Figure 1.

Factor Xa-dependent NO release by HUVEC. (A) HUVEC were exposed to the indicated increasing concentrations of factor Xa in the presence (▪) or in the absence (□) of the NO synthase inhibitor l-NAME (100 μM) for 15 min at 37°C before cGMP determination. (B) HUVEC were treated with factor Xa (2.5 μg/ml), DEGR-factor Xa (2.5 μg/ml), factor IXa (2.5 μg/ml), fibrinogen (2.5 μg/ml), or factor Xa preincubated with TAP (250 μM) for 15 min on ice before determination of cGMP. Histamine (10 μM) stimulation of NO release by HUVEC was not significantly affected by TAP alone (13.2 ± 2.2 versus 10.4 ± 0.6 pmol/mg of protein, n = 4). (C) Thrombin (1 unit/ml) or factor Xa (2.5 μg/ml) were incubated with hirudin (2.5 unit/ml) for 15 min on ice and added to HUVEC for an additional 15 min of incubation before cGMP determination. (D) HUVEC were incubated with the factor Xa-derived inter-EGF peptide Leu⁸³–Leu⁸⁸, which blocks ligand binding to EPR-1 (20), a control scrambled peptide KFTGRLL(s), anti-EPR-1 monoclonal antibody 13E5 (50 μg/ml), or control nonbinding IgG 14E11 (50 μg/ml). After a 30-min incubation at 37°C, cells were stimulated with factor Xa (2.5 μg/ml) and cGMP was determined. Data are presented as mean ± SEM (n = 3–11; P < 0.05).