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Keywords:

  • acquired activated protein C resistance;
  • anti-prothrombin antibodies;
  • lupus anticoagulant;
  • venous thromboembolism;
  • systemic lupus erythematosus

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Summary. Venous thromboembolism (VTE) is one of the common manifestations in the anti-phospholipid (aPL) syndrome. We examined the levels of IgG antibodies (Abs) to β2-glycoprotein I (β2-GP I) and prothrombin, lupus anticoagulant (LA) activity, activated protein C resistance (APC-R), and factor V Leiden in 96 patients with systemic lupus erythematosus (SLE); 19 with VTE and 77 without VTE. Acquired APC-R, which was not found in any patient with the factor V Leiden mutation, was present in 33 (34·4%) out of the 96 patients with SLE. The presence of acquired APC-R was a strong risk factor for VTE. The SLE patients were divided into four groups according to the results of enzyme-linked immunosorbent assay (ELISA) and LA activity for each aPL Abs: ELISA+, LA+; ELISA+, LA; ELISA, LA+; and ELISA, LA. A significant association was observed between APC-R and the co-existence of anti-β2-GP I Abs and LA activity or of anti-prothrombin Abs and LA activity. There was no association between APC-R and the presence of anti-β2-GP I Abs, anti-prothrombin Abs, or LA activity alone. However, when multivariate logistical regression analysis was performed, it was clear that only the co-existence of anti-prothrombin and LA activity was a significant risk factor for APC-R. These findings indicate that the co-existence of anti-prothrombin Abs and LA activity may be an important factor in the pathogenesis of acquired APC-R in patients with SLE.

Anti-phospholipid (aPL) antibodies (Abs) are a heterogeneous group of antibodies that can be detected as anti-β2-glycoprotein I (β2-GP I) Abs, anti-prothrombin Abs, and lupus anticoagulant (LA) (Roubey, 1994). These antibodies are frequently found in the plasma of patients with systemic lupus erythematosus (SLE) (Cabiedes et al, 1995), and have been reported to be associated with clinical events such as arterial and/or venous thrombosis, thrombocytopenia, and recurrent fetal loss in patients with SLE (Galli et al, 1997a; Greaves, 1999). Although the association between aPL Abs and venous thromboembolism (VTE) in patients with SLE has been established (Ginsberg et al, 1995; Simioni et al, 1996; Schulman et al, 1998; Zanon et al, 1999), the precise mechanism responsible for VTE in SLE patients with aPL Abs remains unclear.

A number of clinical studies has established that the prevalence of VTE is strongly associated with congenital abnormalities of the protein C pathway (Griffin et al, 1981; Comp & Esmon, 1984; Comp et al, 1984). Recently, it was shown that aPL Abs may inhibit phospholipid-dependent reactions of the protein C pathway, for example, the thrombin/thrombomodulin activation of protein C and/or the activated protein C/protein S degradation of factor Va (Oosting et al, 1993; Roubey, 1994). More recently, the presence of LA was reported to be significantly associated with acquired activated protein C resistance (APC-R) in patients with SLE (Male et al, 2001).

However, some investigations showed that LA activity can be caused by anti-β2-GP I Abs and/or anti-prothrombin Abs, and that each of these two types of Abs could be divided into two types; one showing LA activity and the other not (Galli et al, 1995, 1997b, 1998; Horbach et al, 1998; Galli & Barbui, 1999). When we studied the anti-prothrombin Abs and anti-β2-GP I Abs in addition to LA activity in patients with SLE, we found that the co-existence of immunoglobulin (Ig) G anti-prothrombin Abs and LA activity correlated best with a history of VTE in patients with SLE (Nojima et al, 2001a).

