248 We previously reported that particles coated with the tuftsin antagonist peptide TKPPR, or synthetic TKPPR multimers bind to cells expressing neuropilin-1 (NP-1), a dual receptor for VEGF and class 3 semaphorins with multiple functions in the neural, vascular, and immune systems. The TKPPR multimers were also able to block VEGF165 from binding to neuropilin-1-expressing but not VEGFR-2-expressing cells. To firmly establish that the TKPPR sequence binds selectively to neuropilin-1, neuropilin-1-negative A293H cells were transfected with a neuropilin-1 expression vector. Binding assays with TKPPR-conjugated particles (e.g. lipid microbubbles) demonstrated that neuropilin-1-expression was necessary and sufficient for the particles to bind these cells. Homogenous fluorescence polarization assays with recombinant extracellular receptor domains confirmed that a synthetic tetrameric TKPPR bound with nanomolar affinity to NP-1, but had much lower or no affinity for VEGF, VEGFR-1, VEGFR-2, or NP-2 (NP-1, VEGFR-2, and NP-2 polarization data reported previously). After noting that 125I-VEGF165 prebound to HUVEC could only be partially displaced with even 100 nM unlabeled VEGF, selective peptide competitors of VEGF binding to VEGFR-2 and NP-1 were tested on HUVEC with 125I-VEGF165 prebound. The VEGFR-2-binding peptide was only able to block a fraction of the labeled VEGF binding, and only when administered simultaneously with the ligand. There was no displacement of 125I-VEGF165 prebound to HUVEC. When tetrameric TKPPR was used as an NP-1 antagonist, it was able to block or displace the same fraction of labeled VEGF regardless of whether the 125I-VEGF was prebound or added simultaneously. This fraction was essentially the same as that which cold VEGF could displace from 125I-VEGF165 prebound to HUVEC, suggesting that, once bound, VEGF165 on endothelial cells can be displaced from NP-1 but not VEGFR-2.