(49e) Biophysical Characterization of CD44v-Counter Receptor Interactions Using Force-Spectroscopy

Cell adhesion molecules such as P-selectin and fibrin(ogen) play indispensable roles in cancer and thrombosis. Recently, CD44v was identified as the major functional P-selectin ligand and fibrin receptor in LS174T metastatic colon carcinoma cells. We aimed to provide a mechanistic interpretation at the molecular level for the transient rolling interactions of P-selectin-CD44v and the firm adhesion of fibrin-CD44v using single-molecule force-spectroscopy with purified proteins incorporated in polymer cushioned lipid bilayers. The longer unstressed lifetime and a higher tensile strength of P-selectin-CD44v bond indicated that the higher affinity is responsible for the tethering/rolling interactions observed in flow based adhesion assays at higher shear stresses and low substrate concentrations. When the contact duration was increased from 2 ms to 200 ms, the tensile strength of the P-selectin-CD44v bond did not change notably whereas the adhesion strength of the fibrin-CD44v bond increased significantly. The maturation of the fibrin-CD44v bond is supported by the decrease in the unstressed dissociation rate and the increase in the depth of the free energy barrier of interaction. Hence, the stabilization of the fibrin-CD44v bond with increasing time explains the firm adhesion observed in flow based adhesion assays under the low shear regime and with high substrate concentrations, from a single molecule perspective. Thus, our results provide a rationale basis for the design of novel therapeutic strategies to combat thrombotic complications associated with cancer metastasis.