The main objective of the present study was to clarify which aPL Abs were associated with acquired APC-R, anti-prothrombin Abs or anti-β2-GP. Furthermore, we determined whether or not acquired APC-R caused by these aPL Abs was related to the pathogenesis of VTE in patients with SLE.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Study population.  We studied plasma samples from 96 patients (90 women, six men; 21–72 years of age; mean 43·8 years) with SLE. Diagnosis of SLE was made according to the revised criteria of the American Rheumatism Association. Clinical history revealed that VTE was observable in 19 out of the SLE patients: deep vein thrombosis (DVT, 10 cases) and pulmonary embolism (PE, nine cases). Diagnosis of DVT and PE was made based on clinical manifestations and findings by duplex scanning, radioisotope venography, contrast venography and radioisotope lung scanning. For the control group, we studied plasma samples from 80 normal healthy volunteers (74 women, six men; 22–60 years of age; mean 39·8 years) who worked at the Osaka University Hospital. None of them had any history of thrombotic complications, and no abnormality was detected in the blood tests (blood cell counts, coagulation tests, liver function tests and examinations for autoimmunity). Blood samples were taken into vacuum tubes (5·0 ml total volume, Sekisui, Japan) containing 0·5 ml of 3·13% trisodium citrate (Na3C6H5O7·2H2O), and platelet-poor plasma was prepared by double centrifugation at 2800 g for 10 min at 18°C. The plasma sample was frozen at −80°C until batch assays could be performed. Informed consent was obtained from all patients and control subjects.

APC resistance assay and factor V Leiden test.  APC-R assay was performed four times for 3 months in all patients with SLE using an activated partial thromboplastin time (APTT)-based assay on the KC-10 coagulometer (Amelung, Germany). APTT was measured in the presence and absence of APC using a Coatest Activated Protein C Resistance Kit (Coatest, Chromogenix, Molndal, Sweden). The test was performed with undiluted patient plasma. The results were expressed as the ratio of APTT in the presence and absence of APC (APTT with APC:APTT without APC). The APC sensitivity ratio in 80 healthy controls was 2·68 ± 0·34 (mean ± SD). The APC sensitivity ratio of less than 2·0, which was the mean −2SD in healthy controls, was determined as the normal cut-off value in the APC-R study. Factor V Leiden status was determined by extracting genomic DNA from the plasma of the patients, as reported previously (Fujimura et al, 1995).

Measurement of protein C, protein S and anti-thrombin.  Pro-tein C activity was measured by use of a chromogenic substrate assay kit employing PGPA-MNA (Berichrom, Behring-Werke, Marburg, Germany), as reported using a homogeneous enzyme immunoassay as reported previously (Shimamoto et al, 1988). Protein S activity was determined by a clotting assay (Staclot Protein S, Diagnostica Stago, Asnieres, France), as reported previously (Nomura et al, 2000). Plasma levels of total and free protein S antigens were measured using enzyme-linked immunosorbent assay (ELISA) kits (Asserachrom Total Protein S and Asserachrom Free Protein S; Diagnostica Stago, Asnieres, France). Anti-thrombin activity was measured with a chromogenic substrate (S-2238, Kabi Vitrium, Stockholm, Sweden) used in an assay kit (Testzym, Daiichi Pure Chemicals, Tokyo, Japan), as reported previously (Nomura et al, 2000). Anti-thrombin antigen was assayed by single radial immunodiffusion in M-Partigen plates (Behring-Werke, Marburg, Germany).

