(336e) Fibrin-Cd44v Binding Interferes with Platelet-Colon Carcinoma Cell Adhesion under High Shear Conditions

The molecular adhesive interactions between platelets and free flowing tumor cells have been shown to facilitate the process of blood borne metastasis. We have previously shown that hydrodynamic shear induced collisions augment platelet-LS174T colon carcinoma cell binding and that a transition in the molecular recognition events mediating heteroaggregation is observed, with a P-selectin-independent/RGD-dependent process in the low shear regime to a P-selectin/alphaIIb beta3 dependent process at high shear rates. However, the role of exogenous plasma proteins on these interactions has not been investigated. Therefore, we have studied the role of various plasma proteins on binding in an effort to elucidate the dynamics and molecular constituents involved in platelet-tumor cell adhesion. We have used a cone-and-plate rheometer to apply a uniform shear field on suspensions of platelets and tumor cells and analyzed the levels of heterotypic aggregation by a dual-color flow cytometric methodology. To specifically identify the role(s), if any, of exogenous proteins, purified fibrinogen, von Willebrand factor, IgG, and human serum albumin (HSA) were added to the suspension of activated washed platelets and LS174T cells. We have shown that at high shear rates plasma proteins, specifically fibrinogen/fibrin, have an inhibitory effect on these heterotypic adhesive events. To further characterize the fibrinogen/fibrin interaction with platelets and tumor cells, we immobilized either fibrinogen or fibrin (fibrinogen treated with thrombin and GPRP-NH2) onto plates and studied the interactions of LS174T cells with the monolayer in a parallel plate flow chamber. Additionally, polystyrene beads coated with the P-selectin ligand, CD44v, were perfused over the fibrin/fibrinogen surfaces and the binding interactions were quantified in shear flow. Our data indicate that CD44v binding to immobilized fibrin occurs at shear stresses up to 1 dyn/cm2. Moreover, sialylation of CD44 is essential for binding to fibrin. CD44v deficient LS174T cells, acquired using siRNA technology, are currently being tested for their ability to mediate tumor cell-fibrin interactions under physiologically relevant shear conditions. In addition, atomic force microscopy technology is being used to characterize the biophysical and kinetic parameters governing the binding of these tumor cells to fibrin.