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Biomedical subjects

D Borgel

Publications and source records attributed to D Borgel.

At least 19 recordsLinked to original sources
PubMed2006

Molecular bases of type II protein S deficiency: the I203-D204 deletion in the EGF4 domain alters GLA domain function.

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.

Adult↗
PubMed2005

Sepsis and coagulation.

PURPOSE OF REVIEW: There is considerable evidence that dysregulation of the coagulation and fibrinolytic systems plays a major role in the pathophysiology of severe sepsis, with a special focus on the protein C system. Conversely, there is an approval for use of recombinant human activated protein C in the more severe patients. This review highlights recent findings about the biology of the protein C system and of other important coagulation components such as tissue factor, platelets, and protein S, with an effort to link fundamental data and recent clinical findings. RECENT FINDINGS: There is a better comprehension of the biology of the thrombomodulin-protein C-endothelial protein C receptor complex, and mainly of its cellular effects via the protease activated receptor 1 receptor and of its implication in the generation of anticoagulant microparticles. The implications of other important agents such as platelets and von Willebrand factor, tissue factor, and protein S are also becoming increasingly evident, both from experimental and clinical studies. From a clinical point of view, the more immediately promising approach could be the ability to identify the fraction of severe sepsis patients exhibiting an impaired ability to activate protein C, raising the possibility to select the better candidates for activated protein C infusion. SUMMARY: The comprehension of the protein C pathway is undoubtedly progressing both in experimental and clinical settings. In parallel, some promising other coagulant pathways are also under investigation in the sepsis context, with a hope for major clinical implications in the future.

Animals↗
PubMed2003

Familial thrombophilia is an oligogenetic disease: involvement of the prothrombin G20210A, PROC and PROS gene mutations.

Population-based case-control studies and cases previously published suggest that the prothrombin G20210A mutation is a weak risk factor for thrombosis, leading to clinical expression mainly in the presence of other risk factors. We report the results of plasma and genetic analyses performed in a 13-year-old symptomatic boy homozygous for the 20210A allele and in his family, which are in accordance with this suggestion. These analyses demonstrated the presence of several PROC (R-5W, R87H) and PROS (R60C, T103N) gene mutations in this family. These additional mutations have modulating effects on clinical expression of the G20210A mutation. The present family study illustrates the concept of 'mild' mutation and the hypothesis that familial thrombophilia is a multifactorial disease.

Adolescent↗
PubMed2001

Implication of protein S thrombin-sensitive region with membrane binding via conformational changes in the gamma-carboxyglutamic acid-rich domain.

In the vitamin K-dependent protein family, only protein S (PS) contains a thrombin-sensitive region (TSR), located between the domain containing the gamma-carboxyglutamic acid and the first epidermal growth factor-like domain. To better define the role of TSR in the PS molecule, we expressed a recombinant human PS (rHPS) and its analogue lacking TSR (rTSR-less), and prepared factor Xa- and thrombin-cleaved rHPS. A peptide reproducing TSR (TSR-peptide) was also synthesized in an attempt to obtain direct evidence of the domain involvement in PS anticoagulant activity. In a coagulation assay, both rTSR-less and factor Xa-cleaved PS were devoid of activated protein C cofactor activity. The TSR-peptide did not inhibit rHPS activity, showing that TSR must be embedded in the native protein to promote interaction with activated protein C. The binding of rHPS to activated platelets and to phospholipid vesicles was not modified after factor Xa- or thrombin-mediated TSR cleavage, whereas the binding of rTSR-less was markedly reduced. This suggested a role for TSR in conferring to PS a strong affinity for phospholipid membranes. TSR-peptide did not directly bind to activated platelets or compete with rHPS for phospholipid binding. The results of the present study show that TSR may not interact directly with membranes, but probably constrains the gamma-carboxyglutamic acid-rich domain in a conformation allowing optimal interaction with phospholipids.

1-Carboxyglutamic Acid↗
PubMed2001

Three-dimensional model of the SHBG-like region of anticoagulant protein S: new structure-function insights.

Protein S (PS) is a vitamin K-dependent glycoprotein that consists of several modules including a C-terminal sex hormone-binding globulin (SHBG)-like domain that has been subdivided into two laminin LG-type domains. The SHBG-like region of PS is known to bind to a complement regulator molecule, C4b-binding protein (C4BP), coagulation factor Va (FVa) and receptor tyrosine kinases. Inherited PS deficiency has been associated with thromboembolic disease. Yet, study of the mechanisms by which the SHBG-like region of PS serves its essential functions has so far been hampered because of the lack of structural information. Recently, the three-dimensional (3D) structure of LG domains from plasma SHBG, laminin and neurexin have been reported and were found related to the pentraxin family. We used these X-ray structures to build homology models of the SHBG-like region of human PS. We then analyzed previously reported experimental/clinical data in the light of the predicted structures. A potential calcium-binding site is found in the first LG domain of PS and D292 could play a role in this process. This region is close to the interface between the two LG domains and is also surrounded by segments that have been suggested by synthetic peptide studies to be important for C4BP or FVa binding. The 39 point mutations linked to PS deficiencies or reported as neutral variants were rationalized in the 3D structure. Proteins 2001;43:203-216.