ELISA for aPLs.  Recent studies have indicated that anti-β2-glycoprotein I and anti-prothrombin do not recognize the native forms of β2-GP I and prothrombin on plain polystyrene ELISA plates, but do bind to the conformationally changed structures of these molecules coated on γ-irradiated polystyrene ELISA plates (Igarashi et al, 1996; Galli & Barbui, 1999; Nojima et al, 2001b). In this study, we used a specific ELISA system for detecting the antibodies to prothrombin or β2-GP I, in which human prothrombin or β2-GP I (Diagnostica Stago) was immobilized directly on γ-irradiated polystyrene ELISA plates. Such plates (Nunc-Immunoplate, Maxi-Sorp, Kamstrup, Roskilde, Denmark) were coated overnight at 4°C with 50 µl per well of human prothrombin or β2-GP I suspended at a concentration of 10 µg/ml in Tris-buffered saline (TBS: 50 mmol/l Tris-HCl, 0·1 m/mol NaCl, pH 7·4). The wells were blocked for 60 min at room temperature with 50 µl of TBS containing 1·0% bovine serum albumin (BSA; Sigma, St Louis, MO, USA), and then washed three times with TBS containing 0·1% Tween 20 (TBS Tween). Thereafter, 50 µl of plasma sample (diluted 101 times with 1·0% BSA-TBS, 0·1% Tween 20) was added to each well. After 60 min of incubation at room temperature, the wells were washed with TBS-Tween. Horseradish peroxidase-conjugated goat anti-human IgG (γ-chain specific, A-2290) F(ab′)2 fragment of affinity isolated Ab (Sigma) was used, and the colour was developed by means of tetramethylbenzidine (TMB) solution (Moss, MD, USA). The OD was measured at 450 nm. Monoclonal anti-human β-2-GP I and anti-human prothrombin (MBL, Nagoya, Japan) were used in each assay as a positive control and selected normal plasma samples were used as a negative control. First, we determined the levels of anti-β2-GP I and anti-prothrombin Abs in the 80 normal healthy control subjects. Each of these antibody levels detected by ELISA was log transformed to approximate normality using the stat flex program (ARTECH, Osaka, Japan) before performing statistical analysis. The mean + 3SD of each antibody in the 80 healthy controls subjects was chosen as the cut-off point. The cut-off values (mOD) for anti-prothrombin and anti-β2-GP I were 500·8 and 392·1 milliabsorbence. A result was regarded as positive when the log-transformed absorbence exceeded each cut-off value. To assess ELISA assay performance, both intra-assay and interassay coefficient of variations (CV) were calculated. The intra-assay CV was established from 24 measurements of a control sample within an assay run and was calculated to be 5·4% and 4·2% for anti-prothrombin and anti-β2-GP I respectively. Interassay CV was calculated from measurements of a control sample assayed on 10 different assay runs and was 9·6% and 8·8% for anti-prothrombin and anti-β2-GP I respectively.

Detection of lupus anticoagulant (LA) activity.  The LA activity was detected using both the diluted Russell Viper Venom time (dRVVT) and Staclot LA test. The dRVVT (Gradipore, Sydney, Australia) and Staclot LA (Diagnostica Stago) tests were performed using commercially available screening and confirmatory tests as reported previously (Nojima et al, 2001a).

dRVVT-Gradipore.  Initially, 200 µl of plasma sample was incubated at 37°C. After 5 min of incubation, 200 µl of LA-screen reagent (simplified dRVVT reagent), which had been preincubated at 37°C, was added and the clotting time was then measured with an Amelung KC-10 A (Heinrich Amelung GmbH, Lemgo, Germany). This time was designated as LA-screen. The LA confirm time was determined by incubating a 200-µl volume of plasma sample at 37°C for 5 min before the addition of 200 µl of prewarmed LA-confirm reagent (phospholipid-rich reagent) and measurement of the clotting time. The ratio of the clotting time (LA-screen/LA-confirm) was calculated and a value > 1·3 was defined as dRVVT-positive.

Staclot LA.  Initially, 50 µl of plasma sample was incubated at 37°C with 50 µl of buffer in Tube 1. In Tube 2, a similar volume of sample was incubated with 50µl of hexagonal phase phosphatidylethanolamine (HPE). After 9 min of incubation at 37°C, 50 µl of normal human platelet-poor plasma was added to each tube. Then, 1 min later, 100 µl of activated partial thromboplastin time (APTT)-reagent was added to Tube 1 and Tube 2, and these tubes were then incubated for 5 min at 37°C. Next, 100 µl of 0·0025 mol/l CaCl2, which had been preincubated at 37°C, was added to Tube 1 and Tube 2. The clotting time for Tube 1 and Tube 2 was measured with the Amelung KC-10 A, and when the clotting time in Tube 2 was greater (8 s or more) than that in Tube 1, LA was defined as positive.

