CHO cells expressing the high affinity αIIbβ3T562N integrin demonstrate enhanced adhesion under shear

Background: alpha(IIb)beta(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 alpha(IIb)beta(3) affinity state on cell attachment under flow, we compared Chinese hamster ovary cells expressing the low affinity alpha(IIb)beta(3) wild-type (wt) receptor to those expressing the high affinity alpha(IIb)beta(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), alpha(IIb)beta(3)T562N supported higher cell adhesion to fibrinogen (63.3 +/- 2.9 cells/field) than alpha(IIb)beta(3)wt (38.7 +/- 2.4 cells/field, P < 0.0001). At 100 s(-1), alpha(IIb)beta(3)T562N mediated cell adhesion (40.5 +/- 3.8 cells/field), while alpha(IIb)beta(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 mu M fibrinogen gamma-chain dodecapeptide [HHLGGAKQAGDV (H12)], while Arg-Gly-Asp-Ser (RGDS) peptide (10-1000 mu 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 alpha(IIb)beta(3)T562N. Conclusions: Our data show that alpha(IIb)beta(3) activation is associated with a stabilization of integrin binding to fibrinogen or VWF under shear.