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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 523-531
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Laboratory Medicine, Division of
Clinical Chemistry, Lund University, University Hospital,
Malmö, Sweden, and Centre de Genètica
Mèdica i Molecular, Institut de Recerca Oncològica,
Barcelona, Spain.
In protein S Heerlen, an S-to-P (single-letter amino acid codes)
mutation at position 460 results in the loss of glycosylation of N458.
This polymorphism has been found to be slightly more prevalent in
thrombophilic populations than in normal controls, particularly in
cohorts of patients having free protein S deficiency. This suggests
that carriers of the Heerlen allele may have an increased risk of
thrombosis. We have now characterized the expression in cell cultures
of recombinant protein S Heerlen and investigated the anticoagulant
functions of the purified recombinant protein in vitro. Protein S
Heerlen was synthesized and secreted equally well as wild-type protein
S by transiently transfected COS-1 cells. The recombinant protein S
Heerlen interacted with conformation-dependent monoclonal antibodies
and bound C4b-binding protein to the same extent as wild-type protein
S. Protein S Heerlen displayed reduced anticoagulant activity as
cofactor to activated protein C (APC) in plasma-based assays, as well
as in a factor VIIIa-degradation system. In contrast, protein S
Heerlen functioned equally well as an APC cofactor in the degradation
of factor Va as wild-type protein S did. However, when
recombinant activated factor V Leiden (FVa:Q506) was used as APC
substrate, protein S Heerlen was found to be a poor APC cofactor as
compared with wild-type protein S. These in vitro results suggest a
possible mechanism of synergy between protein S Heerlen and factor V
Leiden that might be involved in the pathogenesis of thrombosis in
individuals carrying both genetic traits.
(Blood. 2000;96:523-531)
In hereditary thrombophilia, different genetic factors
predispose to the development of thromboembolic events.1
Resistance to activated protein C (APC) is a highly prevalent inherited
hypercoagulable state caused by a single-point mutation in the factor V
(FV) gene (FV R506Q,[single-letter amino acid codes] FV:Q506, or FV
Leiden). This mutation is found in 2% to 15% of different healthy
white populations.2 The high prevalence of FV Leiden
mutation facilitates studies of multiple gene-gene and gene-environment
interactions among thrombosis patients.3-6 Although not as
prevalent as APC resistance, many mutations in the genes of
antithrombin, protein C, and protein S have been described as
pathogenic risk factors of thrombosis. In the heterozygous form, these
gene modifications have a mild clinical expression by themselves, but
the likelihood of thrombosis increases when they are associated with a
second prothrombotic genetic defect.7 Recently, the concept
that thrombophilia is a multigenic disorder has been reinforced by
several studies of genetic risk factors in thrombotic
individuals.8-12
Congenital protein S deficiency is found in 1% to 5% of patients with
thrombosis.13,14 The important anticoagulant role of
protein S is dramatically illustrated by the severe thrombotic tendency
in homozygous or compound heterozygous cases. Heterozygous carriers
have a fivefold to tenfold increased frequency of thrombosis, as
compared with their healthy relatives.15,16 Protein S is a
plasma glycoprotein that exerts its major role via the APC
anticoagulant pathway.17 It works as a nonenzymatic
cofactor to APC, enhancing the degradations of activated factor V (FVa)
and activated factor VIII (FVIIIa), thereby limiting thrombin
generation. Full anticoagulant activity of APC in the degradation of
FVIIIa requires not only protein S but also FV, with the 2 proteins
functioning as synergistic APC cofactors.18 Thus, FV has
the potential of working as both procoagulant and anticoagulant
protein. Protein S has also been reported to regulate hemostasis by
APC-independent inhibition of tenase and
prothrombinase.19-22
Human protein S is a single-chain multimodular protein, composed of a
Protein S Heerlen contains an S460P mutation that results in the loss
of the N-linked glycosylation site at N458,35 which is
probably the same variant protein S reported by Schwarz et al.37 Protein S Heerlen was reported as an immunologic
polymorphism detected during the screening of thrombophilic patients
with a monoclonal antibody-based enzyme-linked immunosorbent assay
(ELISA). In that study, no association was observed between the
presence of protein S Heerlen and an increased risk of
thrombosis,35 and loss of this glycosylation has not been
found to affect the anticoagulant function of protein S or the C4BP
binding.38 These facts, together with the prevalence of the
S460P mutation in healthy populations, which has been found to range
between 0.5% and 0.8%, have led to the mutation's being considered a
neutral polymorphism or protein variant. However, subsequent family
studies have shown that the Heerlen allele is significantly more
frequent in thrombophilic families diagnosed with protein S deficiency
than in the normal population.36,39,40 Nevertheless, in
families having the Heerlen variant and type III protein S deficiency,
the 2 traits do not always cosegregate,37,40 calling into
question the mutation-phenotype relationship between the Heerlen
polymorphism and protein S deficiency.
