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Acquired free protein S deficiency is associated with antiphospholipid antibodies and increased thrombin generation in patients with systemic lupus erythematosus
Acquired Free Protein S Deficiency Is Associated With Antiphospholipid Antibodies and Increased Thrombin Generation in Patients With Systemic Lupus Erythematosus Jeffrey S. Ginsberg, MD, Christine Demers, MD, Patrick Brill-Edwards, MD, Robert Bona, MD, Marilyn Johnston, ART, Albert Wong, DM, Judah A. Denburg, MD, Hamilton, Canada In order to determine whether there is a relationship between acquired free protein S deficiency and increased thrombin generation, we performed a cross-sectional study of patients with systemic lupus arythematosus (SLE). Plasma samples were assayed for free protein S and were correlated to levels of prothrombtn fragments (F1+2j; an elevated level of Fl+Z was used as a surrogate marker for a prothrombotic state. Assays for anlicardiolipin antibodies (ACA) and lupus anticoagu’bnt &A] were performed on two separate blood samplas taken at least 3 months apart in order to detect the presence of antiphospholipid antibodies. Of the 36 subjMs, 9 had reduced free protein S levels compared to 0 of 21 controls (P = 0.01) and the mean free protein S level was significantiy lower in the SLE population than in controls (0.30 t 0.08 U/mL versus 0.43 * 0.10 U/mL, P
ree circulating protein S is a cofactor for protein C-mediated inhibition of factors Va and VIIIa.112 Congenital deficiency of protein S is characterized by reduced plasma levels of free protein S and is associated with an increased risk of venous thrombosis.” Subjects with congenital protein S deficiency have been reported to have higher plasma levels of prothrombin fragments (F1+2) than their nondeficient family members. c6 F1+2 is a peptide that is cleaved from prothrombin by the prothrombinase complex during thrombin formation.7 Elevated levels of Fl+Z, have been reported in patients with other prothrombotic states.6~s~gIt has been suggested that such levels identify individuals at high risk for thrombosis. Acquired deficiency of free protein S has been reported in patients with antiphospholipid antibodies (APLA).‘O The clinical significance of this finding is not known. Some investigators have suggested that acquired protein S deficiency contributes to the increased risk of thrombosis in individuals with APIA”‘; however, it has not been established that patients with protein S deficiency had increased thrombin generation or increased risk of thrombosis. In order to determine the relationship of acquired free protein S deficiency to thrombin generation, we performed a cross-sectional analysis of patients with systemic lupus erythematosus (SLE). We selected patients with SLE because a substantial proportion30% to 50%~~11~12-haveAPLA. Our population would thus include a reasonable number of patients with protein S deficiency whom we could identify and evaluate. Plasma samples were assayed for free protein S and correlated to levels of F1+2, in search of a relationship between reduced free protein S levels and thrombin generation.
F
PATIENTS AND METHODS Study Population
From the Departments of Medicine and Laboratory Medicme, McMaster University, HamIlton, Ontario, Canada. Dr. Ginsberg’s participation in this study IS supported by a Research Scholarship from the Heart and Stroke Foundation of Canada. Supported by a grant from the Lupus Society of Hamilton. Requests for reorrnts should be addressed to Dr. J. Ginsbere. McM&ter University Medical Centre, 1200 Main Street West, iSC3W11, Hamilton, Ontario, Canada L8N 325. Manuscrtpt submItted February 14, 1994 and accepted in revised form July 5, 1994.
The study population consisted of consecutive patients referred to an SLE clinic in Hamilton, Canada, between June 1,199O and December 341991. All participants fulfilled the 1982 revised American Rheumatism Association criteria for SLE.‘” Clinical characteristics and the results of anticardiolipin antibodies (ACA) and F1+2 assays for this cohort have been published.<’ This study excludes 8 patients from the published study. Five were receiving warfarin therapy, April 1995 The American Journal of Medicine*
Volume 98
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PROTEIN S IN SYSTEMIC LUPUS ERYlIiEMATOSUS/GINSBERG
ET AL
TABLE I Levels of Naturally Occurring Anticoagulants in Normal Controls and Systemic Lupus Erythematosus (SLE) Population TPS FPS Proc ATIII 38 Normal controls (n) 21 21 0.81 0.43 0.97 Mean (U/mL) Normal range (U/mL) 0.52-1.10 0.24-0.62 0.65-l .29 36 36 SLE patients (n) 36 0.30* 1.10 Mean (U/mL) 0.83 0.08 0.31 SD 0.25 ‘Significantly lower than the normal controls (P ~0.001). TPS = total protein S; FPS = free protein S; ProC = protein C; ATIII = antithrombin
and 3 had not given sufficient plasma for assay of free protein S.
Intervention The study was approved by the Institutional Review Board of Chedoke-McMaster Hospitals. All patients who underwent laboratory and clinical evaluation gave informed consent. Each was scheduled to be seen on two separate occasions at least 3 months apart. Patients who were seen on only one occasion were excluded from the analysis.
