Search

Type III protein S (PS) deficiency, characterized by low levels of free PS and normal total PS levels, is often associated with the Ser 460 to Pro substitution. However, some patients bearing this mutation have normal PS levels, suggesting that another gene defect may account for this phenotype. We postulated that this defect was located in the C4b-BP β-chain gene (C4BPB) and searched for a mutation in the coding regions of this gene in 35 propositi with type III PS deficiency and bearing the Ser 460 to Pro mutation. No mutations explaining the phenotype of type III PS deficiency were identified. We did, however, find two frequent nucleotide changes, one being located in the donor splice site of intron d and the second in the codon corresponding to Asn 137. We used these two polymorphisms to establish C4BPB gene haplotype in five informative type III PS-deficient families and exclude a role of the C4BPB gene in this phenotype of three of them. Finally, increased C4b-BP β-chain levels were not responsible for the phenotype of type III PS deficiency as the C4BPB haplotype did not correlate with C4b-BP β-chain levels. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: 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. Materials and 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. Results and conclusions: Conformational 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±1.6nM, rPS217S 146.0±16.1nM and rPSDelI203D204 234.1±28.1nM) was substantially increased by membrane oxidation (10.9±0.6, 38.2±3.5 and 81.4±6.0nM), 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. [Copyright &y& Elsevier]