Membrane binding and anticoagulant properties of protein S natural variants |
| |
Authors: | Marcello Baroni Giulia Pavani Tahar Kaabache Sophie Gandrille Cristina Legnani Mirko Pinotti |
| |
Institution: | a Department of Biochemistry and Molecular Biology, ICSI, University of Ferrara, Ferrara, Italy b Coagulation Service and Thrombosis Research Unit, IRCCS H S. Raffaele, Milano, Italy c INSERM Unité de Recherche 765, Paris, France d Univ Paris-Sud, IFR 141, Faculté de Pharmacie, Orsay, F-91405, Paris, France e AP-HP Hôpital Européen Georges Pompidou, Service d'Hématologie Biologique, Paris; Université Paris Descartes, UFR des Sciences Pharmaceutiques et Biologiques, Paris, France f Dept. Angiology and Blood Coagulation, University Hospital S. Orsola-Malpighi, Bologna, Italy |
| |
Abstract: | IntroductionProtein S (PS) is a vitamin K-dependent plasma glycoprotein with a key role in the control of coagulation pathway on phospholipid membranes. We compared anticoagulant and membrane binding properties of PS altered by natural mutations (N217S, DelI203D204) affecting the epidermal growth factor like-domain 4 (EGF4) and causing PS deficiency.Materials and methodsBinding of recombinant, immunopurified PS (rPS) to several conformation-specific antibodies, to C4BP and to phospholipid liposomes was investigated by ELISA. PS binding to cells was analysed by flow cytometry. PS inhibitory activities were studied in plasma and purified systems.Results and conclusionsConformational changes produced by mutations were revealed by mapping with calcium-dependent antibodies. The immunopurified recombinant mutants (rPS) showed at 200-800nM concentration reduced inhibition of coagulation (rPS217S, 10.2-17.3%; rPSDelI203D204, 5.8-8.9% of rPSwt) in FXa 1-stage clotting assay with APC. In thrombin generation assays the inhibition of ETP was reduced to 51.6% (rPS217S) and 24.1% (rPSDelI203D204) of rPSwt. A slightly shortened lag time (minutes) was also observed (rPS217S, 2.58; rPSDelI203D204, 2.33; rPSwt, 3.17; PS deficient plasma, 2.17).In flow cytometry analysis both mutants efficiently bound apoptotic cells in adhesion or in suspension. The affinity for phosphatidylserine-rich vesicles (apparent Kd: rPSwt 27.7 ± 1.6 nM, rPS217S 146.0 ± 16.1 nM and rPSDelI203D204 234.1 ± 28.1 nM) was substantially increased by membrane oxidation (10.9 ± 0.6, 38.2 ± 3.5 and 81.4 ± 6.0 nM), which resulted in a virtually normal binding capacity of mutants at physiological PS concentration.These properties help to define the molecular bases of PS deficiency, and provide further elements for PS-mediated bridging of coagulation and inflammation. |
| |
Keywords: | Thrombophilia protein S deficiency protein S domains protein S activity protein S-membrane binding oxidized phospholipids |
本文献已被 ScienceDirect 等数据库收录! |
|