Premium
CHO cells expressing the high affinity α IIb β 3 T562N integrin demonstrate enhanced adhesion under shear
Author(s) -
LEGENDRE P.,
SALSMANN A.,
RAYES J.,
TRASSARD O.,
KIEFFER N.,
BARUCH D.
Publication year - 2006
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2005.01653.x
Subject(s) - chinese hamster ovary cell , integrin , rgd motif , cell adhesion , chemistry , fibrinogen , adhesion , microbiology and biotechnology , receptor , platelet , biophysics , biochemistry , biology , immunology , organic chemistry
Summary. Background: α IIb β 3 ‐mediated platelet adhesive interactions in the vasculature, which are dependent on the functional state of this receptor, may be sensitive to shear forces. Objectives: To evaluate the influence of the α IIb β 3 affinity state on cell attachment under flow, we compared Chinese hamster ovary cells expressing the low affinity α IIb β 3 wild‐type (wt) receptor to those expressing the high affinity α IIb β 3 T562N receptor. Materials and methods: We designed a real‐time videomicroscopy adhesion assay for von Willebrand factor (VWF) or fibrinogen under flow conditions. Results: At 50 s −1 , α IIb β 3 T562N supported higher cell adhesion to fibrinogen (63.3 ± 2.9 cells/field) than α IIb β 3 wt (38.7 ± 2.4 cells/field, P < 0.0001). At 100 s −1 , α IIb β 3 T562N mediated cell adhesion (40.5 ± 3.8 cells/field), while α IIb β 3 wt did not (5.3 ± 1.4 cells/field, P < 0.001), allowing to discriminate the efficiency of each receptor. Similar findings were observed for adhesion to VWF. Complete inhibition of cell adhesion to fibrinogen was achieved with 800 μ m fibrinogen γ ‐chain dodecapeptide [HHLGGAKQAGDV (H12)], while Arg‐Gly‐Asp‐Ser (RGDS) peptide (10–1000 μ m ) induced a dose‐dependent cell detachment. These results suggest that the H12 motif allows initial attachment, in contrast to the RGDS site, which strengthens the stability of adhesion. Interestingly, compared with wt, a 10‐fold lower concentration of RGDS was required to reach a similar reduction of cell adhesion mediated by α IIb β 3 T562N. Conclusions: Our data show that α IIb β 3 activation is associated with a stabilization of integrin binding to fibrinogen or VWF under shear.