Cleaved protein S (PS), total PS, free PS, and activated protein C cofactor activity as risk factors for venous thromboembolism.
As for other vitamin K-dependent proteins involved in coagulation, the N-terminal part of PS contains a [gamma]-carboxyglutamic acid-containing domain involved in phospholipid binding and four epidermal growth factor-like domains. PS also contains a protease-sensitive loop, called the thrombin-sensitive region, between the [gamma]-carboxyglutamic acid-containing domain and the first epidermal growth factor-like domain. Under physiologic conditions, the thrombin-sensitive region can be cleaved after [Arg.sup.60] (11), and this cleavage can be reproduced in vitro by factor Xa (FXa) (12). The resulting two-chain disulfide-linked protein can no longer potentiate APC activity (13). Instead of the serine protease domain found in other vitamin K-dependent proteases involved in coagulation, the C-terminal part of PS is homologous to sex-hormone-binding globulin; this domain allows PS to bind to C4b-binding protein (C4b-BP), a protein of the complement system comprising seven [beta]-chains and one or no [beta]-chain (14). PS binds to the [beta]-chain of C4b-BP with such affinity that all of the C4b-BP isoforms with a [alpha]-chain circulate as PS/C4b-BP complexes having no cofactor activity, whereas only ~0% of PS remains free. Therefore, in plasma, PS circulates as four different molecular species, comprising intact or cleaved free PS and intact or cleaved C4b-BP-bound PS. Measurement of free PS has been recognized as the appropriate assay to detect hereditary PS deficiency in thrombophilic families (15), but to date, it has not been clear whether phenotypic variations in PS concentrations are associated with a risk of VTE (16-18). As recently pointed out, the assays designed to quantify free PS, which are based on the isolation of the free molecules by their ability to bind a specific monoclonal antibody (mAb) or to bind C4b-BP, may yield variable results depending on assay conditions (19).
The primary objective of our study was to reevaluate the putative risk for VTE of phenotypic PS deficiency. To this aim, we designed a case-control study including 87 patients with VTE explored at the end of anticoagulant treatment and 174 healthy individuals. Patients and controls were matched for age, sex, and hormonal treatment to minimize physiologic or acquired factors that influence PS concentrations. We took advantage of a recently developed mAb assay to measure cleaved PS and evaluate its significance in terms of risk of VTE in comparison with total PS and free PS measured in two different assays based, respectively, on the specific binding of PS to a mAb or to C4b-BP.
Materials and Methods
The cases were consecutive patients with at least one episode of symptomatic deep venous thrombosis diagnosed by compression ultrasonography or venography and/or with a pulmonary embolism diagnosed by perfusion and ventilation scanning, conventional pulmonary angiography, or computer tomographic angiography. Patients with recent thrombosis (<3 months) and patients receiving anticoagulant treatment were excluded. Blood samples were drawn at least 1 month after discontinuation of anticoagulant treatment. The median delay between exploration and the last event was 10 months (range, 3-304 months); the delay was at least 5 months in 90% of cases. This study was a case-control study, and all cases from the original case-control study [PATHROS study (20)] fulfilling these criteria were included. Between September 1997 and November 1999, among the 296 new patients referred to the vascular medicine department who were included in the PATHROS study, 87 met the criteria mentioned above. Of these, 56% had deep vein thrombosis, 29% had a pulmonary embolism and deep vein thrombosis, and 15% had a pulmonary embolism. In 46% VTE was spontaneous, and 24% had recurrent events. Fort[gamma]eight percent had a family history of VTE.
Controls were randomly selected from a population of 1224 healthy individuals after stratification for age, sex, oral contraception, and hormone replacement therapy in postmenopausal women; two controls were matched with one case. The healthy controls were recruited in a healthcare center to which they had been referred for a routine check-up. Individuals with a history of VTE, arterial disease (stroke, myocardial infarction, angina pectoris, or peripheral vascular disease), or known malignancy were excluded on the basis of a medical questionnaire. All participants gave their informed consent, and the local ethics committee approved the study.
Venous blood was collected into tubes containing 0.11 mol/L trisodium citrate (1:10) and centrifuged within 2 h. Plasma was obtained by two centrifugation steps at 12[degrees]C for 15 min at 23008; we then distributed the plasma into aliquots and stored the aliquots at -40[degrees]C until analysis. DNA was prepared from white blood cells by a standard technique (21) and stored at 4[degrees]C until analysis. The FV Arg506G1n and prothrombin G20210A mutations were identified as described previously (22).