The frequent association between protein S Heerlen and the FV Leiden
mutation among thrombophilic patients suggests a cooperative effect
between these 2 traits.39,41 However, even though this association has been found in particular cases and FV Leiden is known
to increase the risk of thrombosis in protein S
deficiency,4,5 a direct biochemical basis for a synergism
between the 2 traits is lacking. The aim of the present study was
to understand the possible basis of thrombosis in symptomatic carriers
of the protein S Heerlen allele. We expressed recombinant protein S
Heerlen in mammalian cell lines and analyzed its secretion
profile. The purified recombinant protein S Heerlen was characterized
for functional anticoagulant activity. Furthermore, we studied the in
vitro effect of the combination between protein S Heerlen and FV Leiden
and demonstrate a possible synergistic prothrombotic effect of the 2 mutations.
Protein S assays
Site-directed mutagenesis
Cell culture and transient expression The protein S Heerlen variant and wild-type protein S were transiently expressed in monkey kidney COS-1 cells, and the expression levels were determined with an ELSA as described.43 Pulse-chase experiments of recombinant protein S including radioactive labeling with [35S] methionine and [35S] cysteine, immunoprecipitation, and electrophoresis were performed essentially as previously described,46 except that a transient expression system was used instead of a stable expression system.45 In brief, the expression vectors were transiently transfected into COS-1 cells, and the cells were divided into several dishes on the following day. On the third day after transfection, the cells were pulse-labeled with [35S] methionine and [35S] cysteine and then chased for the indicated times. Radiolabeled protein S in the culture media and cell lysates were immunoprecipitated with anti-protein S polyclonal antibodies (Dako, Glostrup, Denmark) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were dried and a Phospho Imager (Molecular Dynamics, Sunnyvalle, CA) system was used to quantify the radioactivity of the bands on the gels. Wild-type FV and R506Q mutant FV were also transiently expressed, and the expression levels were determined by an ELISA as described.47Stable expression of protein S, purification and characterization of recombinant proteins The protein S Heerlen variant and wild-type protein S were purified from conditioned media of stably transformed human kidney cell line 293, with the use of methods described previously.25,45 The expression levels were approximately 1.5 mg/L. The concentration of the purified protein S was determined by amino acid composition analysis, with the use of methods as previously described.45 Determination of the content of Gla residues was performed as described.45 The purified recombinant proteins were subjected to 10% SDS-PAGE and stained with Coomassie Brilliant Blue R-250 (BDH, Poole, England) to check their purity. The recognition of the protein S Heerlen variant and wild-type protein S by monoclonal antibodies24 directed against Gla domain, TSR, and EGF1 and the ability of the protein S Heerlen variant to interact with C4BP were analyzed with the ELSA technique.43 The stability at 55°C of the wild-type protein S and the Heerlen variant was evaluated by following the decay of protein S activity.48 In brief, 100 µL aliquots of wild-type or Heerlen protein S (10 nmol/L) diluted in 50 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.5 (TBS), containing 0.1% bovine serum albumin (BSA), were incubated at 55°C in a water bath, removed at 30-minute intervals, and kept on ice. After 2.5 hours, all aliquots were analyzed for their APC-cofactor activity in an APC-dependent FVIIIa-degradation assay, as described below.Coagulation assays APC-cofactor activity of protein S in an activated partial thromboplastin time (APTT)-based analysis. A previously described procedure was followed with minor modifications.45 In this assay, increasing concentrations of the protein S variants were added to protein S-deficient plasma. The