Amino Acid Sequence↗
PubMed2000

Expression and characterization of recombinant protein S with the Ser 460 Pro mutation.

To characterize the putative biochemical modifications induced by the Ser 460 to Pro (Heerlen) mutation in protein S (PS), we expressed both wild-type (wt) and mutated recombinant PS in HEK cells. In SDS-polyacrylamide gels, r-PS Heerlen migrated at 71 kDa whereas r-wt PS migrated at 73 kDa, a difference abolished after deglycosylation by N-glycosidase, suggesting that the Ser 460 Pro mutation abolishes N-glycosylation of Asn 458. The affinity of r-wt PS and r-PS Heerlen for C4b-binding protein (C4b-BP) and for phospholipid vesicles was similar. Neither the enhancement of APC-dependent prolongation of the APTT, nor the specific enhancement of FVa and FVIIIa proteolysis by APC in purified systems was affected by the mutation. However, the Ser 460 Pro mutation induced a slight conformational change in the SHBG domain of the PS molecule, as shown by reduced binding affinity for monoclonal antibodies. The type III phenotype associated with the Heerlen mutation might thus result from a slightly modified rate of synthesis or catabolism. The resulting moderate decrease in the circulating PS concentration may modify the equilibrium between free PS and C4b-BP/PS complexes.

Amino Acid Substitution↗
PubMed2000

Characterization of cleaved plasma protein S with a monoclonal antibody-based assay.

A monoclonal antibody (mAb 5A5G2) recognized cleaved plasma protein S (PS) but not uncleaved PS. Interestingly, mAb 5A5G2 did not recognize thrombin-cleaved recombinant PS. Microsequencing of cleaved plasma PS showed a Q-S-T-N amino-terminal sequence, inferring cleavage after the Arg 60 residue. The mAb epitope was located within the sequence encompassing residues 61 to 73, i.e. the carboxy-terminal part of the thrombin-sensitive region (TSR). We used this mAb to develop an ELISA assay to quantify in vivo cleaved PS. In plasma from 10 normal subjects, about 10% of PS was cleaved (7.1% to 15.4%), with a more than 2-fold increase in the corresponding sera. We found increased levels of cleaved PS in 8 patients with disseminated intravascular coagulation (DIC) and decreased levels in 22 patients on long-term oral anticoagulant therapy, whereas cleaved PS levels were similar in 8 hemophiliacs and the 10 normal subjects. Cleaved PS levels did not correlate with prothrombin fragment 1+2 levels released after cleavage by FXa in any of the groups, suggesting that circulating FXa is not the main factor involved in the production of cleaved PS in vivo.

Antibodies, Monoclonal↗
PubMed1997

Protein S deficiency.

The protein C (PC) pathway, with its cofactor protein S (PS), is an important natural antithrombotic mechanism. Patients with phenotypic PS deficiency may develop recurrent thrombosis during adulthood, with a probability of remaining free of thrombosis of about 50% at age 45. The molecular basis for hereditary PS deficiencies is highly heterogeneous, with a large spectrum of mutations that have various effects on the expression of the relevant allele.

Anticoagulants↗
PubMed1997

Protein C and protein S deficiencies.

The protein C (PC) pathway, with its cofactor protein S (PS), is an important natural antithrombotic mechanism. Both PC and PS deficiencies have been implicated in thrombophilia. The molecular basis for hereditary PC and PS deficiencies is highly heterogeneous, with a large spectrum of mutations that have various effects on the expression of the relevant allele. A small subset of patients who are homozygous or compound heterozygous for a PC gene mutation have severe thrombotic complications at birth, whereas onset occurs later in the other cases. Patients heterozygous for a PC or PS gene abnormality may develop recurrent thrombosis during adulthood, with a probability of remaining free of thrombosis of about 50% at age 45. A PC or PS gene defect is associated with the factor V Arg 506 to Gln mutation in 10% to 30% of symptomatic patients, suggesting that clinical expression is controlled by several genes in heterozygous patients.

