Cleaved high molecular weight kininogen binds directly to the integrin CD11b/CD18 (Mac-1) and blocks adhesion to fibrinogen and ICAM-1 by Nijing Sheng, Michael B. Fairbanks, Robert L. Heinrikson, Gabriela Canziani, Irwin M. Chaiken, David M. Mosser, Hong Zhang, and Robert W. Colman Blood Volume 95(12):3788-3795 June 15, 2000 ©2000 by American Society of Hematology

Transfection of HEK 293 with Mac-1 Transfection of HEK 293 with Mac-1.Flow cytometry analysis and FACSorting of HEK 293 cells transfected with Mac-1 (A to D). Transfection of HEK 293 with Mac-1.Flow cytometry analysis and FACSorting of HEK 293 cells transfected with Mac-1 (A to D). (A) Immunofluorescence assay using mAb LM2/1 with control transfected HEK 293 cells as a control. (B) Immunofluorescence assay using mAb LM2/1 with Mac-1 transfected HEK 293 cells. (C) Low level of expression of Mac-1, after FACSorting of Mac-1 transfected HEK cells. (D) High level of expression of Mac-1, after FACSorting of Mac-1 transfected HEK cells. Nijing Sheng et al. Blood 2000;95:3788-3795 ©2000 by American Society of Hematology

Specific binding of HKa to HEK 293 cells transfected with Mac-1 (Mac-1–HEK 293).FITC–HKa (at indicated concentrations) was incubated with HEK 293 cells either transfected with Mac-1 or control transfected in binding buffer containing 50 μmol/L ZnCl2 for 30 ... Specific binding of HKa to HEK 293 cells transfected with Mac-1 (Mac-1–HEK 293).FITC–HKa (at indicated concentrations) was incubated with HEK 293 cells either transfected with Mac-1 or control transfected in binding buffer containing 50 μmol/L ZnCl2 for 30 minutes at room temperature, as described in “Materials and methods.” The same reaction was also performed in the presence of 10 mmol/L EDTA to determine the nonspecific binding. Concentrations of FITC–HKa used were 0, 15, 30, 60, and 120 nmol/L. Reactions were carried out in wells of a filtration 96-well plate containing polyvinylidene difluoride membrane. After filtration, the fluorescence of cell-bound FITC–HKa was measured on a Cytofluor 2350 system. (•) HKa binding to Mac-1 transfected HEK 293 cells. (▾) HKa binding to untransfected HEK 293 cells. (○) HKa binding to Mac-1 transfected HEK 293 cells in the presence of 10 mmol/L EDTA. (▿) HKa binding to untransfected HEK 293 cells in the presence of 10 mmol/L EDTA. The data are the mean ± SE of triplicate reactions. Nijing Sheng et al. Blood 2000;95:3788-3795 ©2000 by American Society of Hematology

Inhibition of biotin-HKa binding to Mac-1 transfected cells by HKa Inhibition of biotin-HKa binding to Mac-1 transfected cells by HKa.Biotin–HKa (10 nmol/L) was incubated with Mac-1 transfected HEK 293 cells (4 × 106/mL) in binding buffer containing 50 μmol/L ZnCl2 for 45 minutes at room temperature in the presence of indi... Inhibition of biotin-HKa binding to Mac-1 transfected cells by HKa.Biotin–HKa (10 nmol/L) was incubated with Mac-1 transfected HEK 293 cells (4 × 106/mL) in binding buffer containing 50 μmol/L ZnCl2 for 45 minutes at room temperature in the presence of indicated HKa concentrations. The fluorescence of cell-bound biotin–HKa was measured, and the percentage of biotin–HKa bound to the cells in the presence of HKa was determined by comparing that with biotin–HKa alone, which was 100%. Data are the mean ± SE of 3 separate experiments. Nijing Sheng et al. Blood 2000;95:3788-3795 ©2000 by American Society of Hematology

Inhibition of biotin–HKa binding to Mac-1 transfected cells by antibody and fibrinogen.Biotin–HKa (10 nmol/L) was incubated with Mac-1 transfected or control transfected HEK 293 cells (4 × 106/mL) in binding buffer containing 50 μmol/L ZnCl2, 1 mmol/L MgCl2... Inhibition of biotin–HKa binding to Mac-1 transfected cells by antibody and fibrinogen.Biotin–HKa (10 nmol/L) was incubated with Mac-1 transfected or control transfected HEK 293 cells (4 × 106/mL) in binding buffer containing 50 μmol/L ZnCl2, 1 mmol/L MgCl2, and 1 mmol/L CaCl2 for 45 minutes at room temperature. The reactions were carried out either in the presence or absence of antibody, 2LPM19c, or fibrinogen as indicated. The relative amount of biotin–HKa bound to the Mac-1 transfected cells in the presence of inhibitors was determined by comparing that with biotin–HKa alone. Data are the mean ± SE of 3 separate experiments. Nijing Sheng et al. Blood 2000;95:3788-3795 ©2000 by American Society of Hematology