38 0.97 0.80-1.15 36 1.07 0.17 Ill,
Protein C (Behring Diagnostics, Montreal, Canada). The controls for the assays were derived from normal healthy laboratory volunteers. For the primary anaIysis, the means of the two results obtained on the two separate dates were calculated and used.
Antiphospholipid
Antibodies
The presence or absence of APIA was ascertained by performing an ELISA for ACA and three different assays for lupus anticoagulant (LA). A commercially available, standardized ELISA kit (Cambridge Life Sciences, Laboratory Imports, Toronto, Canada) was used to quantitate IgG ACA. A Laboratory Methods normal range was established and levels 211 IgG For the laboratory assays, blood was drawn into a vacutainer tube (Becton-Dickinson, Mountain View, phospholipid (GPL) U/mL were considered to be abCalifornia) containing 0.105 M buffered citrate. normal. This cutoff was three standard deviations Plasma was immediately separated from cellular ele- above the mean of controls. ments by centrifugation at 1,700~ for 15 minutes at Three tests were used to detect the presence of LA: room temperature, with maximal removal of platelets (1) dilute Russell viper venom time (DRVVT); (2) the being assured by the use of a silicone-based gel (Sure- dilute one-stage prothrombin time (DPT); and (3) the sep, Organon Teknica, Scarborough, Canada). Subse- activated partial thromboplastin time (aPIT). For the quently, plasma was frozen at -70°C until batch assays DPT and the aPTI’, patient samples were run neat and were performed. in a 1:l mix of normal pooled plasma derived from 20 normal hospital personnel. Values for the 1:1 mix Total Protein S, Free Protein S, above the upper limit, of normal for each test were Antithrombin III, and Protein C Assays considered to be positive for LA. The DRWT was perTotal protein S was quantified by enzyme-linked im- formed as previously described.‘” If the test was abmunoelectrodiffusionn assay (ELISA) as previously normal, ionophore-treated platelets were substituted described using antibodies from Affinity Biologicals for bovine phospholipid and the test was repeated; a (Yarker, Canada).14 Free protein S was separated from shortening of the DRWT was considered to be diagcomplexed prot.ein S by the polyetheline glycol (PEG) nostic of LA. The DPT was performed using a l/500 precipitation t,echnique of Comp et al” and measured dilution of rabbit brain thromboplastin (Dade Thromby ELISA. A standard was prepared by PEG precipiboplastin C, Dade International, Miami, Florida) in a tation of a pool of normal plasma and this was consaline CaCl, mixture.‘” The aPTT was performed ussidered to represent a plasma value of 0.40 U/mL. The ing a commercially available reagent (Dade Actin FSL, pool of normal plasma was derived from 10 male and BaxterKanlab, Mississauga, Canada), as previously 20 female healthy volunteers, none of whom were tak- described.” Patients were classified as positive for LA ing oral contraceptives. Antithrombin III and protein if one or more of the assays was positive. C were measured by chromogenic assays using commercially available kits. Antithrombin III was assayed F1+2 Assay using a thrombin inhibition assay (Stachrom ATIII, F1+2 levels were measured on two occasions usDiagnostica Stage, Wellmark Diagnostics, Guelph, ing a commercially available ELISA kit (Behring, Canada). Protein C was measured using Berichrom Marburg, Germany). The normal range for the F1+2 380
April 1995 The American Journal of Medicine@ Volume 98
PROTEIN S IN SYSTEMIC LUPUS ERYTHEMATOSUS/GINSW?G
assay is cl.1 nmol/L. For the analysis, the means of the two results were calculated and used.
Clinical Evaluation The presence or absence of prior thromboembolic disease was ascertained by interview, critical review of objective tests, and the performance of venous Doppler of the leg veins as a specific indicator of previous venous thrombosis. l8,lg Details of the criteria for the diagnosis of venous and arterial thromboembolism have been previously published.“,”
Statistics Review of the F1+2 data indicated that the distribution was skewed since the standard deviation increased with the mean. Hence, each data point was
ET AL
subjected to a logarithmic transformation before comparing means using unpaired t-te~ts.~~ Proportions were compared using Fisher’s exact test. A twosided P value of ~0.05 was considered to be statistically significant.
