Research Article

Ecotin-Like ISP of L. major Promastigotes Fine-Tunes Macrophage Phagocytosis by Limiting the Pericellular Release of Bradykinin from Surface-Bound Kininogens: A Survival Strategy Based on the Silencing of Proinflammatory G-Protein Coupled Kinin B2 and B1 Receptors

Figure 4

Effect of ISP2 on DXS-induced contact phase activation of human plasma. Citrated human platelet free plasma diluted 1 : 20 in buffer (described in methods) was supplemented with (i) 4 μM Abz-MTEMARRPQ-EDDnp (a, c) or 4 μM Abz-GFSPFRSVTVQ-EDDnp (b, d), intramolecular quenched fluorescent substrates whose sequences span the N-terminal or the C-terminal flanking sites (resp.) of BK in mHK (ii) dextran sulfate 500 kDa (DXS; 20 nM). The substrate was also tested in the absence of DXS (Control). Assays with the elastase inhibitor (NEi—MeOSuc-AAPV-CMK-10, 20, and 30 μM), the synthetic PKa inhibitor (PKSI-527—5 μM), and the inhibitor of serine peptidase 2 (ISP2—142, 177, 240, and 355 nM) were performed using two different schemes. (a, b) PKSI or the elastase inhibitor was added to the plasma together with DXS and the substrate. (c, d) PKSI or ISP2 was preincubated with plasma for 15 min, at 37°C, prior to the addition of DXS and the substrate. Hydrolysis was followed by measuring the fluorescence at  nm and  nm (up to 1800 seconds). The plot shows the increase of fluorescence with time, reflecting substrate hydrolysis. The values in the figures represent the mean ± SE of duplicate determinations performed within 1 representative experiment of 2.