PREPARATION OF PS AND FXa-CLEAVED PS
Plasma PS and recombinant PS were obtained as described previously (11,13). The method described by Long et al. (12) was used to prepare FXa-cleaved PS, with the following minor modifications: PS (final concentration, 700 nmol/L) was allowed to react for 3 h at 37[degrees]C with human FXa [alpha]0 nmol/L; Diagnostica Stago, Asnieres, France) in the presence of phospholipid vesicles in 50 mmol/L Tris, 150 mmol/L NaCl, 2 mmol/L CaC[l.sub.2], pH 7.4. The absence of uncleaved PS was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions.
For Western blot analysis experiments, plasma PS and FXa-cleaved PS were subjected to SDS-PAGE on a 10% SDS polyacrylamide gel under reducing conditions. After electrophoretic transfer of proteins from the gel to nitrocellulose membranes, membranes were blocked for 1 h with 50 g/L nonfat dry milk in Tris-buffered saline, pH 7.4, and incubated with either mAb 5A5G2 or mAb 4A10H6, followed by peroxidase-conjugated rabbit anti-goat IgG. An enhanced chemiluminescence system was used for signal detection.
PROTEIN S MEASUREMENT
Five different assays were performed to evaluate PS concentrations: two commercial assays and three homemade assays. APC cofactor activity was measured with the Staclot PS[R] assay (Diagnostica Stago), according to the manufacturer's instructions.
Free PS was measured in plasma either by a specific ELISA (Asserachrom free protein S[R] assay; Diagnostica Stago), according to the manufacturer's instructions, or by the enzyme-linked ligand sorbent assay (ELSA) method described by Giri et al. (23) with a minor modification: a two-step revelation system was used, with a first incubation using a mAb (mAb 14C10H10; bioMerieux, Marcy FEtoile, France) directed against PS that does not interfere with PS binding to C4b-BP. The bound mAb was then detected with an anti-IgG polyclonal peroxidase-conjugated antibody (Sigma Aldrich). A frozen plasma from a single individual was used for quality control on each plate. The interassay CV was 6%.
Previously described ELISAs for total and cleaved PS were used (11). For the cleaved PS assay, plasma was diluted 1:10 (final volume, 100 [micro]L) in 50 mmol/L Tris, 150 mmol/L NaCl, 5 mmol/L EDTA, pH 7.4; for the total PS assay, plasma was diluted 1:500 (final volume, 100 [micro]L) in 50 mmol/L Tris, 150 mmol/L NaCl, 2 mmol/L Ca[Cl.sub.2], 3 nmol/L hirudine, pH 7.4. The between-assay CVs were 5% for the total PS assay and 6% for the cleaved PS assay.
For the three homemade assays, the calibration curves were constructed with a pool of plasma from 20 healthy individuals. To evaluate the percentage of cleaved PS vs total PS, we quantified the two molecular forms of PS in the pool vs calibration curves constructed, respectively, by adding purified PS and purified FXa-cleaved PS to PS-depleted plasma (Diagnostica Stago). This allowed us to express total and cleaved PS in nmol/L. Free PS and APC cofactor activity are expressed as percentages of plasma calibrator values.
All tests were performed at room temperature. Those performing and interpreting the tests were not blinded to the diagnosis.
Results are presented as means (SD) for continuous variables and as numbers and percentages for categorical variables. Variables followed a gaussian distribution. Groups, consisting of controls and patients with VTE, were compared using the Student unpaired t-test for continuous variables and a [chi square] or Fischer exact test for categorical variables. Relationships between variables were assessed by the correlation coefficient (r), and the proportion of variability was quantified as [r.sub.2]. The influence of the concentrations of the various forms of PS on the risk of VTE was assessed with use of the control 10th percentiles. The odds ratios (ORs) for VTE associated with values below the 10th percentiles were derived from 2 x 2 tables (univariate OR) or from a logistic regression model that included VTE as the dependent variable and age, sex, hormone treatment, the FV Arg506Gln mutation, and the prothrombin G20210A mutation as independent covariables. Statistics were computed with the StatView[R], Ver. 5 (SAS Institute).
Plasma samples from 87 patients with a history of VTE and from 174 controls were analyzed for cleaved PS, total PS, free PS evaluated by ELISA and ELSA, and PS APC cofactor activity.
[FIGURE 1 OMITTED]
Cleaved PS was measured (11) with a homemade ELISA based on a mAb (mAb 5A5G2) specific for cleaved PS. The specificity of the mAb was further confirmed by an immunoblot experiment showing that only cleaved PS was recognized by mAb 5A5G2 (Fig. 1).