RESULTS Thirty-six patients (33 women) with a mean age of 39 years (range 19 to 75) were enrolled. Table I summarizes the levels of free and total protein S, protein C, and antithrombin III in the study population and controls. Table II shows the results of APLA, protein S (total and free), and Fl+Z assays for each participant. None of the women was taking oral contraceptives. Two (patients 3 and 26) had been pregnant within 6 weeks of one or both blood samples. One pa-
TABLE II
Pt# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Age (y)/Sex 39F 25F 28F 34F 44F 54M 67F 34F 24F 58F 19F 36M 42F 29F 25F 41F 52F 21F 33F 54F 48F 50F 35F 41F 29F 34F 30F 48F 46F 20F 31F 48F 75M 34F 33F 20F
ACA +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/;: $1 +/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-
LA 2+/2+/3+ 3+/3+ -/3+/3+ -/l+ 3+/3+ l+/-/1+/l+ -/1 +/-/-/-/l+ 1+/l+ 2+/-/-/3+/3+ -/2+ -/1+/l+ l+/-/-/-/-/* -/-/-/-/-/-/-/-/-
Summary of Results TPS FPS PJ/mLl W/mLl 0.69 0.18 (0.15/0.20) 0.68 0.19 (0.19/0.19) 0.63 0.21 (0.17/0.25) 0.69 0.22 (0.19/0.24) 0.48 0.22 (0.20/0.24) 0.88 0.22 (0.22/0.22) 0.51 0.24 (0.21/0.27) 0.76 0.27 (0.34/0.20) 0.49 0.28 (0.27/0.28) 0.66 0.29 (0.31/0.26) 0.80 0.30 (0.35/0.25) 0.60 0.34 (0.30/0.37) 0.75 0.37 (0.41/0.32) 1.22 0.39 (0.37/0.40) 0.73 0.39 (0.45/0.36) 0.92 0.51 (0.47/0.54) 0.40 0.12 (0.10/0.13) 0.50 0.20 (0.20/0.20) 0.98 0.33 (0.30/0.35) 1.04 0.33 (0.38/0.27). 1.34 0.36 (0.36/0.35) 0.65 0.38 (0.38/0.37) 0.56 0.23 (0.24/0.21) 1.00 0.38 (0.46/0.30) 0.91 0.24 (0.24/0.24) 0.64 0.25 (0.25/0.25) 1.12 0.29 (0.28/0.30) 0.88 0.29 (0.29/0.29) 0.96 0.30 (0.28/0.31) 0.94 0.32 (0.30/0.34) 1.29 0.35 (0.25/0.45) 1.00 0.35 (0.35/0.35) 1.02 0.35 (0.37/0.34) 1.11 0.36 CO.26/0.46) 0.77 0.40 (0.43/0.37) 1.28 0.43 (0.47/0.38)
F1+2 (nmW) 1.34 (0.79,1.89) 1.75 (2.20,1.30) 0.93 (1.54,0.32) 1.72 (1.10,2.33) 0.66 (0.84,0.48) 1.76 (1.61,1.91) 0.77 (0.70,0.84) 1.13 (0.96,1.30) 0.74 (0.85,0.63) 0.55 (0.30,0.80) 0.64 (1.24,0.04) 0.81 (0.95,0.67) 1.02 (1.14,0.90) 0.99 (0.93,1.04) 1.20 (1.35,1.04) 0.96 (1.05,0.87) 1.50 (1.80,1.19) 0.80 (0.97,0.63) 0.50 (0.52,0.47) 0.87 (0.83,0.91) 0.79 (0.75,0.82) 0.58 (0.57,0.58) 0.51 (0.50,0.51) 0.52 (0.50,0.53) 0.40 (0.36,0.43) 1.25 (1.24,1.26) 1.00 U.30,0.70) 1.39 (2.10,0.68) 0.73 (0.76,0.70) 0.49 (0.46,0.52) 0.43 (0.40,0.46) 0.55 (0.62,0.47) 0.68 (0.80,0.56) 0.48 (0.49,0.46) 0.78 (0.84,0.72) 0.84 (0.87,0.81)
TE A V A V A VA V A V VA -
For ACA and LA results, the numerator provides test results on the first occasron and the denominator provides test results on the second occasion. FPS and F1+2 are expressed as means of the results in brackets. ACA = antrcardioiipin antibodies; LA = lupus anticoagulant; TPS = total protein S; FPS = free protein S; TE = thromboembolic episode; + = abnormal; = negative; l+, 2t, or 3+ = 1, 2, or 3 of 3 tests abnormal; A = previous artercal thromboembolism (see methods); V = previous venous thromboemboltsm (see methods).