Statistical analysis.  Fisher's exact probability test was used to compare the prevalence of each APC-R between the SLE patients with or without VTE. The non-parametric Mann–Whitney U-test was used to compare the values of the APC ratio among groups. As an approximation of the relative risk, the odds ratios (OR) and 95% confidence intervals (CI) were calculated for several putative risk factors using univariate logistical regression analysis with the statistical program stat flex. An OR was considered statistically significant when the lower limit of the 95% confidence intervals (CI) was > 1·0 or if the upper limit was < 1·0. The variable that achieved statistical significance in the univariate logistical regression analysis was tested by multivariate logistical regression analysis by the stat flex program. In the multivariate logistical regression analysis, a value of P < 0·05 was considered to be statistically significant and indicated a risk factor.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Patients groups with or without VTE, and normal control group

In total, 96 patients with SLE were evaluated with objective tests for diagnosing VTE; 19 patients diagnosed with VTE (10 cases with DVT and nine cases with PI) were assigned to the VTE-positive group. The other 77 subjects, who had no abnormality in the objective tests for diagnosing VTE, were considered as the VTE-negative group. Table I shows the main characteristics of the VTE-positive group, VTE-negative group, and the control group. There was no significant difference in age and sex among these three groups.

Table I.  Characteristics of the systemic lupus erythematosus (SLE) patients groups with or without venous thromboembolism (VTE) and control group.
 VTE-positiveVTE-negativeNormal control
  1. There was no significant difference in age and sex among these three groups.

Number of cases19 cases77 cases80 cases
Female/male (n)17/273/474/6
Mean age44·243·739·8
Age range21–6823–7222–60

It has been established that patients who were deficient in protein C, protein S or anti-thrombin had a markedly increased risk for VTE (Griffin et al, 1981; Comp & Esmon, 1984; Comp et al, 1984; Lane et al, 1996). Therefore, we studied the concentrations of protein C, protein S and anti-thrombin in the 19 patients with VTE; none had any congenital abnormalities in protein C, protein S or anti-thrombin.

Association between acquired APC-R and VTE

APC-R was present in 33 (34·4%) out of the 96 patients with SLE. Factor V Leiden was successfully examined in 25 patients with APC-R, and all of them tested negative for the factor V Leiden mutation. Genetic testing was unsuccessful in the other eight patients with APC-R (one patient with VTE and seven patients without VTE). As APC-R was transient in all eight patients, they were assumed to be factor V Leiden-negative. Therefore, all 33 patients were considered to have acquired APC-R. The prevalence of acquired APC-R was significantly higher in the SLE patients with VTE (12 out of 19 cases, 63·2%, P < 0·01) than in those without VTE (21 out of 77 cases, 27·3%). The results revealed that the presence of acquired APC-R was a strong risk factor for VTE (OR 4·57; 95% CI 1·59–13·2; P = 0·0049; Table II). Furthermore, the mean value of APC sensitivity ratios was significantly lower for the SLE patients with VTE (mean ± SD, 1·76 ± 0·43, P < 0·001, Table III) than for those without VTE (2·24 ± 0·41) or for normal control subjects (2·68 ± 0·34).

Table II.  Association between acquired activated protein C resistance (APC-R) and VTE in SLE patients.
VTEPrevalence of APC-RLogistical analysis
OR95% CIP-value
  • *

    Results shown to be statistically significant.

  • APC-R, activated protein C resistance; VTE, venous thromboembolism; OR, odds ratio; CI, confidence interval. An OR was considered statistically significant when the lower limit of the 95% CI was > 1·0.

  • Statistical analysis was performed using logistical analysis. A value of P < 0·05 was considered to be statistically significant and indicated a risk factor.

Positive63·2% (12/19 cases)4·571·59–13·2*0·0049
Negative36·8% (21/77 cases)
Table III.  APC-sensitivity ratios between the VTE-positive group, VTE-negative group and normal control group.
GroupsNumberMean ± SDP-value
  • *

    P-value versus VTE-negative group or normal control group.

  • Statistical analysis was performed using the non-parametric Mann–Whitney U-test.