plasma aliquots (50 µL) were incubated with an APTT reagent (50 µL of the APTT reagent present in the APC resistant kit, Chromogenix, Mölndal, Sweden) for 180 seconds at 37°C before the clotting was initiated by the addition of the APC/CaCl2 mixture (50 µL) from the APC resistance kit. The clotting times were measured by means of an Amelung-Coagulometer KC 10, Lemgo, Germany. APC-cofactor activity of protein S in a prothrombin time-based analysis. Protein S-depleted plasma (50 µL) was incubated at 37°C for 120 seconds in the Amelung-Coagulometer KC-10 sample cups. Mixtures containing 10 µL wild-type or protein S Heerlen (with final concentrations of 0 to 100 nmol/L), 50 µL APC/CaCl2 (from the APC resistance kit), and 50 µL Simplastin Excel (Organon Teknika, RM Boxtel, Netherlands) were prepared at room temperature and added to the wells, and the clotting time was recorded. The Simplastin was diluted 1:10 or 1:100 with TBS containing 0.1% BSA and 25 mmol/L CaCl2. Effects of protein S variants in APC-catalyzed inactivation of
wild-type and mutant FVa.
A previously described procedure was followed with minor
modifications.45 As APC substrate, either plasma-derived or
recombinant FVa was used. In the assay, 30 nmol/L wild-type FVa or FVa
Leiden, phospholipid (20 µmol/L), wild-type protein S
or protein S Heerlen (final concentration from 0 to 80 nmol/L), and
human APC (0.3 nmol/L, final concentration) were incubated at 37°C.
Aliquots of this mixtures were removed at intervals and diluted 1:10 in ice-cold TBS, and the remaining FVa activity was determined in a
standard clotting-based FV assay using FV deficient plasma. The
remaining FVa in the incubation mixture with only APC was considered as
being 100%. The transiently expressed wild-type FV and FV Leiden were
activated prior to each experiment by incubating the FV (100 µL at 45 nmol/L) with 5 nmol/L of Effects of wild-type or mutant protein S in APC-catalyzed
inactivation of FVIIIa.
The FVIIIa-degradation assay was performed as described
previously,50 with minor modifications.45 Human
FVIII, activated factor IX, and phospholipids were incubated with
Free protein S concentrations in carriers of the protein S Heerlen allele as measured by different free protein S methods There is an established association between the presence of the protein S Heerlen allele and an increased prevalence of type III protein S deficiency. To elucidate the possibility that the protein S Heerlen is less efficiently detected than wild-type protein S by different assays, plasma samples of individuals carrying the Heerlen mutation in heterozygous state (n = 14) or in homozygous form (n = 1) were analyzed together with 12 control samples by 3 different protein S assays. The 3 assays were based on different principles of detection of the free form of protein S. In the RIA assay, C4BP-protein S complexes were precipitated by polyethylene glycol, and the free protein S present in the supernatant was measured by a standard RIA, with the use of polyclonal antibodies.42 The free protein S ELISA relied on a monoclonal antibody specific for free protein S as catcher.44 The more recently developed free protein S ELSA used immobilized C4BP as catcher of free protein S and subsequent detection of the bound protein S by a monoclonal antibody.43 The 3 methods were found to give similar results both for protein S Heerlen carriers and for normal controls, suggesting that protein S Heerlen can be detected equally well by the 3 methods (Figure 1). In a pairwise comparison, the correlation coefficients between the 3 methods were high (r2 0.95). In all 3 methods, the mean
concentrations of free protein S among the heterozygous carriers of
protein S Heerlen were significantly lower than those estimated for
normal controls. The single homozygous case of protein S Heerlen
yielded the lowest free protein S values in all 3 methods (around
40%). From these results, it can be concluded that the slightly low
free protein S levels found among carriers of the protein S Heerlen
allele are real and not the result of systematic laboratory errors.