The influence of sex and age on PS concentrations was studied in the 174 controls. Cleaved PS was significantly lower in women, with concentrations of 42 (17) nmol/L in men and of 36 (12) nmol/L in women (P = 0.009). Interestingly, age had no influence on cleaved PS concentrations (r = 0.08; P = 0.3). The relationship between the concentrations of cleaved PS and other molecular forms of PS was studied in the whole population (Table 1). Cleaved PS concentrations were significantly correlated with total PS (r = 0.38) and with free PS concentrations (r = 0.59), respectively, accounting for 14% and 35% of their variations. Because cleaved PS is devoid of APC cofactor activity, we expected to find a negative correlation between cleaved PS and APC cofactor activity, but surprisingly, cleaved PS concentrations correlated positively with APC cofactor activity (r = 0.51). To check that FXa-cleaved PS had no APC cofactor activity in our assay, we compared the APC cofactor activity of purified PS to that of cleaved PS added to PS-depleted plasma. After supplementation with 300 nmol/L uncleaved PS, APC cofactor activity was 120%, whereas the same amount of cleaved PS yielded APC cofactor activity of only 12%. This 90% decrease in the recovery of APC cofactor activity, found in two different experiments, indicates that PS cleaved after [Arg.sup.60] has virtually no activity in our assay.
To study the possible influence of the different terms of circulating PS on the risk of VTE, we compared the 87 cases with the 174 controls, whose main characteristics are shown in Table 2. The cases and controls were well matched in terms of age, sex, and the percentage of women on hormone treatment (oral contraception or replacement therapy). As expected, the prevalences of the FV Arg506G1n and prothrombin G20210A mutations were higher in the cases than in the controls, with respective frequencies of 23% vs 3.5% and 15% vs 6.6%.
As shown in Table 2, cleaved PS concentrations were not significantly different in cases and controls, with respective values of 36 (11) nmol/L [9.7% (2.4%) of total PS] and 39 (14) nmol/L [9.9% [alpha].2%) of total PS]. The concentration of total PS was also similar in the patients and the controls [376 (73) nmol/L vs 390 (73) nmol/L], whereas free PS concentrations determined by ELISA or ELSA and APC cofactor activity were significantly lower in the cases than in the controls: 91% (21%) vs 96% (18%), P = 0.03; 98% (26%) vs 107% (22%), P = 0.006; and 85% (21%) vs 94% (20%), P = 0.001, respectively.
The risk of VTE associated with low PS, defined as below the control 10th percentile, was evaluated by calculating the ORs (Table 3). ORs were 0.9 [95% confidence interval (CI), 0.4-2.3] for cleaved PS and 1.3 (0.6-3.0) for total PS, indicating no significant association. Free PS concentrations were associated with an increased risk of VTE with ORs of 2.4 (1.2-5.0) and 2.1 (1.0-4.4) for the ELISA and the ELSA, respectively, as was APC cofactor activity, with an OR of 2.8 (1.3-5.7). Multivariate analysis including age, sex, hormone treatment, and the FV and FII mutations as covariables confirmed that free PS concentrations, whatever the method used, and APC cofactor activity were independent risk factors for VTE in this population, with ORs of 2.9 (1.3-6.4) and 2.5 (1.1-5.6) for free PS measured by ELISA and ELSA, respectively, and 2.9 (1.3-6.4) for APC cofactor activity. For patients with free PS or APC cofactor activity below the 10th percentile, the frequencies of family history for VTE, recurrent events, or associated pulmonary embolism were not different from the rest of the case population.
Because the cleavage of PS is associated with a 90% decrease in APC cofactor activity, we looked for an association of increased cleaved PS with VTE risk. However, the OR associated with cleaved PS concentrations above the 90th percentile was 0.8 (95% CI, 0.3-2.2), ruling out this hypothesis.
There have been numerous studies showing that PS deficiency, when transmitted hereditarily, is a risk factor for VTE (24-26). However, in contrast to the observation that the risk of VTE increases with decreasing protein C concentrations (16), the implication of phenotypic PS deficiency in VTE has not been clearly established. Actually, both case-control studies performed showed no involvement of total PS concentrations, whereas free PS gave contradictory results (17,18).
To clarify this issue, we designed a new case-control study comparing 87 consecutive patients with objectively diagnosed VTE and 174 healthy controls. Factors known to influence PS concentrations were taken into account by matching the healthy controls for age, sex, and hormone treatment and by excluding patients receiving oral anticoagulants or with acute thrombosis. In addition to total and free PS measurements using ELISAs as in previous studies (17,18), we measured free PS by an ELSA, APC cofactor activity, and cleaved PS to determine which assay(s) would be the most relevant to evaluate the thrombotic risk. Our study had enough power ([beta] = 0.2, 80% power; two-sided test, [alpha] = 0.05) to detect a difference in cleaved PS concentrations of >4.5 nmol/L between groups and an OR >2.7 for VTE in individuals with cleaved PS below the 10th percentile.
