Vitronectin Concentrates Proteolytic Activity on the Cell Surface and Extracellular Matrix by Trapping Soluble Urokinase Receptor-Urokinase Complexes by Triantafyllos Chavakis, Sandip M. Kanse, Barbara Yutzy, H. Roger Lijnen, and Klaus T. Preissner Blood Volume 91(7):2305-2312 April 1, 1998 ©1998 by American Society of Hematology

Presence of suPAR in conditioned media of vascular cells. Presence of suPAR in conditioned media of vascular cells. (A) suPAR was captured by immobilized anti-uPAR MoAb-R4 and subsequently detected by measuring the extent of uPA binding using a plasminogen activation assay. Recombinant suPAR was used to generate the standard curve (rate of plasmin formation; Vmax, mOD/min at 405 nm) against which the unknown samples were quantified. (B) The production of suPAR by HVSMC, HUVEC, HL-60, and U937 cells was measured under basal conditions (hatched bars) or after stimulation by PMA (100 ng/mL) (filled bars). Data are shown as ng/18 h/106 cells (mean ± standard error of mean [SEM] of triplicate wells) and similar results were observed in three separate experiments. Triantafyllos Chavakis et al. Blood 1998;91:2305-2312 ©1998 by American Society of Hematology

Effect of suPAR on the binding of125I-Gly158scuPA to vascular cells. Effect of suPAR on the binding of125I-Gly158scuPA to vascular cells. (A) The specific binding of 125I-Gly158scuPA to HVSMC was determined in the absence and presence of increasing suPAR concentrations as indicated. Binding experiments were performed with untreated cells (•) or with piPLC-treated cells (○). Data represent mean ± SEM (cpm/well) of triplicate wells from a typical experiment. Similar results were obtained in three separate experiments on HVSMC or HUVEC, respectively. (B) The effects of MoAb-13H1 against VN (25 μg/mL) and multimeric VN (20 μg/mL) on the binding of 125I-Gly158scuPA to piPLC-treated HVSMC were tested in the absence (hatched bars) or presence (filled bars) of suPAR (1 μg/mL). Data are expressed as percentage of control (mean ± SEM) of three different experiments, where 100% (control) is represented by the specific binding of125I-Gly158scuPA in the absence of suPAR. Similar results were obtained in experiments with HUVEC. Triantafyllos Chavakis et al. Blood 1998;91:2305-2312 ©1998 by American Society of Hematology

Binding of uPA/suPAR-complex to piPLC pretreated vascular cells and their isolated extracellular matrix. Binding of uPA/suPAR-complex to piPLC pretreated vascular cells and their isolated extracellular matrix. The binding of 125I-Gly158scuPA to piPLC pretreated HVSMC (A) and HUVEC (B) (filled bars) and their respective extracellular matrix preparations (hatched bars) is compared in the absence or presence of suPAR (1 μg/mL) or MoAb-13H1 (25 μg/mL). For both cell types, data represent mean ± SEM of a typical experiment in triplicate where the maximal binding to piPLC pretreated cells in the presence of suPAR is set at 100%. Similar results were obtained in three separate experiments. Triantafyllos Chavakis et al. Blood 1998;91:2305-2312 ©1998 by American Society of Hematology

Binding of uPA/suPAR-complex to LM-TK−cells. Binding of uPA/suPAR-complex to LM-TK−cells. (A) The binding of 125I-suPAR to LM-TK− cells was tested in the absence or presence of increasing concentrations of unlabeled Gly158scuPA as indicated. No specific binding of 125I-suPAR alone was observed. (B) The binding of 125I-Gly158scuPA to LM-TK− cells was tested in the absence or presence of increasing concentrations of unlabeled suPAR (•) or the truncated, domain 1-lacking suPAR (○) as indicated. Data represent mean ± SEM (cpm/well) of triplicate wells. Similar results were observed in five separate experiments. Triantafyllos Chavakis et al. Blood 1998;91:2305-2312 ©1998 by American Society of Hematology

Effect of different competitors on the binding of uPA/suPAR-complex to LM-TK− cells. Effect of different competitors on the binding of uPA/suPAR-complex to LM-TK− cells. MoAb-13H1 against VN (•), multimeric VN (○), monomeric VN (▴), an MoAb against thrombospondin-1 (□), or soluble thrombospondin-1 (▪) were tested for their effect on the binding of the uPA/suPAR-complex to LM-TK− cells. Data are expressed as percentage of control (mean ± SEM) from three different experiments. The binding of125I-Gly158scuPA in the presence of suPAR and in the absence of any competitor served as the 100% control, and the binding of 125I-Gly158scuPA alone was about 20% of the binding of the complex. Triantafyllos Chavakis et al. Blood 1998;91:2305-2312 ©1998 by American Society of Hematology

Binding of 125I-Gly158scuPA to serum-free cultures of LM-TK− cells and their extracellular matrix. Binding of 125I-Gly158scuPA to serum-free cultures of LM-TK− cells and their extracellular matrix. Cells were grown for 14 days in completely serum-free medium on gelatin- or fibronectin-coated dishes. Before the binding experiment, the cells were preincubated with buffer only (hatched bars) or with 10 μg/mL multimeric VN for 48 hours (filled bars). Binding of 125I-Gly158scuPA to cells and extracellular matrix in the absence or presence of suPAR (1 μg/mL) was performed as indicated. Results are mean ± SEM (cpm/well) of triplicate wells. Similar results were obtained in three separate experiments. Triantafyllos Chavakis et al. Blood 1998;91:2305-2312 ©1998 by American Society of Hematology

Plasminogen activation on LM-TK− cells. Plasminogen activation on LM-TK− cells. LM-TK− cells (filled bars) and their extracellular matrix (hatched bars) were incubated in the absence or presence of uPA (10 nmol/L), as well as in the absence or presence of suPAR (1 μg/mL) and MoAb-13H1 (25 μg/mL) against VN for 2 to 3 hours at 4°C as indicated. The unbound uPA was then washed away and the rate of plasmin formation was measured (Vmax, mOD/min at 405 nm). Results are mean ± SEM of triplicate wells and similar results were obtained in three separate experiments. Triantafyllos Chavakis et al. Blood 1998;91:2305-2312 ©1998 by American Society of Hematology

Adhesion of LM-TK− cells to immobilized VN Adhesion of LM-TK− cells to immobilized VN. Adhesion of cells to VN-coated wells was performed in the absence of any competitor (−) or in the presence of uPA (100 nmol/L), suPAR (1 μg/mL), cRGD (10 μg/mL), PAI-1 (200 nmol/L), anti-avβ3 MoAb-LM609 (25 μg/mL)... Adhesion of LM-TK− cells to immobilized VN. Adhesion of cells to VN-coated wells was performed in the absence of any competitor (−) or in the presence of uPA (100 nmol/L), suPAR (1 μg/mL), cRGD (10 μg/mL), PAI-1 (200 nmol/L), anti-avβ3 MoAb-LM609 (25 μg/mL), or control MoAb-IgG (25 μg/mL) and measured by crystal violet staining. Results are expressed as percentage of adhesion to VN without competitor (mean ± SEM of triplicate wells). Similar results were obtained in six separate experiments. Triantafyllos Chavakis et al. Blood 1998;91:2305-2312 ©1998 by American Society of Hematology