title: Membrane binding and anticoagulant properties of protein S natural variants.
abstract: BACKGROUND
  Protein 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.
  
  
  METHODS
  Binding 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.
  
  
  CONCLUSIONS
  Conformational changes produced by mutations were revealed by mapping with calcium-dependent antibodies. The immunopurified recombinant mutants (rPS) showed at 200-800 nM 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, ICSI, University of Ferrara, Ferrara, Italy. brnmcl@unife.it
authors.forename: Marcello
authors.initials: M
authors.lastname: Baroni
authors.forename: Giulia
authors.initials: G
authors.lastname: Pavani
authors.forename: Diego
authors.initials: D
authors.lastname: Marescotti
authors.forename: Tahar
authors.initials: T
authors.lastname: Kaabache
authors.forename: Delphine
authors.initials: D
authors.lastname: Borgel
authors.forename: Sophie
authors.initials: S
authors.lastname: Gandrille
authors.forename: Giovanna
authors.initials: G
authors.lastname: Marchetti
authors.forename: Cristina
authors.initials: C
authors.lastname: Legnani
authors.forename: Armando
authors.initials: A
authors.lastname: D'Angelo
authors.forename: Mirko
authors.initials: M
authors.lastname: Pinotti
authors.forename: Francesco
authors.initials: F
authors.lastname: Bernardi
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D001777:Blood Coagulation / Q000235:genetics; D002460:Cell Line; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism*; D002472:Cell Transformation, Viral; D050716:Complement C4b-Binding Protein; D006224:Cricetinae; D005347:Fibroblasts / Q000166:cytology; D004854:Herpesvirus 4, Human / Q000502:physiology; D006649:Histocompatibility Antigens / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D008081:Liposomes / Q000378:metabolism*; D008566:Membranes / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011486:Protein C / Q000378:metabolism; D011487:Protein Conformation; D017293:Protein S / Q000235:genetics* / Q000378:metabolism*; D018455:Protein S Deficiency / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013539:Simian virus 40 / Q000502:physiology
chemical_list: D000925:Anticoagulants; C055609:C4BPA protein, human; D050716:Complement C4b-Binding Protein; D006649:Histocompatibility Antigens; D008081:Liposomes; D010743:Phospholipids; D011486:Protein C; D017293:Protein S; D011994:Recombinant Proteins