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ET AL
tein S deficiency in patients with APLA.‘O AMtough the numbers are relatively small and the 95% con& Between Free Protein S Levels and F1+2 dence interval (CI) on the observed frequency is conF1+2 Levels nmol/L sequently wide, over one third (9 of 24; [38%]; 95% CI Low FPS Normal FPS 19% to 59%) of patients with SLE and APLA had re(co.24 u/mL) (20.24 u/mL) P Value duced free protein S levels. All study 1.22 + 0.50 0.78 ?r 0.27 0.05 The novel observation of this study is that patients (n = 27) participants (n = 9) with reduced free protein S levels had a higher mean 0.06 Study participants 1.36 + 0.47 0.88 f 0.21 F1+2 level than patients with normal free protein S (n = 6) (n = 10) with persistent ACA positivity levels. This parallels findings in patients with congenital protein S deficiency. The mean F1+2 level in subjects with persistently tient (patient 7) had a platelet count of less than 100 positive ACA and normal free protein S levels (0.88 x 10%. The mean free protein S level was lower in the SLE nmol/L) was normal and not significantly different population than the controls (0.30 + 0.08 versus 0.43 from the mean F1+2 level in study subjects with transiently positive or persistently negative ACA (0.75 + 0.10 U/mL; mean difference 0.12, Y
382
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PROTEIN S IN SYSTEMIC LUPUS ERYTHEMATOSUS/GINSBERG
occurring anticoagulants, protein C and antithrombin III, would also be decreased, but this is not the case. The fact that the total protein S levels were reduced or in the low-normal range in the individuals with reduced free protein S levels eliminates the possibility of increased C4BP levels as a cause of the reduced free protein S levels. Therefore, the possibility that a component in the blood of SLE patients binds to protein S, inactivating it in vivo and/or making it inaccessible to assay by ELISA in vitro, must be considered. It is attractive to implicate APLA in such an process, but such a hypothesis requires testing in carefully designed studies. The results of our study are consistent with those of other studies that have reported reduced free protein S levels in patients with APLA.lO Ours is the first study to demonstrate an association between reduced free protein S levels and increased thrombin generation. Purlher studies will be important to determine the clinical importance of our observations and whether the decrement in free protein S is transient or persistent, since we made measurements on one occasion only. The presence of APLA and its dual association with an increased prevalence of thrombosis and reduced free protein S levels lead us to hypothesize that free protein S deficiency is the main cause of thrombosis in patients with SLE. This hypothesis requires testing in a large cohort study.
ACKNQWLElX%ENT The authors are grateful to Dr. J. Hirsh and Dr. M. Gent for their constructive suggestions.
REFERENCES 1. Walker FJ. Regulation of activated protein C by a new protein, A possible function for bovine protein S. J Biol Chem. 1980;255:5521-5524. 2. Dahlback B, Stenflo J. High molecular weight complex in human plasma between vitamin K-dependent protein S and complement component C4bbtnding protein. Proc Nat/ Acad Sci USA. 1981;78:2512-2516, 3. Comp PC, Doray D, Patton D, et al. An abnormal plasma distribution of protein S occurs in funchonal protein S deficiency. Blood. 1986;67:504-508. 4. Leroy-Matheron C, Lamare M, Levent M, et al. Markers of coagulabon activation in inherited protein S deficiency. Jhromb Res. 1992;67:607-612.
ET AL
5. Borg J, Vasse Y, Thirion M, et al. Congenital protein S deficiency: clinical and biological data in 22 families. Thrombotic risk evaluation using prethrombotic markers. Thromb Haemost. 1991;65:1043. Abstract. 6. Mannucci PM, Tripodi A, Botasso A, et al. Markers of procoagulant imbalance in patients with inherited thrombophilic syndromes. Thromb Haemost. 1992;67:200-202. 7. Aronson DL, Stevan L, Ball AP, et al. Generation of the combined activation peptide (Flt2) during the clotting of blood and plasma. J Clin Invest. 1977;60:1410-1418. 8. Demers C, Ginsberg JS, Henderson P, et al. Measurement of markers of activated coagulation in antithrombin III deficient subjects. Thromb Haemost. 1992;67:542-544. 9. Ginsberg JS, Demers C, BrillEdwards P, et al. Increased thrombin generation and activity in patients with systemic lupus erythematosus and anticardiolrpin antibodies; Evidence for a prothrombotic state. Blood 1993;81:2958-2963. 10. Parke AL, Weinstein RE, Bona RD, et al. The thrombotic diathesis associated with the presence of phospholipid antibodies may be due to low levels of free protein S. Am J Med. 1992;93:49-56. 11. Long AA, Ginsberg JS, Brill-Edwards P, et al. The relationship of antiphospholipid antibodies to thromboembolic disease in systemic lupus erythematosus: a cross-sectional study. Thromb Haemost. 1991;66:520-524. 12. Ginsberg JS, Brill-Edwards P, Johnston M, et al. Relationship of anhphospholipid antibodies to pregnancy loss in patients with systemic lupus erylhematosus: a cross-sectional study. Bbod. 1992;80:975-980. 13. Tan EM, Cohen AS, Fries JF, et al. The 1982 revised criteria for classificabon of systemic lupus erythematosus. Arthritis Rheum. 1982;25: 1271-1277. 14. Deutz-Terlouw PP, Ballering L, Wijngaarden A, et al. Two ELlSA’s for the measurement of protein S, and their use In the laboratory diagnosis of protein S deficrency. Clin Chim Acta. 1989;186:321-334. 15. Thiagarajan P, Pengo V, Shapiro SS. The use of the dilute Russell viper venom time for the diagnosis of lupus anticoagulants. Blood. 1986;68: 869-874. 16. Schleider MA, Nachman RL, Jaffe EA, et al. A clinical study of the lupus anticoagulant. Blood. 1976;48:499-509. 17. Proctor RR, Rapaport SI. The partial thromboptastin time with kaolin. Am J Clin Pathol. 1%1;36:212-219. 18. Lindner DJ, Edwards JM, Phinney ES, et al. Long term haemodynamic and clinical sequelae of lower extremity deep vein thrombosis. J Vast Surg. 1986;4:436-442. 19. Ginsberg JS, Shin A, Turpie AGG, et al. Detection of previous proximal venous thrombosis with doppler ultrasonography and photoplethysmography. Arch Intern Med. 1989;149:2255-2257. 20. Colton T. Probabrlii. in: Colton T, ed. Statrshcs in Medicme. Boston: Lathe Brown &Co; 1974: 63-96. 21. Svensson PJ, Dahlback B. Resistance to activated protein C as a basis for venous tbrombosa. NEJM. 1994;330:517-522.
