Activation of recombinant alphaIIbbeta3 expressed in Chinese hamster ovary cells exposes different binding sites for fibrinogen or von Willebrand factor: evidence using monoclonal antibodies to alphaIIbbeta3

We have investigated the interaction of von Willebrand factor (VWF) and fibrinogen (Fg) with recombinant integrin alphaIIbbeta3 expressed in Chinese hamster ovary (CHO) cells either in its native conformation or following partial reduction by dithiothreitol (DTT). We found that DTT-treated cells aggregated in the presence of soluble VWF as well as Fg, whereas non-treated cells did not. Furthermore, we demonstrated that DTT was required to specifically induce alphaIIbbeta3-dependent cell adhesion to immobilized VWF, while Fg-dependent cell adhesion occurred independently of the activation state of alphaIIbbeta3. By comparing the effects of two potent platelet alphaIIbbeta3 inhibitors, monoclonal antibodies (mAbs) AP2 and 10E5, we highlighted the different blocking properties of these mAbs on VWF or Fg binding to activated alphaIIbbeta3. In particular, AP2 prevented VWF-dependent but not Fg-dependent CHO cell aggregation. Furthermore, AP2 inhibited cell adhesion to VWF, but had no effect on adhesion to Fg. In contrast to this distinct effect of AP2 towards these two ligands, mAb 10E5 inhibited activated alphaIIbbeta3-dependent aggregation completely and adhesion partially, whether in the presence of Fg or VWF. These data provide evidence that interaction of VWF and Fg with DTT-activated alphaIIbbeta3 relies on distinct contact sites exposed on the activated receptor that can be selectively blocked by monoclonal antibodies.