(656c) Distinct Characteristics of CD44s Binding to Fibrin(ogen) Investigated Using Force Spectroscopy At the Single Molecule Level

CD44 and fibrin(ogen) play crucial roles in the hematogenous dissemination of tumor cells. The standard isoform of CD44, CD44s, from HL60 human myeloid leukemia cells binds to both immobilized fibrin and fibrinogen under flow, mediating firm adhesion in the low shear regime and at high substrate concentrations. We utilized single molecule force spectroscopy to delineate the distinct kinetic and micromechanical properties of CD44s binding to fibrin and fibrinogen. To this end, fibrin or fibrinogen immobilized on the cantilever tip was brought in contact with immunopurified CD44s incorporated into lipid vesicles and layered on a polyethyleneimine (PEI)-cushioned glass slide. The CD44s-fibrin pair has a lower unstressed dissociation rate and a higher tensile strength than the CD44s-fibrinogen bond, which may explain the ability of CD44s coated microspheres to bind immobilized fibrin more efficiently than fibrinogen at a shear stress level of 0.5 dyne/cm². Incubating the CD44s incorporated lipid vesicles with function blocking anti-CD44 monoclonal antibody Hermes-1 did not affect the frequency of binding to fibrin and fibrinogen, indicating the presence of an alternate binding site on CD44s for fibrin(ogen). Increasing the duration of receptor-ligand contact from 2 to 200 milliseconds did not affect the micromechanical properties of CD44s-fibrin bond. Furthermore, the molecular recognition of CD44s by fibrin(ogen) requires the presence of both N-linked glycans and the glycosaminoglycans namely chondroitin sulfate and dermatan sulfate at all tested contact durations. Thus, our single molecule characterization of the biophysical properties of receptor-ligand pairs in conjunction with biochemical studies provides a strong quantitative understanding to the interactions of CD44, which is an aberrantly expressed protein on human metastatic tumor cells, with fibrin and fibrinogen that play vital roles in the facilitation of cancer metastasis.