). In three independent experiments, the density of bands was measured, and the cleavage of FXII was calculated as described above and shown as relative band density (ii). ***p < 0.001. (C) Binding of FXII to apoptotic cells mediates its activation. As indicated, FXII (95 nM) was incubated with or without apoptotic cells (AC; 2 × 10⁵), PK (30 nM) plus HK (30 nM), and corn trypsin inhibitor (CTI) (2 µM) at 37°C for 30 min. After centrifugation at 2,500 rpm for 5 min, the supernatant was collected and factor XIIa (FXIIa) activity was analyzed as the hydrolysis of a chromogenic substrate as described in the Section “Materials and Methods” (n = 3). ***p < 0.001.