Biomedical subjects

Marie-Christine Bouton

Publications and source records attributed to Marie-Christine Bouton.

6 recordsLinked to original sources

PubMed2006

Modulation of protease nexin-1 activity by polysaccharides.

Protease nexin-1 (PN-1) is a non-circulating pericellular serpin expressed by vascular cells. PN-1 inhibits different proteases but when associated with glycosaminoglycans, its activity is mainly directed towards thrombin. Fucoidans are sulphated polysaccharides which can interact with several serpins and have antithrombotic and anticoagulant properties in vivo with a lower hemorrhagic risk than heparin. The purpose of this study was to compare the effects of low (LMW) or high molecular weight (HMW) fucoidans to those of standard heparin and LMW heparin on PN-1 properties. Using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and affinity coelectrophoresis, we observed that polysaccharides bound to thrombin, PN-1 and the thrombin/PN-1 complex. Progress curve kinetics showed that LMW and HMW fucoidans accelerate thrombin inhibition by PN-1 (111 and 402 fold, respectively) whereas the acceleration by LMW heparin and standard heparin was only of 36- and of 307-fold, respectively. Moreover, the formation of PN-1/(125)I-thrombin complex was increased in the presence of heparin, HMW and LMW fucoidans, but barely by LMW heparin. The dose response followed a bell shape curve, again suggesting the formation of ternary complexes between thrombin, PN-1 and polysaccharides. We also investigated the ability of polysaccharides to remove PN-1 bound to the cell membrane of smooth muscle cells in culture. PN-1 was detached by fucoidans and heparins and was still able to inhibit thrombin. In conclusion, fucoidans reduce cell-associated PN-1 and thrombin/PN-1 complexes and increase the antithrombin activity of PN-1. The capacity of PN-1 to regulate the pericellular activity of thrombin amongst other proteases reinforces the therapeutical interest of fucoidans.

Amyloid beta-Protein Precursor↗

PubMed2004

A paradoxical pro-apoptotic effect of thrombin on smooth muscle cells.

Whereas thrombin (below 10 nM) is a potent mitogen, recent studies report that exposure to higher doses of thrombin could lead to apoptosis of neurons and tumor cells. Our results show that prolonged exposure (> or = 24 h) to thrombin (50-100 nM) exerts a pro-apoptotic effect on cultured vascular smooth muscle cells (VSMCs). This phenomenon depends on thrombin serine-protease activity but is independent of PAR-1 and -4 activation and subsequent signaling. The parallel occurrence of cell retraction and cleavage of fibronectin suggests that thrombin-induced apoptosis is consecutive to pericellular proteolysis. These data point to a new potential action of thrombin in the cardiovascular system.

Animals↗

PubMed2004

Protease nexin-1: a cellular serpin down-regulated by thrombin in rat aortic smooth muscle cells.

Protease nexin-1 (PN-1), a potent inhibitor of serine proteases, is present in vascular cells and forms complexes with thrombin, plasminogen activators, and plasmin. We examined the effect of thrombin on PN-1 expression by rat aortic smooth muscle cells (RASMCs). PN-1 expression was determined by measuring protein and mRNA levels, using respectively immunoblotting and semi-quantitative reverse transcriptase polymerase chain reaction (PCR). Thrombin down-regulated PN-1 expression in a dose- and time-dependent manner. This effect was mediated via the interaction of thrombin with its receptor protease activated receptor (PAR-1) since the peptide thrombin receptor activating peptide (TRAP) reduced PN-1 expression. PN-1 secreted by smooth muscle cells remained essentially associated to cell-surface glycosaminoglycans and was released from the cell surface by heparin. A lower amount of PN-1 was released by heparin from TRAP-stimulated versus unstimulated cells and correlated with a decreased capacity to inhibit thrombin. In addition, the ability to generate peri-cellular plasmin was increased in cells with a low PN-1 expression. Pre-treatment of smooth muscle cells with cycloheximide abolished the reduction of PN-1 expression by thrombin. Furthermore, conditioned media from thrombin-treated cells reproduced the effect of thrombin, suggesting that thrombin acted via the induction of auto/paracrine mediator(s). We observed that fibroblast growth factor-2 (FGF-2)-neutralizing antibodies abolished thrombin effect whereas FGF-2 reproduced it, indicating that FGF-2 is one of the involved mediator. Together, these results indicate that (i) PN-1 modulates the activity of endogenous and exogenous serine proteases in RASMCs, (ii) thrombin down-regulates PN-1 expression and thus may increase its own activity on cells.

