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Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 173-179
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Clinical Chemistry, Lund University,
University Hospital, Malmö, Sweden; and Centre de Genètica
Mèdica i Molecular, Institut de Recerca Oncològica (IRO),
Barcelona, Spain.
To elucidate the molecular background for the heterogeneity in
protein S plasma concentrations observed in protein S deficient individuals, the in vitro synthesis of recombinant protein S missense mutants was investigated. Six different naturally occurring mutations identified in the protein S gene (PROS1) of thrombosis patients were reproduced in protein S cDNA by site directed mutagenesis. Two
mutants, G441C and Y444C (group A), were associated with low total
plasma concentration of protein S. Modestly low protein S was found in
families with R520G and P626L (group B) mutants. T57S and I518M (group
C), which was associated with marginally low protein S, did not
segregate with protein S deficiency in the respective families, raising
doubts as to whether they were causative mutations or rare neutral
variants. The 6 protein S mutants were transiently expressed in COS 1 cells. The Y444C mutant showed the lowest level of secretion (2.5%)
followed by the G441C mutant (40%). Group B demonstrated around 50%
reduction in secretion, whereas group C mutants showed normal
secretion. Pulse-chase experiments demonstrated impaired protein S
processing with intracellular degradation and decreased secretion into
the culture media of group A and B mutants. Interestingly, there was a
good correlation between in vitro secretion and the concentration of
free protein S in the plasma of heterozygous carriers. These results
demonstrate impaired protein S secretion to be an important mechanism
underlying hereditary protein S deficiency and that variations in
protein secretion is a major determinant of the phenotypic
heterogeneity observed in protein S deficiency. (Blood.
2000;95:173-179)
Hereditary thrombophilia is a complex disorder in which
different genetic factors predispose to the development of
thromboembolic events.1-3 Among known genetic risk factors
of thrombosis, many mutations in the genes of antithrombin, protein C
and protein S have been described.4,5 In some cases,
specific mutations or sets of mutations in a certain gene confer
different risks of thrombosis. For instance, in antithrombin
deficiency, mutations affecting the heparin-binding site associate with
a lower prevalence of thrombosis than other types.6 In
general, it is logical to assume that mutations leading to a complete
null allele will be more deletereous than mutations leading to reduced
expression of a functional protein.
Protein S is a plasma glycoprotein with anticoagulant properties,
acting as cofactor to activated protein C (APC) in the degradation of
factor Va and factor VIIIa.7 Protein S has also
prothrombinase inhibitory properties because of its high affinity for
negatively charged phospholipid membranes.8 In human
plasma, protein S forms an equimolar complex with the complement
regulatory protein C4b-binding protein (C4BP).9 The
formation of this complex affects protein S function, as only the free
protein S is active as APC cofactor.10 The protein S-C4BP
interaction is of very high affinity, especially at physiological
calcium concentration.11 In plasma, free protein S
represents the molar excess of protein S concentration to C4BP binding
sites,12 which are found in the beta chain of
C4BP.13,14 Beta chain containing C4BP and protein S
concentrations seem to be regulated coordinately to maintain a fairly
constant concentration of free protein S in plasma.15,16
Protein S is a multimodular protein composed of a Congenital protein S deficiency is an autosomal dominant disease
present in 2% to 6% of patients with thrombosis. The important anticoagulant role of protein S is dramatically illustrated by the
severity of homozygous or compound heterozygous cases
reported.22-24 Heterozygous carriers have an increased
frequency of thrombosis close to 10-fold of that of their healthy
relatives.25,26 In contrast, in population-based studies,
the relative risk of thrombosis associated with low plasma protein S
values is only 2-fold or less.27,28 Protein S deficiency is
diagnosed using laboratory tests for both antigen and activity, and
specific tests for the free-form of protein S have been
developed.29 On the basis of these measurements, protein S
deficiency is classified into 3 classes. Type I deficiency is
characterized by a decrease in the total protein S antigen and,
concomitantly, of free protein S. Type II or qualitative deficiency, is
characterized by normal antigen levels and reduced protein S activity
due to a dysfunctional protein S in plasma. Although a few type II
deficiencies have been reported,30-33 they seem to be
rare.34 Most of the cases initially reported were found to
be caused by resistance to APC.35-37 Type III deficiency,
in turn, is characterized by low free protein S levels, whereas the
total plasma concentration of protein S is normal. The distinction
between type I and type III could be of clinical importance to assess
the risk of thrombophilia in a given individual, but its biologic basis
and significance has been controversial.4,25,38-42 In fact,
it has been proposed that the 2 types of deficiencies may be phenotypic
variants of the same genetic disease.16 However, a
causative mutation in the protein S gene (PROS1) is more
frequently found in type I PS deficient patients/families than in type
III.40,43
To clarify the causes underlying protein S deficiency and the
heterogeneity observed in its manifestation, it is necessary to know
more on the particular effects of naturally occurring PROS1
mutations. To date, only 1 study has characterized a missense protein S mutation causative of type I deficiency, protein S Nagoya, where Arg 474 is changed to Cys.44 In the current study, to further analyze the molecular basis of protein S deficiency, we tested
the particular effects of other naturally occurring missense mutations
identified in thrombophilic protein S-deficient families. We have
produced 6 naturally occurring protein S mutants and studied their
expression in cultured mammalian cells. The results shed light on the
mechanisms involved in the observed phenotypic heterogeneity of protein
S deficiency.
