Inhibition of PDGF-BB by Factor VII-activating protease (FSAP) is neutralized by protease nexin-1, and the FSAP-inhibitor complexes are internalized via LRP

Abstract

FSAP (Factor VII-activating protease) can inhibit neointima formation and VSMC (vascular smooth-muscle cell) proliferation by cleavage of PDGF-BB (platelet-derived growth factor-BB). Negatively charged polyanions lead to autoactivation of the FSAP, but no information is available concerning the potential regulation of FSAP activity and its metabolism in the vessel wall. In the present study, we demonstrate that the enzymatic activity of FSAP can be inhibited by the serine protease inhibitor, PN-1 (protease nexin-1), that is found in the vasculature. This leads to the loss of the inhibitory effect of FSAP on PDGF-BB-mediated DNA synthesis and mitogen-activated protein kinase phosphorylation in VSMCs. The FSAP-PN-1 complexes bind to the LRP (low-density lipoprotein receptor-related protein) and are subsequently internalized. This binding is inhibited by receptor-associated protein, an antagonist of LRP, as well as heparin. While PDGFbetaR (PDGFbeta receptor) is internalized by an LRP-dependent mechanism after stimulation of cells by PDGF-BB, the FSAP-PN-1 complex neither influenced PDGF-BB-mediated phosphorylation of PDGFbetaR nor its internalization via LRP. Hence, PN-1 inhibits the enzymatic activity of FSAP and neutralizes its effect on PDGF-BB-mediated VSMC proliferation. The FSAP-inhibitor complexes are internalized via LRP without influencing the PDGF-BB signal transduction pathway.

Figure 1. Inhibition of FSAP by PN-1 and complex formation

(A) FSAP (1 μg/ml) was pre-incubated with 0.5–2.0 μg/ml PN-1 in the absence or presence of 10 μg/ml heparin for 30 min. FSAP activity was measured with the conversion of 0.2 mM specific chromogenic substrate S-2288. The results are shown as means±S.D. (n=3). (B) FSAP (30 ng/lane) was pre-incubated with 10–80 ng of PN-1, in the presence of 10 μg/ml heparin. Western blot analysis was performed using two monoclonal antibodies against FSAP. These experiments were repeated three times with similar results. Molecular-mass markers are shown to the right-hand side of the Figure in kDa.

Figure 2. FSAP–PN-1 complex binding to LRP

(A) FSAP (1 μg/ml) was pre-incubated for 30 min with 2 μg/ml PN-1 to allow complex formation, and the binding to immobilized LRP (1 μg/ml) was measured in absence (grey bars) or presence (open bars) of 10 μg/ml heparin using a monoclonal antibody against FSAP. The results are shown as means±S.D. (n=3). (B) FSAP (1 μg/ml) was pre-incubated with 0.05–2 μg/ml PN-1 for 30min to allow complex formation. Binding of the FSAP–inhibitor complexes to immobilized LRP (1 μg/ml) and BSA was measured using a specific monoclonal antibody against FSAP. The results are shown as means±S.D. (n=3). (C) FSAP (1 μg/ml) was pre-incubated with 2 μg/ml PN-1 for 30 min to allow complex formation. Binding of the FSAP–inhibitor complexes to immobilized LRP (1 μg/ml), without (grey bars) or with (striped columns) 10 μg/ml RAP, and control buffer containing BSA (2 μg/ml) was measured using a specific monoclonal antibody against FSAP. The results are shown as means±S.D. (n=3). These experiments were repeated three times with similar results.

Figure 3. Binding of FSAP–PN-1 complex to LRP and the influence of this complex on PDGFβR distribution in VSMCs

Buffer control (a and e), FSAP (1 μg/ml) alone (b and f) and PN-1 (2μg/ml) alone (c and g) or FSAP and PN-1 together (d and h) were pre-incubated for 30 min to allow complex formation and added to VSMC without (a–d) or with RAP (10 μg/ml) (e–h) for 45 min. FSAP was detected by FITC-labelled secondary antibody (green), LRP by Rhodamine Red-X labelled secondary antibody (red) and nuclei by DAPI staining (blue) (a–h). The yellow colour indicates co-localization of FSAP and LRP and is highlighted by white arrows. PDGF-BB (20 ng/ml) was pre-incubated with buffer (i), FSAP (1 μg/ml) (j), PN-1 (2 μg/ml) (k) or FSAP–PN-1 complex (l) for 60 min at 37 °C. VSMCs were incubated with these mixtures for 30 min on 37 °C. PDGFβR was detected by Rhodamine Red-X labelled secondary antibody (red) and nuclei were stained by DAPI (blue) (i–l). The white arrows highlight examples of intracellular accumulated PDGFβR. To examine the co-localization of FSAP and PDGFβR cells were prepared as in (l), and FSAP was detected by FITC-labelled secondary antibody (green), PDGFβR with Rhodamine Red-X labelled secondary antibody (red) and nuclei were stained by DAPI (blue). Yellow colour and white arrows highlight examples of intracellular co-localization of FSAP–PN-1 complex with PDGFβR (m). Scale bar=20 μm. This staining pattern was observed in 80% of cells on a slide and this experiment was repeated twice.

Figure 4. Influence of LRP on the inhibitory effect of FSAP on PDGF-BB-dependent VSMC activation

(A) In serum-free medium containing 10 μg/ml heparin, VSMCs were stimulated with PDGF-BB (20 ng/ml), PDGF-BB pre-incubated with FSAP (1 μg/ml) or PDGF-BB pre-incubated with FSAP–PN-1 complex in the absence or presence of RAP (10 μg/ml) for 15 min. Western blot analysis was performed using a monoclonal antibody against phospho-42/44^MAPK p42/44 MAPK). As a loading control, a polyclonal antibody against total MAPK was used on the stripped membrane. Molecular-mass markers are shown to the right-hand side of the Figure in kDa. (B) As in the above experiment, VSMCs were stimulated in the absence (grey bars) or presence (open bars) 10 μg/ml RAP. DNA synthesis was measured using a kit to measure BrdU incorporation into newly synthesized DNA. The results are shown as means±S.D. (n=3), and similar results were obtained in three independent experiments.