Amyloid beta-Protein Precursor↗

PubMed2003

Protease nexin-1 inhibits plasminogen activation-induced apoptosis of adherent cells.

Degradation of adhesive glycoproteins by plasmin is implicated in cell migration. In this study, we further explored the role of plasminogen activation in cell adhesion and survival and show that uncontrolled plasminogen activation at the cell surface may induce cell detachment and apoptosis. We hypothesized that this process could be prevented in adherent cells by expression of protease nexin-1, a potent serpin able to inhibit thrombin, plasmin, and plasminogen activators. Using two- and three-dimensional culture systems, we demonstrate that Chinese hamster ovary fibroblasts constitutively express tissue-type plasminogen activator and efficiently activate exogenously added plasminogen in a specific and saturable manner (K(m) = 46 nm). The formation of plasmin results in proteolysis of fibronectin and laminin, which is followed by cell detachment and apoptosis. Protease nexin-1 expressed by transfected cells significantly inhibited the activity of plasmin and tissue-type plasminogen activator via the formation of inhibitory complexes and prevented cell detachment and apoptosis. In conclusion, protease nexin-1 may be an important anti-apoptotic factor for adherent cells. This cell model could be a useful tool to evaluate therapeutic agents such as serpins in vascular pathologies involving pericellular protease-protease inhibitor imbalance.

Amyloid beta-Protein Precursor↗

PubMed2003

The serpin protease-nexin 1 is present in rat aortic smooth muscle cells and is upregulated in L-NAME hypertensive rats.

OBJECTIVE: Protease-nexin 1 (PN-1) belongs to the serpin superfamily and behaves as a specific thrombin inhibitor in the pericellular environment. Little is known about PN-1 expression and its regulation in the vascular system. In this study, we examined the expression of functionally active PN-1 in vitro in rat aortic smooth muscle cells and in vivo in rat arterial media and its regulation in hypertensive rats. METHODS AND RESULTS: The vascular PN-1 formed specific covalent complexes with thrombin involving the catalytic site of the protease, and heparin increased the formation of these complexes. We also demonstrated PN-1 in rat arterial media by immunohistochemical staining. Moreover, we examined in vivo vascular expression of PN-1 in a model of chronic hypertension induced by long-term administration of N(G)-nitro-L-arginine methyl ester (L-NAME). Marked increases in PN-1 mRNA (3-fold) and protein (2-fold) were observed after 2 months of hypertension. Increased expression of PN-1 in the vascular wall was associated with an increase in the formation of complexes between radiolabeled-thrombin and PN-1, indicating that PN-1 was functional. CONCLUSIONS: PN-1 may thus participate in the mechanisms that regulate thrombin activity in the vessel wall.

Administration, Oral↗

PubMed2003

Thrombin interaction with platelet membrane glycoprotein Ib alpha.

The interaction of thrombin with platelet glycoprotein Ibalpha (GPIb alpha) is required for optimal platelet activation. The crystal structures of platelet GPIb alpha bound to thrombin reported by Dumas et al. and Celikel et al. both reveal the simultaneous interaction of GPIb alpha with thrombin exosites I and II but differ markedly regarding how the two proteins interact. The possible consequences on thrombus formation of thrombin interacting with GPIb alpha are discussed in light of these new data.

Animals↗