Mutations analyzed
Site-directed mutagenesis and construction of expression vectors
Transient expression of recombinant protein S
Electrophoretic and blotting techniques Media of transiently transfected cells were concentrated 10-fold by filtration with CENTRICON-50 (Amicon, Beverly, MA). The proteins were separated on 7.5% SDS-PAGE gels and transferred to nylon membranes. Protein S was detected by immunoblotting with a rabbit polyclonal antibody and detected colorimetrically with a goat antirabbit IgG antibody conjugated with alkaline phosphatase (DAKO, Glostrup, Denmark).Quantification of protein S secretion Protein S antigen concentration in the conditioned media of transfected cells was measured by an enzyme-linked immunosorbent assay (ELISA), essentially following a described method.19 Culture medium diluted in 50 mmol/L Tris-HCl, 150 mmol/L NaCl, and 2 mmol/L CaCl2; pH 7.5 with 0.1% (w/v) BSA was added to microtiter wells containing immobilized antihuman protein S rabbit polyclonal antibodies. After 2 hours incubation at room temperature (RT), the plates were washed and bound protein S was detected using the HPS67 antihuman protein S monoclonal antibody,19 followed by horseradish peroxidase-coupled antimouse IgG antibodies (DAKO). The plates were developed with 0.65 mg/ mL 1,2-phenylenediamine (DAKO) and 0.012% H2O2 (v/v) diluted in 0.1 mol/L citrate buffer, pH 5.0. After 5 minutes, the reaction was stopped by the addition of 1 mol/L H2SO4 and the absorbance measured at 490 nm. A calibration curve was constructed using purified recombinant wild type human protein S of known concentration diluted in expression media.Pulse-chase experiments Pulse-chase experiments of recombinant protein S by radioactive labeling, immunoprecipitation, and electrophoresis were performed essentially as previously described.46 Eight micrograms of the expression vectors were transiently transfected into COS 1 cells by a Lipofectin (GIBCO-BRL)-mediated method on 10-cm dishes, then divided into several 6-cm dishes on the next day. On the third day after transfection, the cells were incubated with 1 mL of Met/Cys free DMEM, supplemented with glutamine and vitamin K, for 30 minutes to deplete intracellular Met and Cys, then radiolabeled with 800 µL of 100 µCi/mL [35S] Met and [35S] Cys for 30 minutes disolved in the previous medium. After washing twice with 2 mL of phosphate-buffered saline (PBS), the cells were chased for the indicated times with 1 mL of Optimem medium supplemented with 2 mmol/L cold Met and 2 mmol/L cold Cys, and vitamin K. Culture media were harvested and centrifuged at 10 000 rpm for 3 minutes at room temperature to remove cell debris. Labeled cells were washed once with 2 mL of PBS, then lysed on ice with 1 mL of 10 mmol/L Tris pH 7.5, 150 mmol/L NaCl, 2 mmol/L EDTA, 1% NP-40 (v/v), 0.1% SDS (w,v), and 1 mmol/L PMSF for 15 minutes. The supernatants of the cell lysates were collected by centrifugation at 14 000 rpm for 5 minutes at RT. Culture media and supernatants of the cell lysates were precleared with 50 µL of 10% suspension of Pansorbin (Calbiochem, La Jolla, CA) for 2 hours with rocking at RT. After centrifugation at 14 000 rpm for 1 minute at RT, the supernatants were incubated with 3 µg of antiprotein S polyclonal antibody (DAKO) for 16 hours at 4°C. The immune complexes were precipitated by incubation with 50 µL of a 10% suspension of Pansorbin for 1 hour with shaking at RT, then centrifuged at 14 000 rpm for 1 minute. Precipitates were washed 5 times with 1 mL of 10 mmol/L Tris pH 7.5, 150 mmol/L NaCl, 2 mmol/L EDTA, 0.5% NP-40 (v/v), and 0.1% SDS (w/v) to remove nonspecifically bound proteins. Bound proteins were eluted by boiling for 5 minutes in 25 µL of 2 × SDS sample buffer containing 2% mercaptoethanol, then centrifuged at 14 000 rpm for 1 minute. Supernatants were separated by 7.5% SDS-PAGE. Gels were treated with 10% acetic acid and 30% methanol for 15 minutes and dried for 1 hour at 80°C, and analyzed with a Phosphor Imager (Molecular Dynamics, Sunnyvalle, CA) to quantify the radioactivity of the bands on the gels. The total radioactivity immunoprecipitated at time zero was not significantly different among the different mutants, with the exception of Y444C that demonstrated a level of approximately 50% of that observed for wild type protein S.
