CHO cells expressing the high affinity alpha(IIb)beta3 T562N integrin demonstrate enhanced adhesion under shear.
BACKGROUND
Alpha(IIb)beta3-mediated platelet adhesive interactions in the vasculature, which are dependent on the functional state of this receptor, may be sensitive to shear forces.


OBJECTIVE
To evaluate the influence of the alpha(IIb)beta3 affinity state on cell attachment under flow, we compared Chinese hamster ovary cells expressing the low affinity alpha(IIb)beta3 wild-type (wt) receptor to those expressing the high affinity alpha(IIb)beta3 T562N receptor.


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)beta3 T562N supported higher cell adhesion to fibrinogen (63.3 +/- 2.9 cells/field) than alpha(IIb)beta3 wt (38.7 +/- 2.4 cells/field, P < 0.0001). At 100 s(-1), alpha(IIb)beta3 T562N mediated cell adhesion (40.5 +/- 3.8 cells/field), while alpha(IIb)beta3 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 microM fibrinogen gamma-chain dodecapeptide [HHLGGAKQAGDV (H12)], while Arg-Gly-Asp-Ser (RGDS) peptide (10-1000 microM) 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)beta3 T562N.


CONCLUSIONS
Our data show that alpha(IIb)beta3 activation is associated with a stabilization of integrin binding to fibrinogen or VWF under shear.
Legendre
P
P
INSERM U143, Le Kremlin-Bicetre, France.
Salsmann
A
A
Rayes
J
J
Trassard
O
O
Kieffer
N
N
Baruch
D
D
D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion / Q000235:genetics*; D006224:Cricetinae; D005340:Fibrinogen / Q000378:metabolism / Q000494:pharmacology; D018715:Microscopy, Video; D020125:Mutation, Missense; D009842:Oligopeptides / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D013314:Stress, Mechanical; D014841:von Willebrand Factor / Q000378:metabolism
D009842:Oligopeptides; C427756:P2-C protein, fibrinogen; D010446:Peptide Fragments; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D014841:von Willebrand Factor; D005340:Fibrinogen; C046617:arginyl-glycyl-aspartyl-serine