Monoclonal antibodies against receptor-induced binding sites detect cell-bound plasminogen in blood

Abstract

IntroductionPlasminogen binds to nucleated blood cells and platelets in a specific, saturable, and reversible manner. 1,2 Based on the plasminogen concentration in plasma (2M) 3 and the affinity for its receptors (ϳ 1M), 4 approximately 50% of plasminogen binding sites on peripheral blood cells within the vasculature are predicted to be occupied by plasminogen. Recently, we developed antiplasminogen receptor-induced binding site (RIBS) mAbs that preferentially recognize cell-associated plasminogen in the presence of soluble plasminogen. 5 Therefore, we have investigated whether the representative antiplasminogen RIBS mAb, mAb49, can detect plasminogen bound to live cells in blood. Here we demonstrate that plasminogen binds to peripheral blood cells in normal whole blood and that modulation of cell-associated plasminogen occurs during inflammation and blood cell diatheses, including acute promyelocytic leukemia (APL). Methods ProteinsGlu-plasminogen was from Chromogenix (Mölndal). mAb49 against plasminogen was raised and characterized in our laboratory. 5 mAbs to CD15, CD14, CD33, CD19, CD2, GPIIb-IIIa, Glycophorin A, and HLDR were from Immunotech or Coulter. FITC-and PE-conjugated goat antimouse mAbs were from Sera-Lab. CellsNeutrophils, monocytes, lymphocytes, and red blood cells were isolated from blood collected into heparin (5 U/mL), theophylline (10mM), and prostaglandin E 1␣ (10 U/mL; Sigma-Aldrich) as described. 6 Blast cells from peripheral blood were analyzed from a patient with acute nonlymphoblastic leukemia, categorized according to the FrenchAmerican-British classification. 7 Blood drawing was approved by the Institute Català de la Salut Institutional Review Board. NB4 cells were provided by Dr M. Lanotte (Hô pital St Louis, Paris, France). The human cell line Nalm6 was provided by Dr J. Inglés-Esteve (IDIBELL, Barcelona). Other cell lines were from the ATCC and cultured as described. 8 FACS analysisCells were washed with PBS containing 1% BSA and 0.1% sodium azide (PBA), incubated with PBA containing 10% heat-inactivated normal rabbit serum, washed again, and incubated with mAb49 (130nM) or isotype control, washed, and then stained with FITC-goat anti-mouse IgG, which was detected in a flow cytometry analyzer (Coulter EPICS XL-MCL).Plasminogen binding to cells in whole peripheral blood collected into EDTA was determined as in the preceeding paragraph with the following exceptions. Cells were incubated in 10% heat-inactivated human AB serum in PBS, washed with PBA, and incubated with anti-mouse IgG conjugated to PE, washed and incubated with FITC-conjugated antibodies to specific leukocyte antigens. Cells were incubated in Ortho-mune Lysing Reagent (Ortho Diagnostic Systems), centrifuged, and resuspended in PBA containing 7-aminoactynomycion D (Invitrogen) at 1 mg/mL. Radiolabeled antiplasminogen RIBS mAb binding to cellsmAb49 was radiolabeled as described 9 and incubated with cells. Samples were centrifuged through 20% sucrose to separate bound from free ligand as described. For personal use only. ...

New Insights into the Role of Plg-RKT in Macrophage Recruitment

“…This suggests that Glu-plasminogen on the cell surface adopts a conformation distinct from its conformation in solution. Direct evidence for such a conformational change was obtained recently using monoclonal anti-plasminogen antibodies that recognize receptor-induced binding sites (RIBS) in Glu-plasminogen upon its interaction with cells, but react poorly with soluble Glu-plasminogen (Han et al, 2011b; Felez et al, 2012; Jardi et al, 2012). Previously, it had generally been accepted that Glu-plasminogen adopts a Lys-plasminogen-like open conformation when bound to the cell surface, to account for enhancement of activation of cell-associated Glu-plasminogen (Dejouvencel et al, 2010).…”

Plasminogen Receptors: The First Quarter Century

“…Direct evidence for such a conformational change was obtained recently using monoclonal antiplasminogen antibodies that recognize receptor-induced binding sites in Glu-plasminogen upon its interaction with cells but react poorly with soluble Glu-plasminogen. [56][57][58] Previously, it has generally been accepted that Glu-plasminogen adopts a Lys-plasminogen-like open conformation when bound to the cell surface to account for enhancement of activation of cell-associated Glu-plasminogen. 59 However, soluble Lys-plasminogen did not compete for the interaction of antiplasminogen receptor-induced binding sites monoclonal antibodies (mAbs) with surfaceassociated Glu-plasminogen, suggesting that the conformation induced when Glu-plasminogen binds to cells is distinct from the conformation of Lys-plasminogen.…”

Characterization of Plasminogen Binding to NB4 Promyelocytic Cells Using Monoclonal Antibodies against Receptor-Induced Binding Sites in Cell-Bound Plasminogen

“…After incubation, supernatants were removed by centrifugation, incubated with mAb49 (130 nM) for 30 minutes a 4°C, washed and then stained with FITC-goat anti-mouse IgG, which was detected by FACS analyses. This research was originally published in [ 28 ].…”

“…(c) FACS analysis using mAb49 (red tracing) or isotype control (turquoise tracing) of blood from a patient with an M1 leukemia. This research was originally published in [ 28 ].…”