Figure 4.

Inhibition of NEDD9 with monospecific polyclonal Ab (msAb)–NEDD9 peptide sequence 2 (N9-P2) decreases NEDD9–P-Sel (P-selectin) complex formation. Anti-NEDD9 antibodies raised against the NEDD9 target of P-Sel were developed in rabbits (msAb–NEDD9 peptide sequence 1 [N9-P1] and msAb–N9-P2). (A) The inhibitory effect of msAb–N9-P1 and msAb–N-P2 on NEDD9–P-Sel complex formation in vitro was analyzed (n = 3). V, a mixture of msAb–N9-P1 and msAb–N9-P2, was incubated without beads as an additional negative control (immunoblot data not shown). (B) In human pulmonary artery endothelial cells (HPAECs) treated with hypoxia (0.2%) for 24 hours, msAb–N9-P2 inhibited TRAP (thrombin receptor agonist peptide) (25 μM)–stimulated platelet–endothelial adhesion significantly (n = 3). IgG₁ was the negative control. (C) Under normoxic conditions, HPAECs were treated with IL-6 (25 ng/ml) for 24 hours, and the effect of msAb–N9-P2 (20 μg/ml) on platelet–endothelial adhesion was compared with treatment with anti–P-Sel Ab (anti–P-Sel) (10 μg/ml), PSGL-1 (anti–P-Sel glycoprotein ligand-1 Ab from a KPL-1 clone) (15 μg/ml), and t-PA (tissue plasminogen activator) (15 ng/ml) (n = 3). Similar experiments were performed in untreated HPAECs after stimulation of platelets with TRAP. Data are presented as mean ± SEM. Representative immunoblots are shown. Ab = antibody; ND = not detected; PBS = phosphate-buffered saline; V = vehicle control.