Chromosome Mapping↗
PubMed1996

Molecular basis for protein S hereditary deficiency: genetic defects observed in 118 patients with type I and type IIa deficiencies. The French Network on Molecular Abnormalities Responsible for Protein C and Protein S Deficiencies.

Circulating protein S (PS) is partly bound to C4b-binding protein, and only free PS can act as a cofactor for protein C (PC), a natural anticoagulant. Two types of PS deficiencies are commonly observed in patients with unexplained thrombosis, and they are characterized by having both a low total PS level and a low free PS level (type I) or by having only a low free PS level (type IIa). To elucidate the genetic mechanisms responsible for these two plasma phenotypes, we screened 118 symptomatic patients with type I or type IIa PS deficiency for a PS gene coding sequence variation. A total of 34 mutations, 17 of which were novel, were identified in 65 propositi (70% in type I and 44% in type IIa). In type I deficiency, 29 different mutations were distributed throughout the coding sequence. In type IIa deficiency, five different missense mutations were clustered in exons XII and XIII, with a Ser 460 to Pro mutation accounting for most cases (82%). This points to a role of the domain encoded by exons XII and XIII in the distribution between bound and free PS. The Ser 460 to Pro mutation was associated with the factor V Arg 506 to Gin mutation or a PC gene mutation in about half the patients, suggesting a cooperative effect on clinical expression.

DNA Mutational Analysis↗
PubMed1996

Five novel mutations of the protein S active gene (PROS 1) in 8 Norman families.

To further elucidate the molecular basis for hereditary thrombophilia, we screened the protein S active gene in 11 families with type I deficiency, using a strategy based on denaturing gradient gel electrophoresis (DGGE) of all the coding sequences. Fragments with an abnormal DGGE pattern were sequenced, and 5 novel mutations were identified in 8 families. The mutations were a 7-nucleotide deletion in exon II, a 4-nucleotide deletion in exon III, a T insertion in exon VII, a C to T transition transforming Leu 259 into Pro and a T to C transition transforming Cys 625 into Arg in 4 families. These mutations were the only sequence variations found in the propositus' gene exons and co-segregated with the plasma phenotype. A total of 28 members of these 8 families were heterozygous for one of the 5 mutations. Twenty-four (58,5%) of the 41 deficient subjects over 18 years of age had clinical thrombophilia, whereas the 13 subjects under 18 were asymptomatic. Of the 28 subjects, 6 (21,5%) were also found to bear the factor V Arg 506 Gln mutation.

Adolescent↗
PubMed1996

First case of sporadic protein S deficiency due to a novel candidate mutation, Ala 484-->Pro, in the protein S active gene (PROS1).

In a series of 16 propositi with symptomatic protein S deficiency and a protein S gene mutation, we identified a sporadic case of a novel mutation that probably affects gene expression. The mutation, a G to C transversion leading to the substitution of Ala 484 by Pro, was not found in the protein S gene of the patient's parents. Transmission of the paternal and maternal protein S alleles was apparently normal, on the basis of the frequent polymorphism in exon XV. We also checked the transmission of chromosomal material by analysing protein C gene polymorphisms, beta-globin gene frameworks and four variable number of tandem repeats (VNTRs). By combining the results of these analyses, we were able to rule out nonpaternity and to confirm the de novo nature of the mutation.

Alanine↗
PubMed1995

The Ser 460 to Pro substitution of the protein S alpha (PROS1) gene is a frequent mutation associated with free protein S (type IIa) deficiency.

A Ser 460 to Pro mutation of protein S (PS), involving a T to C transition in exon XIII of the protein S alpha (PROS1) gene and known as the Heerlen polymorphism, was found in 16 of 85 symptomatic patients with PS deficiency (18.8%) and only 1 of 113 healthy subjects (0.8%). Another frequent polymorphism was described in exon XV of the PROS1 gene, in the codon for Pro 626 (CCA/CCG). We found that Heerlen polymorphism was associated with allele CCA and not with allele CCG, suggesting a probable transmission by a common ancestor. Most subjects bearing the Ser 460 to Pro mutation were deficient in free PS, but had normal total PS levels. Normal levels of the C4b-binding protein (C4b-BP) isoform containing a beta chain (C4b-BP beta +) ruled out increased C4b-BP beta + as a cause of the free-PS deficiency. The binding curves of the mutated (Heerlen) PS on C4b-BP immobilized on microplates were biphasic, suggesting that one molecule of C4b-BP can bind two molecules of Heerlen PS. Because normal PS binds to C4b-BP with 1:1 stoichiometry, this may explain the free-PS deficiency observed in patients carrying the Ser 460 to Pro mutation.

Carrier Proteins↗