Effect of divalent cation on the HK-Mac-1 interaction Effect of divalent cation on the HK-Mac-1 interaction.To determine the effect of divalent cations on the HK–Mac-1 interaction, binding assays of FITC–HKa (60 nmol/L) to Mac-1 transfected (open bars) or untransfected (black bars) HEK cells were performed in ... Effect of divalent cation on the HK-Mac-1 interaction.To determine the effect of divalent cations on the HK–Mac-1 interaction, binding assays of FITC–HKa (60 nmol/L) to Mac-1 transfected (open bars) or untransfected (black bars) HEK cells were performed in the presence of different divalent cations as indicated. Cells were preincubated with 10 mmol/L EDTA for 10 minutes at 37°C, then washed with binding buffer without any divalent cation (−mol/L). One of the divalent cations was added to each assay as follows: MgCl2, MnCl2, and CaCl2 at 1 mmol/L, and ZnCl2 was added at 50 μmol/L. Nijing Sheng et al. Blood 2000;95:3788-3795 ©2000 by American Society of Hematology

A real-time observation of HK-Mac-1 interaction A real-time observation of HK-Mac-1 interaction.(A) Overlay sensor-gram from the IAsys showing the binding of Mac-1 to immobilized HKa. At “1” purified human Mac-1 was added to the cuvette at the following concentrations: 5, 10, 20, 40, 60, 80, 120, and 160... A real-time observation of HK-Mac-1 interaction.(A) Overlay sensor-gram from the IAsys showing the binding of Mac-1 to immobilized HKa. At “1” purified human Mac-1 was added to the cuvette at the following concentrations: 5, 10, 20, 40, 60, 80, 120, and 160 nmol/L. At “2” the cuvette was washed with buffer (10 mmol/L HEPES, 137 mmol/L NaCl, 4 mmol/L KCl, 50 μmol/L ZnCl2, and 0.05% Tween-20). (B) dR/dt plots of association data according to equation 3. Straight lines were fitted to the initial linear portion of the association phase data to obtain ks. (C) Plot of ks versus Mac-1 concentration. The slope of the line gives the association rate constant. (D) Dissociation phase data from 160 nmol/L Mac-1 sensor-gram plotted according to equation 5. A straight line was fitted to the first 20 seconds of the plot for phase 1, and the other straight line was fitted to the data between 20 and 70 seconds for phase 2. Nijing Sheng et al. Blood 2000;95:3788-3795 ©2000 by American Society of Hematology

HEK 293 cells transfected with Mac-1 or untransfected binding to immobilized HKa using the IAsys optical sensor.At “1” cells (105) were added, and at “2” they were washed with buffer (10 mmol/L HEPES, 137 mmol/L NaCl, 4 mmol/L KCl, 50 μmol/L ZnCl2, and 0.5%... HEK 293 cells transfected with Mac-1 or untransfected binding to immobilized HKa using the IAsys optical sensor.At “1” cells (105) were added, and at “2” they were washed with buffer (10 mmol/L HEPES, 137 mmol/L NaCl, 4 mmol/L KCl, 50 μmol/L ZnCl2, and 0.5% BSA). (A) Mac-1 transfected HEK 293 cells. (B) Untransfected HEK 293 cells. (C, D) Untransfected and Mac-1 transfected HEK 293 cells in the presence of 10 mmol/L EDTA, respectively. Data are representative of 3 separate experiments. Nijing Sheng et al. Blood 2000;95:3788-3795 ©2000 by American Society of Hematology

Effect of HKa on cellular adhesion to fibrinogen and ICAM-1 Effect of HKa on cellular adhesion to fibrinogen and ICAM-1.HEK 293 cells transfected with Mac-1 were labeled with CMFDA, added to wells precoated with the ligands ICAM-1 (▾), fibrinogen, (○) or fibronectin (•), and incubated in the presence or absence of i... Effect of HKa on cellular adhesion to fibrinogen and ICAM-1.HEK 293 cells transfected with Mac-1 were labeled with CMFDA, added to wells precoated with the ligands ICAM-1 (▾), fibrinogen, (○) or fibronectin (•), and incubated in the presence or absence of indicated HKa for 60 minutes. As a control, untransfected HEK 293 cells labeled with CMFDA were added to fibrinogen-coated (▪) or ICAM-1–coated (□) wells. After they were washed 3 times, adherent cells were lysed. Plates were read on a Cytofluor 2350 system. Percentage of adherent cells to each ligand in the presence of HKa was determined by comparing that with in the absence of HKa, which was calculated as 100%. Data are the mean ± SE of 3 separate experiments. Nijing Sheng et al. Blood 2000;95:3788-3795 ©2000 by American Society of Hematology