Membrane binding and anticoagulant properties of protein S natural variants.
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.
Baroni
Marcello
M
Department of Biochemistry and Molecular Biology, ICSI, University of Ferrara, Ferrara, Italy. brnmcl@unife.it
Pavani
Giulia
G
Marescotti
Diego
D
Kaabache
Tahar
T
Borgel
Delphine
D
Gandrille
Sophie
S
Marchetti
Giovanna
G
Legnani
Cristina
C
D'Angelo
Armando
A
Pinotti
Mirko
M
Bernardi
Francesco
F
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
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