April
1995
The American
Journal
of MedicineQ
Volume
98
383
ree circulating protein S is a cofactor for protein C-mediated inhibition of factors Va and VIIIa.112 Congenital deficiency of protein S is characterized by reduced plasma levels of free protein S and is associated with an increased risk of venous thrombosis.” Subjects with congenital protein S deficiency have been reported to have higher plasma levels of prothrombin fragments (F1+2) than their nondeficient family members. c6 F1+2 is a peptide that is cleaved from prothrombin by the prothrombinase complex during thrombin formation.7 Elevated levels of Fl+Z, have been reported in patients with other prothrombotic states.6~s~gIt has been suggested that such levels identify individuals at high risk for thrombosis. Acquired deficiency of free protein S has been reported in patients with antiphospholipid antibodies (APLA).‘O The clinical significance of this finding is not known. Some investigators have suggested that acquired protein S deficiency contributes to the increased risk of thrombosis in individuals with APIA”‘; however, it has not been established that patients with protein S deficiency had increased thrombin generation or increased risk of thrombosis. In order to determine the relationship of acquired free protein S deficiency to thrombin generation, we performed a cross-sectional analysis of patients with systemic lupus erythematosus (SLE). We selected patients with SLE because a substantial proportion30% to 50%~~11~12-haveAPLA. Our population would thus include a reasonable number of patients with protein S deficiency whom we could identify and evaluate. Plasma samples were assayed for free protein S and correlated to levels of F1+2, in search of a relationship between reduced free protein S levels and thrombin generation.
F
PATIENTS AND METHODS Study Population
From the Departments of Medicine and Laboratory Medicme, McMaster University, HamIlton, Ontario, Canada. Dr. Ginsberg’s participation in this study IS supported by a Research Scholarship from the Heart and Stroke Foundation of Canada. Supported by a grant from the Lupus Society of Hamilton. Requests for reorrnts should be addressed to Dr. J. Ginsbere. McM&ter University Medical Centre, 1200 Main Street West, iSC3W11, Hamilton, Ontario, Canada L8N 325. Manuscrtpt submItted February 14, 1994 and accepted in revised form July 5, 1994.
The study population consisted of consecutive patients referred to an SLE clinic in Hamilton, Canada, between June 1,199O and December 341991. All participants fulfilled the 1982 revised American Rheumatism Association criteria for SLE.‘” Clinical characteristics and the results of anticardiolipin antibodies (ACA) and F1+2 assays for this cohort have been published.<’ This study excludes 8 patients from the published study. Five were receiving warfarin therapy, April 1995 The American Journal of Medicine*
Volume 98
379
PROTEIN S IN SYSTEMIC LUPUS ERYlIiEMATOSUS/GINSBERG
ET AL
TABLE I Levels of Naturally Occurring Anticoagulants in Normal Controls and Systemic Lupus Erythematosus (SLE) Population TPS FPS Proc ATIII 38 Normal controls (n) 21 21 0.81 0.43 0.97 Mean (U/mL) Normal range (U/mL) 0.52-1.10 0.24-0.62 0.65-l .29 36 36 SLE patients (n) 36 0.30* 1.10 Mean (U/mL) 0.83 0.08 0.31 SD 0.25 ‘Significantly lower than the normal controls (P ~0.001). TPS = total protein S; FPS = free protein S; ProC = protein C; ATIII = antithrombin
and 3 had not given sufficient plasma for assay of free protein S.
Intervention The study was approved by the Institutional Review Board of Chedoke-McMaster Hospitals. All patients who underwent laboratory and clinical evaluation gave informed consent. Each was scheduled to be seen on two separate occasions at least 3 months apart. Patients who were seen on only one occasion were excluded from the analysis.
