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.
Richard
Benjamin
B
INSERM U698, CHU Xavier Bichat, 46 rue Henri Huchard, 75877 Paris, France. brichard@bichat.inserm.fr
Bouton
Marie-Christine
MC
Loyau
Stéphane
S
Lavigne
Damien
D
Letourneur
Didier
D
Jandrot-Perrus
Martine
M
Arocas
Véronique
V
D016564:Amyloid beta-Protein Precursor / Q000187:drug effects* / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006495:Heparin, Low-Molecular-Weight / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000648:ultrastructure; D011134:Polysaccharides / Q000494:pharmacology*; D058255:Protease Nexins; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D011956:Receptors, Cell Surface / Q000187:drug effects* / Q000378:metabolism; D058258:Serpin E2; D013917:Thrombin / Q000037:antagonists & inhibitors
D016564:Amyloid beta-Protein Precursor; D006495:Heparin, Low-Molecular-Weight; D011134:Polysaccharides; D058255:Protease Nexins; D011956:Receptors, Cell Surface; C545486:SERPINE2 protein, human; D058258:Serpin E2; D006493:Heparin; C007789:fucoidan; D013917:Thrombin