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Clinical Chemistry 51: 545-552, 2005. First published January 6, 2005; 10.1373/clinchem.2004.043414
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(Clinical Chemistry. 2005;51:545-552.)
© 2005 American Association for Clinical Chemistry, Inc.

Hemostasis and Thrombosis

Acquired Activated Protein C Resistance Associated with IgG Antibodies against ß2-Glycoprotein I and Prothrombin as a Strong Risk Factor for Venous Thromboembolism

Junzo Nojima1,a, Hirohiko Kuratsune2, Etsuji Suehisa1, Yoshinori Iwatani3 and Yuzuru Kanakura1,4

1 Laboratory for Clinical Investigation, Osaka University Hospital, Osaka, Japan.
2 Department of Health Science, Faculty of Health Science for Welfare, Kansai University of Welfare Science, Osaka, Japan.
3 Division of Biomedical Informatics, Course of Health Science, and 4 Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Osaka, Japan.

aAddress correspondence to this author at: Laboratory for Clinical Investigation, Osaka University Hospital, 2-15 Yamadaoka, Suita, Osaka 565-0871, Japan. Fax 81-6-6879-6635; e-mail nojima{at}hp-lab.med.osaka-u.ac.jp.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Venous thromboembolic events such as deep vein thrombosis and pulmonary embolism are common manifestations of antiphospholipid syndrome. Our aim was to clarify the roles of anti-phospholipid (aPL) antibodies in the pathogenesis of venous thromboembolism (VTE) in patients with systemic lupus erythematosus (SLE).

Methods and Results: We examined anti-cardiolipin/ß2-glycoprotein I (anti-CL/ß2-GPI) antibody concentrations, anti-phosphatidylserine/prothrombin (anti-PS/PT) antibody concentrations, and lupus anticoagulant (LA) activity in 87 patients with SLE (21 with VTE and 66 without thrombosis). Both anti-CL/ß2-GPI and anti-PS/PT antibodies strongly correlated with LA activity. Multivariate logistic analysis confirmed that both anti-CL/ß2-GPI and anti-PS/PT antibodies were significant independent risk factors for VTE (odds ratios = 4.98 and 7.54, respectively; 95% confidence intervals, 1.51–16.4 and 2.30–24.7, respectively). We therefore studied the in vitro effects of IgG fractions containing anti-CL/ß2-GPI or anti-PS/PT antibodies on the anticoagulant activity of activated protein C (APC) and found that purified IgG containing anti-CL/ß2-GPI or anti-PS/PT antibodies significantly hampered the anticoagulant activity of APC. We also studied the ability of IgG fractions to impede the anticoagulant activity of APC before and after complete removal of anti-CL/ß2-GPI or anti-PS/PT antibodies by adsorption. Removal of anti-CL/ß2-GPI or anti-PS/PT antibodies from all positive IgG samples clearly decreased the inhibitory effect of those samples on APC anticoagulant activity.

Conclusions: Anti-CL/ß2-GPI and anti-PS/PT antibodies independently cause APC resistance, which may contribute to risk of VTE in patients with SLE.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Anti-phospholipid (aPL)1 antibodies are a heterogeneous group of autoantibodies that include anti-cardiolipin/ß2-glycoprotein I (anti-CL/ß2-GPI) antibodies, anti-phosphatidylserine/prothrombin (anti-PS/PT) antibodies, and lupus anticoagulant (LA) (1)(2). These antibodies are frequently found in the plasma of patients with systemic lupus erythematosus (SLE) (3)(4)(5) and are reported to be associated with clinical events such as arterial and/or venous thrombosis, thrombocytopenia, and obstetric complications (6)(7)(8)(9)(10)(11)(12). Thromboembolic events are reported to occur in ~30% of SLE patients with aPL antibodies (13). Venous thromboembolic events, such as deep vein thrombosis (DVT) and pulmonary embolism (PE), are common manifestations in SLE patients (2). Although the association between the presence of aPL antibodies and venous thromboembolism (VTE) in patients with SLE has been established, the precise mechanism responsible for VTE in these patients remains unclear.

Several clinical studies have established that the prevalence of VTE is associated with congenital or acquired abnormalities of the protein C pathway (14)(15)(16)(17). Recently, several authors have suggested that acquired abnormalities of the protein C pathway are frequently found in patients with aPL antibodies (18)(19)(20)(21)(22). Other authors have reported that aPL antibodies might inhibit PL-dependent reactivity of the activated protein C (APC) pathway, such as activation of protein C by thrombin/thrombomodulin or degradation of factor Va by activated protein C/protein S (1)(23). More recently, Male et al. (24) reported that the presence of LA was significantly associated with acquired APC resistance (APC-R) in patients with SLE. However, LA activities are heterogeneous with respect to the specificities and functional capacities of the antibodies and include autoantibodies to at least two PL-bound plasma proteins (1)(25)(26). More recently, several studies showed that antibodies against ß2-GPI and PT are independently responsible for the LA activity (2)(27)(28).