38 0.97 0.80-1.15 36 1.07 0.17 Ill,
Protein C (Behring Diagnostics, Montreal, Canada). The controls for the assays were derived from normal healthy laboratory volunteers. For the primary anaIysis, the means of the two results obtained on the two separate dates were calculated and used.
Antiphospholipid
Antibodies
The presence or absence of APIA was ascertained by performing an ELISA for ACA and three different assays for lupus anticoagulant (LA). A commercially available, standardized ELISA kit (Cambridge Life Sciences, Laboratory Imports, Toronto, Canada) was used to quantitate IgG ACA. A Laboratory Methods normal range was established and levels 211 IgG For the laboratory assays, blood was drawn into a vacutainer tube (Becton-Dickinson, Mountain View, phospholipid (GPL) U/mL were considered to be abCalifornia) containing 0.105 M buffered citrate. normal. This cutoff was three standard deviations Plasma was immediately separated from cellular ele- above the mean of controls. ments by centrifugation at 1,700~ for 15 minutes at Three tests were used to detect the presence of LA: room temperature, with maximal removal of platelets (1) dilute Russell viper venom time (DRVVT); (2) the being assured by the use of a silicone-based gel (Sure- dilute one-stage prothrombin time (DPT); and (3) the sep, Organon Teknica, Scarborough, Canada). Subse- activated partial thromboplastin time (aPIT). For the quently, plasma was frozen at -70°C until batch assays DPT and the aPTI’, patient samples were run neat and were performed. in a 1:l mix of normal pooled plasma derived from 20 normal hospital personnel. Values for the 1:1 mix Total Protein S, Free Protein S, above the upper limit, of normal for each test were Antithrombin III, and Protein C Assays considered to be positive for LA. The DRWT was perTotal protein S was quantified by enzyme-linked im- formed as previously described.‘” If the test was abmunoelectrodiffusionn assay (ELISA) as previously normal, ionophore-treated platelets were substituted described using antibodies from Affinity Biologicals for bovine phospholipid and the test was repeated; a (Yarker, Canada).14 Free protein S was separated from shortening of the DRWT was considered to be diagcomplexed prot.ein S by the polyetheline glycol (PEG) nostic of LA. The DPT was performed using a l/500 precipitation t,echnique of Comp et al” and measured dilution of rabbit brain thromboplastin (Dade Thromby ELISA. A standard was prepared by PEG precipiboplastin C, Dade International, Miami, Florida) in a tation of a pool of normal plasma and this was consaline CaCl, mixture.‘” The aPTT was performed ussidered to represent a plasma value of 0.40 U/mL. The ing a commercially available reagent (Dade Actin FSL, pool of normal plasma was derived from 10 male and BaxterKanlab, Mississauga, Canada), as previously 20 female healthy volunteers, none of whom were tak- described.” Patients were classified as positive for LA ing oral contraceptives. Antithrombin III and protein if one or more of the assays was positive. C were measured by chromogenic assays using commercially available kits. Antithrombin III was assayed F1+2 Assay using a thrombin inhibition assay (Stachrom ATIII, F1+2 levels were measured on two occasions usDiagnostica Stage, Wellmark Diagnostics, Guelph, ing a commercially available ELISA kit (Behring, Canada). Protein C was measured using Berichrom Marburg, Germany). The normal range for the F1+2 380
April 1995 The American Journal of Medicine@ Volume 98
PROTEIN S IN SYSTEMIC LUPUS ERYTHEMATOSUS/GINSW?G
assay is cl.1 nmol/L. For the analysis, the means of the two results were calculated and used.
Clinical Evaluation The presence or absence of prior thromboembolic disease was ascertained by interview, critical review of objective tests, and the performance of venous Doppler of the leg veins as a specific indicator of previous venous thrombosis. l8,lg Details of the criteria for the diagnosis of venous and arterial thromboembolism have been previously published.“,”
Statistics Review of the F1+2 data indicated that the distribution was skewed since the standard deviation increased with the mean. Hence, each data point was
ET AL
subjected to a logarithmic transformation before comparing means using unpaired t-te~ts.~~ Proportions were compared using Fisher’s exact test. A twosided P value of ~0.05 was considered to be statistically significant.