Transient expression of protein S mutants in COS 1 cells Eukaryotic expression plasmids carrying either wild type protein S cDNA or the protein S variants found in the different kindreds described in Table 1 were constructed as indicated in the methods section. COS 1 cells were transiently transfected with the different plasmids and their conditioned media analyzed for protein S content. In Western blot analysis under nonreducing conditions, protein S was detected in all media as a single band with the expected molecular weight (Figure 2). However, the amount of protein detected was clearly different between the various conditioned media. To accurately quantify the level of protein S expression of the different mutants, we standarized an ELISA-based protocol for determining the concentration of protein S in media from 8 different transfections. Analyses were carried out in parallel using 2 cell plates per transfected protein S variant. Wild type protein S was expressed at a concentration around 100 ng/mL, as determined by using purified protein S as standard. The concentration of protein S in the media (Figure 2) was determined by using sequential dilutions of conditioned media containing known protein S concentration. Group A mutants showed the lowest concentrations, although the level of secretion observed was very different between both mutants. Although the concentration of G441C mutant in conditioned media was 40% of the wild type, the effect of the Y444C mutation was much more severe, giving the lowest levels of expression detected, 2.5%. In group B mutants, the levels of expression were approximately half of the wild type, being 42% in the case of R520G and 48% in the case of P626L. All these values were significantly different from the concentration of wild type protein S. In contrast, group C mutants did not produce a significant reduction on the protein expressed, T57S and I518 mol/L had a concentration in the media that was 97% and 96% of the concentration of wild type protein S, respectively.
Pulse-chase experiments To analyze the mechanism of the observed reduction in the concentration of protein S in the media, transiently transfected cells were pulse-labeled and the incorporation of radioactive label into protein S was followed for up to 24 hours both in the media and inside the cells (Figure 3). The same effect in the reduction of protein S secreted in the media observed in the previous experiments was confirmed in the pulse-chase experiment. Group A mutants had the lowest secretion levels and group B mutants had an intermediate effect. These 4 mutants were clearly degraded during the experiment, as their total radioactivity at 24 hours was between 23% (Y444C) and 41% (R520G) of the initial radioactivity. In comparison, wild type protein S (72%) and group C mutants (78% for T57S and 77% for I518 mol/L) all had higher levels. The secretion efficiency, measured as the level of protein S in the media at 24 hours and expressed as a percentage of the initial value, was higher in wild type (65%) and group C mutants (69% for T57S and 64% for I518 mol/L), whereas it was decreased to < 30% in the other mutants. Among group A mutations, the difference observed between G441C and Y444C in the transient expression was confirmed. The G441C secretion efficiency was 2.5-fold higher than that of Y444C (15± 3 vs 6 ± 3, P = .14). Y444C had also the longest intracellular half life of all proteins tested, around 8 hours. In the pulse-chase experiment, the results are not affected by the transfection, transcription, and translation efficiency, as they refer at each time point to the protein S detected at the time of the pulse. Thus, the pulse-chase results confirm that the differences shown in Figure 2 are due to the effect of each mutation in the folding and/or secretion of the protein.