Transient expression of recombinant protein S in COS-1 cells Expression vectors (pcDNA3) containing either wild-type or protein S Heerlen cDNA were used for transient expression in COS-1 cells. The protein S concentration in the conditioned media was analyzed by the ELSA method. The concentrations of wild-type and protein S Heerlen were found to be approximately 110 to 120 ng/mL, and there was no statistically significant difference between the concentrations. When the transfected cells were pulse-chased for protein S, the patterns of expression were very similar for the 2 protein S constructs, and the recovery of protein S in the media after 24 hours was approximately 70% for both (Figure 2). In conclusion, the pulse-chase experiments did not indicate any reduced efficiency in secretion of protein S Heerlen; if such a reduction had been found, it could have provided an explanation for the slightly low protein S levels in protein S Heerlen carriers (Figure 1).
Characterization of the purified recombinant protein S variants Protein S Heerlen and wild-type protein S were expressed in stably transfected eucaryotic cell cultures; the proteins were purified to homogeneity; and the final products were visualized on SDS-PAGE stained with Coomassie Brilliant Blue (Figure 3A). The migration of protein S Heerlen in the gel was slightly faster than that of wild-type protein S owing to the lack of N-glycosylation at residue 458. Gla analysis revealed that the -carboxylation of
glutamic acid residues was similar for protein S Heerlen and wild-type
protein S (approximately 10 Gla/molecule). Recognition of the
recombinant protein S Heerlen by 3 different monoclonal antibodies,
against the Gla-domain (HPS 21), the TSR (HPS 67), or the EGF1 (HPS
54), was analyzed with the use of purified protein S bound to
immobilized C4BP-coated microtiter wells. The 3 monoclonal antibodies
recognized conformation-dependent epitopes, and none of them interfered
with the binding of C4BP to protein S.24 All monoclonal
antibodies were found to recognize protein S Heerlen and wild-type
protein S equally well; the result obtained with one (HPS 54) is shown
(Figure 3B). This experiment also demonstrated that recombinant protein
S Heerlen and wild-type protein S bound C4BP equally well, suggesting
that the S460P mutation did not influence the ability of protein S to
bind C4BP. To test whether the stability of protein S in vitro was
affected by the S460P mutation, wild-type protein S and protein S
Heerlen were incubated at 55°C and their activities recorded at
different time intervals (Figure 4). A
small but consistent difference was observed: the Heerlen variant was
denatured slightly faster than the wild-type protein S. Approximately
35% of the APC-cofactor activity of wild-type protein S was lost after
2.5 hours' incubation, while the Heerlen variant had lost 50% of its
activity.
