Journal: Frontiers in Immunology

Article Title: The Procoagulant Activity of Apoptotic Cells Is Mediated by Interaction with Factor XII

doi: 10.3389/fimmu.2017.01188

Figure Lengend Snippet: 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 × 10 5 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 × 10 5 ), 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 “ ” ( n = 3). *** p < 0.001.

Article Snippet: Corn trypsin inhibitor (CTI) was purchased from Merck Chemicals, Ltd. FXII-deficient plasma and pooled human normal plasma were obtained from George King Bio-Medical, Inc. Phosphatidylcholine (PC), PS, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1-oleoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (NBD-PC) were obtained from Avanti-Polar Lipids.

Techniques: Binding Assay, Activation Assay, Incubation, High Molecular Weight, Centrifugation, Western Blot, Software, Liposomes, Activity Assay

Journal: Frontiers in Immunology

Article Title: The Procoagulant Activity of Apoptotic Cells Is Mediated by Interaction with Factor XII

doi: 10.3389/fimmu.2017.01188

Figure Lengend Snippet: Apoptotic cell-mediated procoagulant activity and intrinsic tenase formation is dependent on FXII activation. (A) Tissue factor (TF) expression as determined by flow cytometry. CEM cells treated with (apoptotic cells) or without (viable cells) 10 µM dexamethasone for 24 h were stained with CD142-PE to analyze TF expression on the cell surface. THP-1 cells were used as a positive control. The background staining with an isotype control is shown in black. (B) After viable cells and apoptotic cells (2 × 10 5 ) were suspended in 150 µL of platelet-poor plasma, clotting was triggered by the addition of 20 mM CaCl 2 and measured as described in the Section “ ” ( n = 3). *** p < 0.001. (C) After apoptotic cells (2 × 10 5 ) were suspended in normal plasma (normal), FXII-deficient plasma [FXII(-)] and FXII-deficient plasma supplemented with 375 nM FXII [FXII(-) + FXII], respectively, clotting was triggered by the addition of 20 mM CaCl 2 and measured as described in the Section “ ” ( n = 3). (D) Effect of apoptotic cells on intrinsic tenase complex formation. As indicated, apoptotic or viable cells were incubated with FXII, prekallikrein, high molecular weight kininogen, FXI, FIX, and FVIII. Then FX was added, and tenase complex formation was analyzed using a chromogenic substrate ( n = 3). Some samples were also treated with 2 µM corn trypsin inhibitor (CTI). *** p < 0.001.

Article Snippet: Corn trypsin inhibitor (CTI) was purchased from Merck Chemicals, Ltd. FXII-deficient plasma and pooled human normal plasma were obtained from George King Bio-Medical, Inc. Phosphatidylcholine (PC), PS, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1-oleoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (NBD-PC) were obtained from Avanti-Polar Lipids.

Techniques: Activity Assay, Activation Assay, Expressing, Flow Cytometry, Staining, Positive Control, Control, Clinical Proteomics, Coagulation, Incubation, High Molecular Weight