RESULTS Thirty-six patients (33 women) with a mean age of 39 years (range 19 to 75) were enrolled. Table I summarizes the levels of free and total protein S, protein C, and antithrombin III in the study population and controls. Table II shows the results of APLA, protein S (total and free), and Fl+Z assays for each participant. None of the women was taking oral contraceptives. Two (patients 3 and 26) had been pregnant within 6 weeks of one or both blood samples. One pa-
TABLE II
Pt# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Age (y)/Sex 39F 25F 28F 34F 44F 54M 67F 34F 24F 58F 19F 36M 42F 29F 25F 41F 52F 21F 33F 54F 48F 50F 35F 41F 29F 34F 30F 48F 46F 20F 31F 48F 75M 34F 33F 20F
ACA +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/;: $1 +/-/-/-/-/-/-/-/-/-/-/-/-/-/-/-
LA 2+/2+/3+ 3+/3+ -/3+/3+ -/l+ 3+/3+ l+/-/1+/l+ -/1 +/-/-/-/l+ 1+/l+ 2+/-/-/3+/3+ -/2+ -/1+/l+ l+/-/-/-/-/* -/-/-/-/-/-/-/-/-
Summary of Results TPS FPS PJ/mLl W/mLl 0.69 0.18 (0.15/0.20) 0.68 0.19 (0.19/0.19) 0.63 0.21 (0.17/0.25) 0.69 0.22 (0.19/0.24) 0.48 0.22 (0.20/0.24) 0.88 0.22 (0.22/0.22) 0.51 0.24 (0.21/0.27) 0.76 0.27 (0.34/0.20) 0.49 0.28 (0.27/0.28) 0.66 0.29 (0.31/0.26) 0.80 0.30 (0.35/0.25) 0.60 0.34 (0.30/0.37) 0.75 0.37 (0.41/0.32) 1.22 0.39 (0.37/0.40) 0.73 0.39 (0.45/0.36) 0.92 0.51 (0.47/0.54) 0.40 0.12 (0.10/0.13) 0.50 0.20 (0.20/0.20) 0.98 0.33 (0.30/0.35) 1.04 0.33 (0.38/0.27). 1.34 0.36 (0.36/0.35) 0.65 0.38 (0.38/0.37) 0.56 0.23 (0.24/0.21) 1.00 0.38 (0.46/0.30) 0.91 0.24 (0.24/0.24) 0.64 0.25 (0.25/0.25) 1.12 0.29 (0.28/0.30) 0.88 0.29 (0.29/0.29) 0.96 0.30 (0.28/0.31) 0.94 0.32 (0.30/0.34) 1.29 0.35 (0.25/0.45) 1.00 0.35 (0.35/0.35) 1.02 0.35 (0.37/0.34) 1.11 0.36 CO.26/0.46) 0.77 0.40 (0.43/0.37) 1.28 0.43 (0.47/0.38)
F1+2 (nmW) 1.34 (0.79,1.89) 1.75 (2.20,1.30) 0.93 (1.54,0.32) 1.72 (1.10,2.33) 0.66 (0.84,0.48) 1.76 (1.61,1.91) 0.77 (0.70,0.84) 1.13 (0.96,1.30) 0.74 (0.85,0.63) 0.55 (0.30,0.80) 0.64 (1.24,0.04) 0.81 (0.95,0.67) 1.02 (1.14,0.90) 0.99 (0.93,1.04) 1.20 (1.35,1.04) 0.96 (1.05,0.87) 1.50 (1.80,1.19) 0.80 (0.97,0.63) 0.50 (0.52,0.47) 0.87 (0.83,0.91) 0.79 (0.75,0.82) 0.58 (0.57,0.58) 0.51 (0.50,0.51) 0.52 (0.50,0.53) 0.40 (0.36,0.43) 1.25 (1.24,1.26) 1.00 U.30,0.70) 1.39 (2.10,0.68) 0.73 (0.76,0.70) 0.49 (0.46,0.52) 0.43 (0.40,0.46) 0.55 (0.62,0.47) 0.68 (0.80,0.56) 0.48 (0.49,0.46) 0.78 (0.84,0.72) 0.84 (0.87,0.81)
TE A V A V A VA V A V VA -
For ACA and LA results, the numerator provides test results on the first occasron and the denominator provides test results on the second occasion. FPS and F1+2 are expressed as means of the results in brackets. ACA = antrcardioiipin antibodies; LA = lupus anticoagulant; TPS = total protein S; FPS = free protein S; TE = thromboembolic episode; + = abnormal; = negative; l+, 2t, or 3+ = 1, 2, or 3 of 3 tests abnormal; A = previous artercal thromboembolism (see methods); V = previous venous thromboemboltsm (see methods).