Recombinant protein S Heerlen demonstrated decreased APC-cofactor activity in plasma-based coagulation assays To investigate whether the Heerlen mutation had any effect on the APC-cofactor function of protein S, the activities of wild-type protein S and protein S Heerlen were tested in plasma-based assays with the use of APC and protein S-deficient plasma supplemented with the purified recombinant protein S preparations. In an APTT-based assay, where clotting was triggered via the intrinsic pathway, protein S prolonged the clotting time in a dose-dependent manner in the presence of APC (Figure 5A). In the absence of protein S, the addition of APC yielded a clotting time of 77 ± 1 seconds, as compared with 35.4 ± 0.2 seconds in the absence of added APC. In the presence of both APC and 100 nm wild-type protein S, the clotting time was 165.3 ± 3.5 seconds. Under similar conditions but with protein S Heerlen, the mean clotting time was 138.6 ± 3.2 seconds. The assay did not allow an accurate estimation of the exact activity loss, because the 2 dose-response curves were not parallel. However, it was apparent that protein S Heerlen demonstrated reduced APC-cofactor activity as compared with wild-type protein S, a difference that was statistically significant in an unpaired Student t test (P = .011). In the absence of APC, the presence or absence of protein S (100 nm) did not affect the clotting times (36.5 ± 0.2 seconds versus 35.4 ± 0.2 seconds). Next, we tested the APC-cofactor activities of the expressed protein S variant in a clotting assay based on the tissue-factor pathway. APC was unable to prolong the clotting time after induction of coagulation by undiluted tissue-factor reagent (Simplastin). To be able to record the anticoagulant activity of APC, we therefore diluted the Simplastin reagent 1:10 and 1:100. In the absence of APC, the presence or absence of protein S (100 nmol/L) did not affect the clotting time (19.4 ± 0.2 seconds versus 20 ± 0.5 seconds) when the 1:10 dilution was used. In the presence of APC, the clotting time was 26.5 ± 1 seconds in the absence of protein S and 40.5 ± 1.5 seconds in the presence of 100 nmol/L wild-type protein S. The protein S Heerlen was essentially equally efficient as wild-type protein S (Figure 5B). However, when the Simplastin reagent was diluted 1:100, the Heerlen variant demonstrated approximately 50% of the activity of wild-type protein S (Figure 5B). Under these conditions, neither of the recombinant proteins demonstrated APC-independent anticoagulant activity. Thus, the clotting time was prolonged by 34 seconds, and protein S (100 nmol/L) alone did not have any independent effect. Addition of APC alone yielded a clotting time of approximately 51 seconds.
APC-cofactor activities of recombinant protein S in degradation of wild-type FVa or FVa Leiden Protein S Heerlen was found to be equally efficient as wild-type protein S in supporting APC-mediated inactivation of plasma-purified FVa (data not shown). Approximately 80% of the initial FVa activity was lost in the presence of 0.3 nmol/L APC and 10 nmol/L wild-type protein S after 5 minutes' incubation. Subsequently, we tested the influence of protein S on the APC-mediated degradation of FVa Leiden, which carries the R506Q mutation. In this experiment, recombinant wild-type FV and FV Leiden were produced in parallel in a transient expression system. The conditioned media were collected and concentrated and the FV levels were then determined with an ELISA for FV. Wild-type FV and FV Leiden were activated by thrombin and thereafter incubated with APC and the protein S variants in the presence of phospholipid vesicles. Wild-type protein S and protein S Heerlen were equally active as APC cofactors in the inactivation of wild-type FVa (Figure 6A), which was in accordance with the experiment described above using plasma-purified FVa. However, when FVa Leiden was used as the APC substrate, protein S Heerlen displayed poor APC-cofactor activity. In the presence of wild-type protein S, approximately 90% of the initial FVa Leiden activity was lost, which was similar to the results obtained with the wild-type FVa. However, under the same conditions, protein S Heerlen failed to support the APC-mediated degradation of FVa Leiden, whereas the degradation of wild-type FVa was efficiently stimulated by protein S Heerlen (Figure 6A).
APC-cofactor activities of protein S variants in APC-mediated inactivation of factor VIIIa Efficient inhibition of FVIIIa by APC requires the synergistic cofactor activities of protein S and FV. To test the effect of the protein S Heerlen mutation in this reaction, increasing concentrations of either wild-type protein S or protein S Heerlen were added in a FVIIIa-inactivation assay. In both the presence and the absence of FV, protein S Heerlen demonstrated approximately 50% APC-cofactor activity as compared with wild-type protein S (Figure 6B). In this assay system, APC alone yielded approximately 10% reduction in FVIIIa activity after 2.5 minutes' incubation, which is consistent with results on record showing that APC alone is an inefficient inhibitor of FVIIIa.50,51 Protein S (10 nmol/L) alone had essentially no APC-independent inhibitory activity in the FVIIIa assay system. In contrast, after 2.5 minutes' incubation in the presence of APC, 10 nmol/L wild-type protein S, and 5 nmol/L FV, approximately 75% of the FVIIIa activity was lost. Under these assay conditions, protein S Heerlen demonstrated reduced APC-cofactor activity, and at 10 nmol/L protein S Heerlen, approximately 50% FVIIIa activity remained (Figure 6B).