Effect of alternative amino acid substitutions in selected mutants Next, we studied alternative amino acid changes at the mutated positions to test whether more conservative substitutions could recover the effect observed in the mutants in groups A and B. For these experiments, we compared the sequences of proteins with structure similar to protein S. All mutations in groups A and B are in residues belonging to the G domains, which form the C-terminal SHBG-like region of protein S. Therefore, in our sequence comparison, we used the 7 mammalian protein S sequences reported, as well as the human sequences of the structurally related proteins Gas6, SHBG, and laminin A.47 First, we changed the residues affected in group A mutations to amino acids with characteristics more similar to the amino acids found in the wild type sequence: G441A and Y444F. In both cases, the concentration of protein S in the media increased to almost normal levels, 84% and 89% respectively (Figure 4). The remaining difference to wild type protein S observed in the case of G441A could be explained by the fact that Gly seems to be a structural feature of the G module at this position, as it is present in G modules distantly related to protein S, such as those in laminin A (Figure 4). At the position of Y444, mouse Gas6 and human laminin have a Phe, whereas human SHBG has a Leu, indicating that several side chains are compatible with the structure at this residue. Group B mutations were also changed to alternative amino acids. When R520 was changed to other charged amino acids, either positive or negative, the concentration in the media increased (R520H, 93%; R520K, 93%; and R520E, 84%) in comparison to that of the R520G mutant. An intermediate effect was produced by changing the residue to Ala (77%), which is more similar to Gly. Finally, at position 626, we located a Gly residue but the levels were only slightly increased compared with the original mutation (58% vs 48%). Interestingly, changing Pro for a stop codon seemed to be comparatively better for the secretion of the mutant protein (68%).
The molecular basis of protein S deficiency is not known for most type I missense mutations. An exception is protein S Nagoya (R474C), where the mutation produces an 8-fold reduction in the secretion of protein S by transfected cells.44 This difference is due to impaired secretion and intracellular degradation of the mutant protein S. Phenotypically, the mutation is manifested as a type I deficiency. In this study we extend this characterization to PROS1 mutations found in different types of protein S deficiencies to analyze the molecular fate of these mutants.
We acknowledge Helena Kruyer for her helpful assistance with the manuscript.
Submitted April 28, 1999; accepted August 27, 1999.
Supported by grants from the Tore Nilson Trust (to P.G. de F.); the Albert Pählsson Trust, and the Fondation Louis-Jeantet de Mèdecine (to B.D.); the Swedish Medical Research Council (grants 07143 and 13000), research funds from the University Hospital, Malmö, the Johan and Greta Kock Trust, the Alfred Österlund Trust (to P.G. de F. and B.D.) and by the Spanish DGICYT (grant PB94-1233) and research funds from the Servei Català de la Salut (to N.S.). Y.E-P. was supported by a Marie Curie fellowship from the EU (grant ERB/4001/GT/97/1535).
Reprints: Björn Dahlbäck, Wallenberg Laboratory, University Hospital, Malmö, S-20502 Malmö, Sweden.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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T. Yamazaki, G. A. F. Nicolaes, K. W. Sorensen, and B. Dahlback Molecular basis of quantitative factor V deficiency associated with factor V R2 haplotype Blood, September 18, 2002; 100(7): 2515 - 2521. [Abstract] [Full Text] [PDF]
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T. K. Giri, T. Yamazaki, N. Sala, B. Dahlback, and P. G. de Frutos Deficient APC-cofactor activity of protein S Heerlen in degradation of factor Va Leiden: a possible mechanism of synergism between thrombophilic risk factors Blood, July 15, 2000; 96(2): 523 - 531. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-carboxyglutamic
acid-containing module (Gla module), a thumb loop sensitive to
thrombin (thrombin-sensitive region, TSR), 4 epidermal growth factor
(EGF)-like modules, and a C-terminal region similar to the sex hormone
binding globulin (SHBG-like region). The SHBG-like region consists of a
spacer region followed by 2 G modules, a structural motive present in
laminin A and other extracellular matrix proteins.
.05) are shown.
>Glu) for the regulation of the blood coagulation system.
Biochim Biophys Acta.
1995;1272:159-167