Christina S. Alves1, Sergiy Yakovlev2, Leonid Medved2, and Konstantinos Konstantopoulos3. (1) Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles St., Baltimore, MD 21218, (2) Biochemistry and Molecular Biology, University of Maryland, Baltimore, Baltimore, MD 21201, (3) Chemical & Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles Street, Maryland Hall Room 223, Baltimore, MD 21218
Fibrin(ogen) plays a critical role in the hematogenous dissemination of tumor cells, including colon carcinoma, presumably by mediating their sustained adhesion and survival in the high shear environment of secondary target organs. However, the fibrin(ogen) ligand(s) on colon carcinoma cells has yet to be defined along with its relative capacity to bind fibrinogen versus fibrin under flow. Using CD44-knockdown LS174T colon carcinoma cells and microspheres coated with CD44 immunopurified from control LS174T cells, and purified fibrin(ogen) as substrate, we demonstrate that CD44 variant isoforms (CD44v) act as the major fibrin, but not fibrinogen, ligands on colon carcinoma cells. In contrast, the standard form of CD44, CD44s, immunoprecipitated from human myeloid cells is capable of binding to both fibrinogen and fibrin in shear flow. CD44v-fibrin binding is primarily dependent on the presence of O-linked glycans on CD44v, whereas the CD44s-fibrin interaction has an absolute requirement for N-linked glycans. Interestingly, both N- and O-linked glycans on CD44s are necessary for optimal CD44s-fibrinogen binding. Enzymatic assays reveal that chondroitin and dermatan sulfates are also implicated in the CD44-fibrin(ogen) molecular recognition. To characterize the CD44 binding site on the fibrin(ogen) molecule, we perfused CD44-coated microspheres over the major fibrin(ogen) proteolytic fragment D-D and (t)-NDSK, and the recombinant Aα221-610 and (β15-66)2 fragments. Our flow-based adhesion assays disclose that CD44-bearing microspheres interact avidly and extensively with the (β15-66)2 and t-NDSK fragments. We verified these results and determined the relative affinity of these fragments for CD44v and CD44s via Surface Plasmon Resonance. Use of the function-blocking monoclonal antibody (mAb) Hermes-1 enabled us to localize the fibrin epitope on the CD44v molecule, which is identical to that of hyaluronic acid. In contrast, this mAb failed to inhibit the adhesive interactions of CD44s-coated microspheres with fibrin, suggesting the existence of an additional fibrin binding epitope on CD44s. Taken together, our data bring together disparate observations about the contributions of fibrin(ogen) and CD44 to hematogenous metastasis in a unifying mechanistic interpretation, and provide a rational basis for the design of novel therapeutic strategies to impede metastasis.