VTE-positive19 cases1·76 ± 0·43< 0·001*
VTE-negative77 cases2·24 ± 0·41 
Normal control80 cases2·68 ± 0·34 

Association of acquired APC-R with anti-β2-GP I Abs, anti-prothrombin Abs and LA activity

The prevalence of anti-prothrombin and anti-β2-GP I in the 96 patients with SLE was as follows: anti-β2-GP I, 28 cases (29·2%); anti-prothrombin, 49 cases (51·0%). Using both dRVVT and Staclot LA tests, we detected LA activity in 45 (46·9%) out of the 96 patients with SLE. All SLE patients were divided into four groups according to the results of ELISA for each aPL Ab (anti-prothrombin and anti-β2-GP I) and LA activity: ELISA+ and LA+; ELISA+ and LA; ELISA and LA+; and ELISA and LA. The results of univariate and multivariate logistical analysis are shown in Table IV. A significant association was observed between acquired APC-R and the co-existence of anti-β2-GP I Abs and LA activity (OR 3·00; 95% CI 1·09–8·24) or of anti-prothrombin Abs and LA activity (OR 3·54; 95% CI 1·45–8·58). In contrast, there was no association between acquired APC-R and the presence of anti-β2-GP I Abs, anti-prothrombin Abs or LA activity alone. Furthermore, it is important to note that theabsence of these aPL Abs (anti-β2-GP I ELISA, anti-prothrombin ELISA and LA) was a significant negative risk factor for the prevalence of acquired APC-R. Multivariate logistical regression analysis indicated that the co-existence of anti-β2-GP I and LA activity was not a significant risk factor for the prevalence of acquired APC-R (OR 1·61; 95% CI 0·49–5·28). It was clear that only the co-existence of anti-prothrombin and LA activity was a significant risk factor for the prevalence of acquired APC-R (OR 2·86; 95% CI 1·02–8·02; Table IV).

Table IV.  Association between the prevalence of acquired APC-R and the presence of each anti-phospholipid (aPL) Ab in SLE patients.
ELISALAnAPC-R positive casesUnivariate analysisMultivariate analysis
OR95% CIOR95% CI
  • *

    Results shown to be statistically significant risk factor for APC-R.

  • Results shown to be statistically significant negative risk factor for APC-R.

  • APC-R, activated protein C resistance; LA, lupus anticoagulant; β2-GP I, β2-glycoprotein I; PT, prothrombin; n, number of cases; OR, odds ratio; CI, confidence interval.

  • Statistical analysis was performed using univariate and multivariate logistical analysis. An OR was considered statistically significant when the lower limit of the 95% CI was > 1·0.

Anti-β2-GP I
 ++20113·001·09–8·24*1·610·49–5·28
 +  – –– + – 8 53·570·80–16·0  
25122·200·86–5·60  
43 50·12 0·04–0·35  
Anti-PT
 ++34183·531·45–8·58*2·861·02–8·02*
 +  – –– + –15 40·650·19–2·23  
11 51·700·48–6·04  
36 60·240·09–0·67  

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Venous thromboembolism (VTE), such as DVT and PE, is a common manifestation in SLE patients with aPL Abs (Ginsberg et al, 1995; Palosuo et al, 1997; Wahl et al, 1997; Galli & Barbui, 1999). However, the precise mechanism responsible for VTE in these patients remains unclear. The present study shows that 52·9% (18 out of 34 cases) of SLE patients with both anti-prothrombin antibodies and lupus anticoagulant (LA) activity had an acquired activated protein C resistance (APC-R) and that this condition was a strong risk factor for VTE.

Resistance to APC is defined as a decreased anticoagulant response to the activated protein C pathway. Hereditary APC-R caused by the factor V Leiden mutation is clearly associated with an increased risk of VTE (Lane et al, 1996; De Stefano et al, 1999; Martinelli et al, 2000). However, factor V Leiden mutation has not been reported in Japan up to now. Acquired APC-R, a phenotypic APC-R that occurs in the absence of the factor V Leiden mutation, has been reported in patients with defined APS (Ehrenforth et al, 1995; Martinuzzo et al, 1996; Ruiz-Arguelles et al, 1996; Aznar et al, 1997; Picillo et al, 1998; Male et al,2001), in patients with VTE (Zivelin et al, 1999; Haim et al, 2001), in pregnancy (Cumming et al, 1995) and in individuals using oral contraceptives (Henkens et al, 1995; Rosing et al, 1997). However, the mechanism by which acquired APC-R is generated in these conditions has not been elucidated.

