Fibrin(ogen) is a CD44 ligand on colon carcinoma cells: Biochemical and biophysical characterization, growth factor involvement, and diagnostic applications

Selectins, CD44, and fibrin(ogen) play critical roles in the hematogenous dissemination of tumor cells, including colon carcinomas. However, the fibrin(ogen) receptor(s) on colon carcinoma cells has yet to be defined along with its relative capacity to bind fibrinogen versus fibrin under flow. Using human CD44-knockdown and control LS174T cells, we demonstrate the pivotal involvement of CD44 in the P-selectin-mediated binding to platelets in shear flow. Using CD44-knockdown LS174T cells and microspheres coated with CD44 immunoprecipitated from LS174T cells, and purified fibrin(ogen) as substrate, we provide the first direct evidence that CD44 also acts as the major fibrin, but not fibrinogen, receptor on LS174T colon carcinomas. Interestingly, binding of fibrin to CD44 on the colon carcinoma cell surface interferes with the P-selectin-CD44 molecular interaction, and diminishes platelet-LS174T heteroaggregation in the high shear regime.;We biochemically characterized this interaction and the roles of CD44 standard, CD44s, versus CD44 variant isoforms, CD44v, in fibrin(ogen) recognition. In contrast to CD44v from colon carcinomas, CD44s binds to immobilized fibrin and fibrinogen under flow. Use of highly specific enzymes and metabolic inhibitors reveals that LS174T CD44 binding to fibrin is dependent on O-glycosylation of CD44, whereas CD44s-fibrin(ogen) interaction has an absolute requirement for N-, but not O-, linked glycans. The presence of chondroitin and dermatan sulfate on CD44 facilitates fibrin recognition. Use of an anti-CD44 function-blocking antibody nearly abolishes binding of LS174T CD44 to fibrin, while it has no effect on CD44s-fibrin(ogen) interaction.;Surface plasmon resonance experiments revealed high-affinity binding of immobilized CD44 with solubilized fibrin, but not fibrinogen and the location of the CD44-binding site within the N-terminal portion of the fibrin beta-chains including amino acid residues beta15-66. Treatment of LS174T cells with PDGF revealed reduced sulfation of chondroitin and dermatan sulfate chains on CD44 that leads to drastically increased adhesion to fibrin under flow. We have utilized single molecule force microscopy and protein-incorporated lipid bilayers to probe the kinetics parameters of CD44-fibrin(ogen) binding. We have successfully micro-patterned proteins on glass substrates for use in cell capture and sorting applications. Our findings may provide a rational basis for designing novel therapeutic strategies to combat metastasis.