Plasma cleaved PS was first evaluated in healthy controls to define the physiologic variations of this PS form. The mean circulating cleaved PS concentration in the 174 controls was 39 nmol/L, corresponding to 10% of total PS. As for free PS concentrations and APC cofactor activity, cleaved PS concentrations were higher in men (18,27-29) but did not increase with age. Evaluation of the relationship between concentrations of cleaved PS and other molecular forms of PS in the whole population showed a positive correlation among the different assays. As expected, we found that APC cofactor activity was highly correlated with free PS. Cleaved PS was poorly correlated with total PS (r = 0.14), suggesting that cleavage is not related to PS synthesis and thus probably occurs after secretion of the protein, although it remains possible that cleaved PS could be cleared from circulation by a different mechanism. Interestingly, the correlation between cleaved and free forms was higher, although in vitro experiments showed that cleavage was not affected by C4b-BP binding (unpublished results). One possible explanation is that binding to C4b-BP might favor collocation of PS and the protease involved in its cleavage. Surprisingly, we found a positive correlation between cleaved PS concentrations and APC cofactor activity (r = 0.51). Indeed, cleaved PS was devoid of APC cofactor activity in a FVIII proteolysis assay (12) and in a global coagulation test (13), and the lack of such activity was confirmed in this assay. This correlation could reflect a shift in total and free PS. Indeed, a decrease in total or free PS concentrations, and thus in APC cofactor activity, could lead to low cleaved PS concentrations. However, correlation coefficients showed that only 14-35% of the variation of cleaved PS might be explained by differences in total and free PS concentrations and thus only partially explain the positive correlation between the concentration of the cleaved PS form and APC cofactor activity.
In the present study, cleaved PS concentrations did not differ significantly between the cases and controls, ruling out a major role of PS cleavage in the variations of circulating PS available for anticoagulant activity. However, the role of PS as an APC cofactor was confirmed by the finding that, when below the 10th percentile, free PS, measured by either ELISA or ELSA, and APC cofactor activity were independent risk factors for thrombosis, with respective ORs of 2.9,2.5, and 2.9. Worth mentioning is that in two case-control studies, the relative risk of thrombosis was not clearly influenced by a deficiency in free PS, with respective ORs of 2.4 (95% CI, 0.8-7.9) and 1.3 (0.5-3.5), inferring that low free PS measured by ELISA may not itself be a risk factor (17,18). In familial studies, PS deficiencies had ORs for thrombosis of 5-11.5 (24-26). The discrepancy between case-control and familial studies might be explained by an additional genetic risk factor for thrombosis that cosegregates with PS deficiency (30-33), but the present study shows that both free PS concentrations and APC cofactor activity are independent risk factors for VTE when below the 10th percentile. Interestingly, in a large study of hemostatic variables in thrombophilic families, household effects were found to account for >20% of free PS variance (34). Because the controls in both previous case-control studies (17,18) were relatives or partners, they may have been subjected to the same environmental factors, attenuating the difference in PS concentrations between cases and controls.
Other differences in study design may account for the link between low PS concentrations and thrombosis found in our study but not in the two previous studies. As underlined by Faioni et al. (17), inclusion of patients with recurrent VTE (24% of cases in our study) may increase the probability of recruiting patients with hereditary PS deficiency compared with the Leiden Thrombophilia Study (LETS), which included only those who had experienced their first VTE event (18). Furthermore, in the two previous case-control studies, the cutoffs were chosen to distinguish between individuals with hereditary PS deficiency and healthy controls. In contrast, we used a cutoff below the 10th percentile, as our purpose was not to identify patients with hereditary deficiencies, which may be difficult on the basis of plasma concentrations with regard to the variability of the phenotypic expression of the PS gene mutation [alpha]5), but rather to seek an association between low PS concentrations and the risk of VTE, independent of a genetic defect. We reanalyzed our data using cutoff values obtained in 400 healthy controls (<67% for men, <49% for women using oral contraceptives, and <57% for women not using oral contraceptives; unpublished data) to identify patients with hereditary deficiencies on the same basis as in the LETS study (2.5th percentile): 2.7% of our cases had values below the reference interval (2.1% in LETS), and 1.4% of our controls had values below the reference interval (1.6% in LETS). The apparent discrepancy between the results reported in the LETS study and our results may be explained by the choice of different definitions of low PS.