In this study, APC-R was present in 34·4% of the patients with SLE, none of whom had the factor V Leiden mutation. The presence of acquired APC-R was significantly associated with the prevalence of VTE, being present in 63·2% of the VTE-positive patients. SLE patients with acquired APC-R had a 4·57 (OR) increased risk of VTE compared with SLE patients without APC-R (95% CI 1·59–13·2; P = 0·0049). Furthermore, the mean value of APC sensitivity ratios was significantly decreased in the SLE patients with VTE (mean ± SD, 1·76 ± 0·43, P < 0·001) than in those without VTE (2·24 ± 0·41) or normal control subjects (2·68 ± 0·34). These results confirmed that acquired APC-R is related to the pathogenesis of VTE in patients with SLE.

Concerning the relationship between aPL Abs and acquired APC-R, anti-β2-GP I Abs and LA were reported to be associated with acquired APC-R in patients with SLE (Malia et al, 1990; Martinuzzo et al, 1996). The prevalence of acquired APC-R induced by these Abs has been hypothesized to be a possible mechanism of aPL Ab-associated thromboembolic complications in patients with SLE. More recently, the presence of LA was reported to be a significantly associated with the prevalence of acquired APC-R in paediatric patients with SLE, and the prevalence of acquired APC-R was significantly more frequent in LA-positive patients (50%) than in LA-negative patients (21%) (Male et al, 2001). Furthermore, some studies suggest that plasma or purified IgG fractions from LA-positive patients impaired APC-mediated factor Va inactivation (Oosting et al, 1993). However, some investigations showed that LA activity could be caused by anti-β2-GP I Abs and/or anti-prothrombin Abs and that both anti-β2-GP I and anti-prothrombin Abs could be divided into two types, one showing LA activity and one not (Galli et al, 1995, 1997b, 1998; Horbach et al, 1998; Galli & Barbui, 1999). It was not previously clear which aPL Abs were associated with acquired APC-R, anti-prothrombin Abs or anti-β2-GP I Abs.

In previous studies, we examined the anti-prothrombin and anti-β2-GP I using a specific ELISA, in addition to LA activity detected by a phospholipid-dependent coagulation assay in 124 patients with SLE, and found that the co-existence of IgG anti-prothrombin Abs and LA activity correlated best with a history of VTE in patients with SLE (Nojima et al, 2001a). In this study, we examined which aPL Abs were associated with acquired APC-R, anti-prothrombin Abs or anti-β2-GP, and whether or not acquired APC-R caused by these aPL Abs was related to the pathogenesis of VTE in patients with SLE.

The univariate logistical regression analysis indicated that the co-existence of anti-prothrombin and LA activity was the most significant risk factor for the prevalence of acquired APC-R (OR 3·53; 95% CI 1·45–8·58) but that the co-existence of anti-β2-GP I and LA activity also appeared to be a significant risk factor for it (OR 3·00; 95% CI 1·09–8·24). However, when multivariate logistical regression analysis was performed, it became clear that only the co-existence of anti-prothrombin and LA activity was a significant risk factor (OR 2·86; 95% CI 1·02–8·02). Furthermore, it is important to note that the absence of these aPL Abs (anti-β2-GP I ELISA, anti-prothrombin ELISA and LA) was a significant negative risk factor for the prevalence of acquired APC-R. These findings indicate that the co-existence of anti-prothrombin and LA activity may be one of the important factors in the pathogenesis of acquired APC-R in patients with SLE; however, the presence of anti-prothrombin Abs, anti-β2-GP I Abs or LA activity alone may not be sufficient to account for this dysfunction in SLE patients.

In this present study, no significant decrease in the plasma concentration of protein S or protein C was observed in relation to the presence of anti-prothrombin Abs with LA activity or of APC-R, or to the prevalence of VTE. These findings suggest that acquired APC-R in SLE patients is due to functional interference of the protein C pathway by the co-existence of anti-prothrombin Abs and LA activity, the action of which may represent an important mechanism to account for the prevalence of VTE in patients with SLE.

Recently, it was suggested that the complexes of phospholipid and plasma proteins such as protein C and protein S were also recognized by aPL Abs and that these Abs may also to be involved in thrombotic complications (Roubey, 1994; Pengo et al, 1996; Galli & Barbui, 1999; Nojima et al, 2001a). Further studies are currently in progress to elucidate the mechanisms by which these Abs inhibit the anticoagulant activity of the protein C pathway.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
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