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tein S deficiency in patients with APLA.‘O AMtough the numbers are relatively small and the 95% con& Between Free Protein S Levels and F1+2 dence interval (CI) on the observed frequency is conF1+2 Levels nmol/L sequently wide, over one third (9 of 24; [38%]; 95% CI Low FPS Normal FPS 19% to 59%) of patients with SLE and APLA had re(co.24 u/mL) (20.24 u/mL) P Value duced free protein S levels. All study 1.22 + 0.50 0.78 ?r 0.27 0.05 The novel observation of this study is that patients (n = 27) participants (n = 9) with reduced free protein S levels had a higher mean 0.06 Study participants 1.36 + 0.47 0.88 f 0.21 F1+2 level than patients with normal free protein S (n = 6) (n = 10) with persistent ACA positivity levels. This parallels findings in patients with congenital protein S deficiency. The mean F1+2 level in subjects with persistently tient (patient 7) had a platelet count of less than 100 positive ACA and normal free protein S levels (0.88 x 10%. The mean free protein S level was lower in the SLE nmol/L) was normal and not significantly different population than the controls (0.30 + 0.08 versus 0.43 from the mean F1+2 level in study subjects with transiently positive or persistently negative ACA (0.75 + 0.10 U/mL; mean difference 0.12, Y
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occurring anticoagulants, protein C and antithrombin III, would also be decreased, but this is not the case. The fact that the total protein S levels were reduced or in the low-normal range in the individuals with reduced free protein S levels eliminates the possibility of increased C4BP levels as a cause of the reduced free protein S levels. Therefore, the possibility that a component in the blood of SLE patients binds to protein S, inactivating it in vivo and/or making it inaccessible to assay by ELISA in vitro, must be considered. It is attractive to implicate APLA in such an process, but such a hypothesis requires testing in carefully designed studies. The results of our study are consistent with those of other studies that have reported reduced free protein S levels in patients with APLA.lO Ours is the first study to demonstrate an association between reduced free protein S levels and increased thrombin generation. Purlher studies will be important to determine the clinical importance of our observations and whether the decrement in free protein S is transient or persistent, since we made measurements on one occasion only. The presence of APLA and its dual association with an increased prevalence of thrombosis and reduced free protein S levels lead us to hypothesize that free protein S deficiency is the main cause of thrombosis in patients with SLE. This hypothesis requires testing in a large cohort study.
ACKNQWLElX%ENT The authors are grateful to Dr. J. Hirsh and Dr. M. Gent for their constructive suggestions.
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5. Borg J, Vasse Y, Thirion M, et al. Congenital protein S deficiency: clinical and biological data in 22 families. Thrombotic risk evaluation using prethrombotic markers. Thromb Haemost. 1991;65:1043. Abstract. 6. Mannucci PM, Tripodi A, Botasso A, et al. Markers of procoagulant imbalance in patients with inherited thrombophilic syndromes. Thromb Haemost. 1992;67:200-202. 7. Aronson DL, Stevan L, Ball AP, et al. Generation of the combined activation peptide (Flt2) during the clotting of blood and plasma. J Clin Invest. 1977;60:1410-1418. 8. Demers C, Ginsberg JS, Henderson P, et al. Measurement of markers of activated coagulation in antithrombin III deficient subjects. Thromb Haemost. 1992;67:542-544. 9. Ginsberg JS, Demers C, BrillEdwards P, et al. Increased thrombin generation and activity in patients with systemic lupus erythematosus and anticardiolrpin antibodies; Evidence for a prothrombotic state. Blood 1993;81:2958-2963. 10. Parke AL, Weinstein RE, Bona RD, et al. The thrombotic diathesis associated with the presence of phospholipid antibodies may be due to low levels of free protein S. Am J Med. 1992;93:49-56. 11. Long AA, Ginsberg JS, Brill-Edwards P, et al. The relationship of antiphospholipid antibodies to thromboembolic disease in systemic lupus erythematosus: a cross-sectional study. Thromb Haemost. 1991;66:520-524. 12. Ginsberg JS, Brill-Edwards P, Johnston M, et al. Relationship of anhphospholipid antibodies to pregnancy loss in patients with systemic lupus erylhematosus: a cross-sectional study. Bbod. 1992;80:975-980. 13. Tan EM, Cohen AS, Fries JF, et al. The 1982 revised criteria for classificabon of systemic lupus erythematosus. Arthritis Rheum. 1982;25: 1271-1277. 14. Deutz-Terlouw PP, Ballering L, Wijngaarden A, et al. Two ELlSA’s for the measurement of protein S, and their use In the laboratory diagnosis of protein S deficrency. Clin Chim Acta. 1989;186:321-334. 15. Thiagarajan P, Pengo V, Shapiro SS. The use of the dilute Russell viper venom time for the diagnosis of lupus anticoagulants. Blood. 1986;68: 869-874. 16. Schleider MA, Nachman RL, Jaffe EA, et al. A clinical study of the lupus anticoagulant. Blood. 1976;48:499-509. 17. Proctor RR, Rapaport SI. The partial thromboptastin time with kaolin. Am J Clin Pathol. 1%1;36:212-219. 18. Lindner DJ, Edwards JM, Phinney ES, et al. Long term haemodynamic and clinical sequelae of lower extremity deep vein thrombosis. J Vast Surg. 1986;4:436-442. 19. Ginsberg JS, Shin A, Turpie AGG, et al. Detection of previous proximal venous thrombosis with doppler ultrasonography and photoplethysmography. Arch Intern Med. 1989;149:2255-2257. 20. Colton T. Probabrlii. in: Colton T, ed. Statrshcs in Medicme. Boston: Lathe Brown &Co; 1974: 63-96. 21. Svensson PJ, Dahlback B. Resistance to activated protein C as a basis for venous tbrombosa. NEJM. 1994;330:517-522.
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