In conclusion, this case-control study confirms that PS with APC cofactor activity plays a role in natural anticoagulant mechanisms, with low free PS concentrations determined by ELISA or ELSA and low APC cofactor activity being associated with an increased risk of VTE.
We thank bioMerieux Society (Marcy l'Etoile, France) for providing mAbs 5G5H9, 4A10H6, and 14C10H10; Diagnostica Stago Society (Asnieres, France) for the APC cofactor and Asserachrom free PS analysis; and Jose Bon-Deguingand for excellent technical assistance.
Received September 4, 2002; accepted January 21, 2003.
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 Service d'Hematologie Biologique A and  Service des Maladies Vasculaires, Hopital Europeen Georges Pompidou, AP-HP, Paris and INSERM 428, UFR de Pharmacie, Universite Paris V, France.
DELPHINE BORGEL,  * JEAN-LUC RENY,  DAVID FISCHELIS,  SOPHIE GANDRILLE,  JOSEPH EMMERICH,  JEAN-NOEL FIESSINGER,  and MARTINE AIACH 
 Nonstandard abbreviations: PS, protein S; VTE, venous thromboembolism; FV and FX, factor V and X; APC, activated protein C; C4b-BP, C4b-binding protein; mAb, monoclonal antibody; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; ELSA, enzyme-linked ligand sorbent assay; OR, odds ratio; CI, confidence interval; and LETS, Leiden Thrombophilia Study.
* Address correspondence to this author at: Service d'Hematologie Biologique A, Hopital Europeen Georges Pompidou, 75908 Paris Cedex 15, France. Fax 33-1-56-09-39-13; e-mail email@example.com.
Table 1. Correlation of cleaved PS with other forms of PS in the whole population (n = 261). Correlation r (95% CI) [r.sup.2] P Cleaved PS with total PS 0.38 (0.27-0.47) 0.14 <0.0001 Cleaved PS with free PS 0.59 (0.51-0.67) 0.35 <0.0001 Cleaved PS with APC 0.51 (0.42-0.60) 0.26 <0.0001 cofactor activity Table 2. Characteristics and PS concentrations of patients with VTE and controls. Controls Cases (n = 174) (n = 87) P Age, years Mean (SD) 48 (14) 48 (14) >0.99 Median (range) 49 (16-77) 49 (16-78) Male, n (%) 64 (37) 32 (37) >0.99 Hormone therapy, n (%) (a) 31 (28) 16 (29) >0.99 Prothrombin G20210A, n (%) 11 (6.6) 13 (15) 0.04 FV Arg506Gln, n (%) 6 (3.5) 20 (23) <0.0001 Mean (SD) cleaved PS, nmol/L 39 (14) 36 (11) 0.15 Mean (SD) total PS, nmol/L 390 (73) 376 (73) 0.13 Mean (SD) free PS, % by ELISA 96 (18) 91 (21) 0.03 by ELSA 107 (22) 98 (26) 0.006 Mean (SD) APC cofactor 94 (20) 85 (21) 0.001 activity, % (a) Percentage of the total number of women in the study. Table 3. Risk of VTE and low PS concentrations. 10th percentile in Patients, % OR, univariate control group (n = 87) analysis (95% CI) P Cleaved PS <24.7 nmol/L 9.2 0.9 (0.4-2.3) 0.9 Total PS <303.1 nmol/L 12.6 1.3 (0.6-3.0) 0.5 Free PS ELISA <75.0% 19.5 2.4 (1.2-5.0) 0.02 Free PS ELSA <75.0% 18.4 2.1 (1.0-4.4) 0.052 APC cofactor activity <70.0% 20.7 2.8 (1.3-5.7) 0.007 OR, multivariate 10th percentile in analysis (a) control group (95% CI) P Cleaved PS <24.7 nmol/L 1.1 (0.4-2.7) 0.9 Total PS <303.1 nmol/L 1.7 (0.7-4.2) 0.2 Free PS ELISA <75.0% 2.9 (1.3-6.3) 0.009 Free PS ELSA <75.0% 2.5 (1.1-5.6) 0.02 APC cofactor activity <70.0% 2.9 (1.3-6.4) 0.009 (a) Logistic regression including deep venous thrombosis as the dependent variable and age, sex hormone treatment, FV Arg506Gln mutation, FII G20210A mutation, and the different forms of PS as independent variables.
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|Title Annotation:||Hemostasis and Thrombosis|
|Author:||Borgel, Delphine; Reny, Jean-Luc; Fischelis, David; Gandrille, Sophie; Emmerich, Joseph; Fiessinger,|
|Date:||Apr 1, 2003|
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