Protein S Heerlen (S460P) has been circumstantially linked to
thrombosis,36 despite not being significantly more
prevalent in populations with venous thrombosis than in the general
population.35 Protein S Heerlen, and other mutations in the
region near residue 460, have been reported to be associated with type
III protein S deficiency, ie, low levels of free protein S despite
normal levels of total protein S.36,39,40 It has been
suggested that the mechanism underlying type III deficiency in these
mutants involves increased affinity for C4BP with a resulting decrease in the level of free protein S, or that each C4BP molecule is able to
bind more than 1 protein S Heerlen molecule.36 However, in
this study, which used recombinant wild-type protein S and protein S
Heerlen produced and purified in parallel, no difference in binding to
C4BP could be detected (Figure 3B). This is in accordance with results
obtained with other recombinant protein S mutants, eg, N458Q or S460G
in the SHBG-like domain of protein S, in which the absence of
carbohydrate at position 460 does not affect binding to
C4BP.38 The SHBG-like region of protein S is known to fully contain the binding site for C4BP, but a detailed molecular
understanding of the binding site is missing.28,52
Recently, we found evidence for the involvement of both laminin-G-type
domains of the SHBG-like region in the formation of the
complex.52 Thus, the binding site in protein S for C4BP is
complex, involving more than 1 domain, and the loss of a carbohydrate
side chain does not appear to affect the binding affinity to any
significant degree. Furthermore, the affinity of protein S for
C4BP is very high,28 and the free protein S
concentration is the molar surplus of protein S over the
We thank Prof R. J. Kaufman for the kind gift of human FV wild-type and Leiden cDNA; and Dr M. Borrell, from Hospital de la Sanat Creu i Sant Pau, and Dr G. Navarro, from Laboratori de Referència de Catalunya, Barcelona, Spain, for plasma samples from carriers of the protein S Heerlen allele and their relatives.
Submitted January 31, 2000; accepted March 2, 2000.
Supported by the Swedish Medical Research Council (Grants 07143, 12561, and 13000), a Senior Investigators Award from the Swedish Foundation for Strategic Research, research funds from the University Hospital in Malmö, the Fondation Louis-Jeantet de Médecine, the Alfred Österlund Trust, and the Albert Påhlsson Trust.
Reprints: Björn Dahlbäck, Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University, University Hospital, S-20502 Malmö, Sweden; e-mail: bjorn.dahlback{at}klkemi.mas.lu.se.
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.
1. Rosendaal FR. Risk factors for venous thrombosis: prevalence, risk, and interaction. Semin Hematol. 1997;34:171-187[Medline] [Order article via Infotrieve]. 2. Dahlbäck B. Resistance to activated protein C as risk factor for thrombosis: molecular mechanisms, laboratory investigation, and clinical management. Semin Hematol. 1997;34:217-234[Medline] [Order article via Infotrieve].
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van Boven HH, Vandenbroucke JP, Briet E, Rosendaal FR.
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Top
Abstract
Introduction
-chain of C4b-binding protein (C4BP), a regulator of the classical
pathway of complement.
70°C before
analysis. Determination of the Heerlen variant was made as described
previously.
thrombin (Haematologic Technologies, Essex
Junction, VT) in the presence of 5 mmol/L CaCl2 and 10 µm phospholipid vesicles at 37°C. After
20 minutes, the reaction was stopped by the addition of 5 nmol/L
PPACK (Calbiochem, La Jolla, CA). The
phosholipid vesicles, composed of 25% (wt/wt) phosphatidylserine,
37.5% (wt/wt) phosphatidylcholine, and 37.5% (wt/wt)
phosphatidylethanolamine, were prepared as described.
); medium, amount of
radioactive protein S in media (
); and total, the sum of protein S
in cell and medium (
). The means ± SEM of 4 independent
experiments are shown.
