Proteina S
Thromb Res. 2010 Feb;125(2):e33-9. Epub 2009 Oct 29.
Membrane binding and anticoagulant properties of protein S natural variants.
Baroni M, Pavani G, Marescotti D, Kaabache T, Borgel D, Gandrille S, Marchetti G, Legnani C, D'Angelo A, Pinotti M, Bernardi F.
Department of Biochemistry and Molecular Biology, ICSI, University of Ferrara, Ferrara, Italy. brnmcl@unife.it
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 200-800 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.
Copyright 2009 Elsevier Ltd. All rights reserved.
PMID: 19878975 [PubMed - indexed for MEDLINE]
Molecular bases of type II protein S deficiency: the I203-D204 deletion in the EGF4 domain alters GLA domain function.
Baroni M, Mazzola G, Kaabache T, Borgel D, Gandrille S, Vigano' D'Angelo S, Marchetti G, di Iasio MG, Pinotti M, D'Angelo A, Bernardi F.
Department of Biochemistry and Molecular Biology, ICSI, University of Ferrara, Ferrara, Italy.
OBJECTIVE: To characterize the first type II protein S (PS) deficiency affecting the epidermal growth factor (EGF)4 domain, a calcium-binding module with a poorly defined functional role. PATIENTS: The proband suffered from recurrent deep vein thrombosis and showed reduced PS anticoagulant activity (31%), and total, free PS antigen and C4bBP levels in the normal range. RESULTS: Reverse transcription-polymerase chain reaction analysis showed the presence of the IVSg-2A/T splicing mutation that, by activating a cryptic splice site, causes the deletion of codons Ile203 and Asp204. Free PS, immunopurified from proband's plasma, showed an altered electrophoretic pattern in native condition or in the presence of Ca2+. The recombinant PS (rPS) mutant showed reduced anticoagulant (<10%) and activated protein C-independent activities (24-38%) when compared with wild-type rPS (rPSwt). Binding of the rPS variant to phospholipid vesicles (Kd 235.7 +/- 30.8 nM, rPSwt; Kd 15.2 +/- 0.9 nM) as well as to Ca2+-dependent conformation-specific monoclonal antibodies for GLA domain was significantly reduced. CONCLUSIONS: These data aid in the characterization of the functional role of the EGF4 domain in the anticoagulant activities of PS and in defining the thrombophilic nature of type II PS deficiency.
PMID: 16409468 [PubMed - indexed for MEDLINE]
Detection and characterization of seven novel protein S (PROS) gene lesions: evaluation of reverse transcript-polymerase chain reaction as a mutation screening strategy.
Formstone CJ, Wacey AI, Berg LP, Rahman S, Bevan D, Rowley M, Voke J, Bernardi F, Legnani C, Simioni P, Girolami A, Tuddenham EG, Kakkar VV, Cooper DN.
Charter Molecular Genetics Laboratory, Thrombosis Research Institute, London, UK.
The molecular genetic analysis of protein S deficiency has been hampered by the complexity of the protein S (PROS) gene and by the existence of a homologous pseudogene. In an attempt to overcome these problems, a reverse transcript-polymerase chain reaction (RT-PCR) mutation screening procedure was developed. However, the application of this mRNA-based strategy to the detection of gene lesions causing heterozygous type I protein S deficiency appears limited owing to the high proportion of patients exhibiting absence of mRNA derived from the mutation-bearing allele ("allelic exclusion"). Nevertheless, this strategy remains extremely effective for rapid mutation detection in type II/III protein S deficiency. Using the RT-PCR technique, a G-to-A transition was detected at position +1 of the exon IV donor splice site, which was associated with type I deficiency and resulted in both exon skipping and cryptic splice site utilization. No abnormal protein S was detected in plasma from this patient. A missense mutation (Asn 217 to Ser), which may interfere with calcium binding, was also detected in exon VIII in a patient with type III protein S deficiency. A further three PROS gene lesions were detected in three patients with type I deficiency by direct sequencing of exon-containing genomic PCR fragments: a single base-pair (bp) deletion in exon XIV, a 2-bp deletion in exon VIII, and a G0to-A transition at position -1 of the exon X donor splice site all resulted in the absence of mRNA expressed from the disease allele. Thus, the RT-PCR methodology proved effective for further analysis of the resulting protein S-deficient phenotypes. A missense mutation (Met570 to Thr) in exon XIV of a further type III-deficient proband was subsequently detected in this patient's cDNA. No PROS gene abnormalities were found in the remaining four subjects, three of whom exhibited allelic exclusion. However, the father of one such patient exhibiting allelic exclusion was subsequently shown to carry a nonsense mutation (Gly448 to Term) within exon XII.
PMID: 7545463 [PubMed - indexed for MEDLINE]
Protein S mRNA in patients with protein S deficiency.
Sacchi E, Pinotti M, Marchetti G, Merati G, Tagliabue L, Mannucci PM, Bernardi F.
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, IRCCS Maggiore Hospital, Italy.
A protein S gene polymorphism, detectable by restriction analysis (BstXI) of amplified exonic sequences (exon 15), was studied in seven Italian families with protein S deficiency. In the 17 individuals heterozygous for the polymorphism the study was extended to platelet mRNA through reverse transcription, amplification and densitometric analysis. mRNA produced by the putative defective protein S genes was absent in three families and reduced to a different extent (as expressed by altered allelic ratios) in four families. The allelic ratios helped to distinguish total protein S deficiency (type I) for free protein S deficiency (type IIa) in families with equivocal phenotypes. This study indicates that the study of platelet mRNA, in association with phenotypic analysis based upon protein S assays in plasma, helps to classify patients with protein S deficiency.
PMID: 7482397 [PubMed - indexed for MEDLINE]
Study of a protein S gene polymorphism at DNA and mRNA level in a family with symptomatic protein S deficiency.
Marchetti G, Legnani C, Patracchini P, Gemmati D, Ferrati M, Palareti G, Coccheri S, Bernardi F.
Centro di Studi Biochimici delle Patologie del Genoma Umano, Università di Ferrara, Italy.
A protein S gene polymorphism, detectable by restriction analysis of amplified exonic sequences, was investigated in a family with members affected by protein S deficiency, deep vein thrombosis and ictus. The clinical laboratory findings as well as RFLP analysis were consistent with the presence of a type WP III protein S deficiency clearly marked by a polymorphic allele, thus enabling us to determine the carrier status in several subjects. The RFLP analysis, extended to platelet mRNA after reverse transcription and amplification, demonstrated that the mRNA produced by the putative defective gene was present in a subject affected by thrombosis.
PMID: 7902733 [PubMed - indexed for MEDLINE]
