This study was undertaken to characterize the adhesion of LS174T colon adenocarcinoma to 4 h TNF- stimulated human umbilical vein endothelial cells (HUVECs) under flow, in the presence and absence of platelets and erythrocytes. Cell binding to HUVECs was significantly enhanced by simultaneous perfusion of thrombin-activated, but not resting, platelets. This increase was achieved via a platelet bridging mechanism whereby a previously tethered cell (primary tether) captures a free-flowing cell (secondary tether) that subsequently attaches to the endothelium downstream of the already adherent cell. The total number of tumor cells tethering to HUVECs and the percentage of secondary tethers relative to the total amount of cell tethering depended on platelet concentration and wall shear stress. At 0.8 dyn/cm² and 25:1 platelet:LS174T cell ratio, the total amount of cell tethering nearly doubled as a result of platelet-induced enhancement compared to the amount without platelet perfusion. Moreover, the percent secondary tethers increased from background levels (<5%) in the absence of platelets to ~60% at the 25:1 platelet:LS174T cell ratio. Platelet-mediated secondary tethering is not limited to LS174T colon carcinoma cells, as THP-1 monocytoid cells also displayed this pattern of interaction. Secondary tethering was dependent on both platelet P-selectin/_IIb3 integrin for LS174T cells and P-selectin alone for THP-1 cells. Furthermore, platelet-mediated secondary tethering of both cell types occurred in the presence of red blood cells. Altogether, these results reveal a novel role for platelets in promoting cell binding to endothelium through a secondary tethering mechanism.