In the present study, we examined the concentrations of anti-CL/ß2-GPI and anti-PS/PT antibodies as well as LA activity in 87 SLE patients with or without VTE to investigate the role of these aPL antibodies in the pathogenesis of acquired APC-R and VTE.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
patients
We studied plasma samples from 87 patients (81 females and 6 males; age range, 22–52 years; mean, 39.8 years) with SLE. Diagnosis of SLE was made according to the revised criteria of the American Rheumatism Association. Clinical history revealed that VTE had occurred in 21 of the SLE patients [DVT (n = 16) and PE (n = 5)]. Diagnosis of DVT and PE was made based on clinical manifestations and findings on duplex scanning, radioisotope venography, contrast venography, and radioisotopic lung scanning. We determined factor V Leiden status by analysis of genomic DNA extracted from the plasma of our 87 patients with SLE and confirmed that all 87 patients with SLE were negative for the factor V Leiden mutation. We also measured the plasma concentrations of anti-thrombin, protein C, and protein S (total and free protein S) in our 87 patients with SLE and found that none of them had any congenital abnormalities in anti-thrombin, protein C, or protein S. Plasma samples from 80 healthy volunteers (staff members of Osaka University Hospital; 74 females and 6 males; age range, 22–60 years; mean, 39.8 years) were used as controls. None of the volunteers had a history of thrombotic complications, and no abnormalities were found by blood testing (blood cell counts, coagulation tests, liver function tests, and examinations for autoimmune activity).

Blood samples were collected into evacuated tubes (5.0 mL total volume; Sekisui) containing 0.5 mL of 31.3 g/L trisodium citrate (Na3C6H5O7 · 2 H2O), and platelet-poor plasma was prepared by double centrifugation at 2800g for 15 min at 15 °C. The plasma samples were frozen at –80 °C until batch assays could be performed. Informed consent was obtained from all patients and controls.

elisa for anti-cl/ß2-gpi antibodies
Anti-CL/ß2-GPI antibody concentrations were measured by a specific ELISA system as described previously (29). Polystyrene ELISA plates (Immulon 1; Kamstrup) were coated with 30 µL (50 mg/L in ethanol) of cardiolipin (Sigma) and dried for 3 h at room temperature. To avoid nonspecific binding of proteins, the wells were incubated overnight at 4 °C with 100 µL of Tris-buffered saline (TBS; 50 mmol/L Tris-HCl, 0.1 mol/L NaCl, pH 7.4) containing 10 g/L bovine serum albumin (BSA; Sigma). After each well had been washed three times with TBS containing 5 g/L BSA and 0.5 mL/L Tween 20, 50 µL (20 mg/L in TBS containing 5 g/L BSA) of human ß2-GPI (Diagnostica Stago) was added to each well. After a 30-min incubation at room temperature, 50 µL of plasma sample diluted 101-fold (2.0 µL of sample plus 200 µL of TBS) with TBS containing 5 g/L BSA was added to each well and incubated for 60 min at room temperature. The wells were then washed with TBS containing 5 g/L BSA and 0.5 mL/L Tween 20 and incubated with 100 µL of affinity-purified peroxidase-conjugated goat anti-human IgG {gamma}-chain specific F(ab')2 fragment (cat. no. A-2290; Sigma) for 60 min at room temperature. The wells were then washed three times with TBS containing BSA and Tween 20, and 100 µL of tetramethylbenzidine solution (Moss Inc.) was added to each well. After a 30-min incubation, the reaction was terminated by addition of 100 µL of 1 mol/L H2SO4, and the absorbance was measured at 450 nm.

elisa for anti-ps/pt antibodies
The concentrations of anti-PS/PT antibodies were measured by a specific ELISA system, as described previously (29). Polystyrene ELISA plates (Immulon 1) were coated with 30 µL (50 mg/L in ethanol) of PS (Sigma) and dried for 3 h at room temperature. To block nonspecific binding, the wells were incubated overnight at 4 °C with 150 µL of TBS containing 10 g/L BSA and 5 mmol/L CaCl2. The wells were subsequently washed three times with TBS containing 5 g/L BSA, 5 mmol/L CaCl2, and 0.5 mL/L Tween 20. Human PT (50 µL at 20 mg/L in TBS containing BSA and CaCl2; Diagnostica Stago) was then added to each well and incubated at room temperature for 60 min.

We added 50 µL of plasma sample prediluted 101-fold (20 µL of sample plus 200 µL of TBS) containing 5 g/L BSA and 5 mmol/L CaCl2 to each well and incubated the wells for 60 min at room temperature.

The wells were then washed with TBS containing BSA, CaCl2, and Tween 20 and incubated with affinity-purified horseradish peroxidase-conjugated goat anti-human IgG F(ab')2 ({gamma}-chain specific) fragment (cat. no. A-2290; Sigma) at room temperature for 60 min. The color reaction was developed with tetramethylbenzidine solution (Moss, Inc.), and the absorbance was measured at 450 nm.

detection of la activity
LA activity was detected by both the dilute Russell viper venom time (Gradipore Ltd) and STACLOT(Diagnostica Stago) LA tests. The dilute Russell viper venom time and STACLOT LA tests were performed with commercially available screening and confirmatory tests as reported previously (30)(31).

IGG purification
The IgG fractions from plasma samples of SLE patients that contained anti-CL/ß2-GPI and/or anti-PS/PT [anti-CL/ß2-GPI+ · anti-PS/PT+ (n = 15); anti-CL/ß2-GPI+ · anti-PS/PT– (n = 11); anti-CL/ß2-GPI– · anti-PS/PT+ (n = 14); anti-CL/ß2-GPI– · anti-PS/PT– (n = 30)] were purified with protein G Sepharose (S-751 82; Pharmacia LKB Biotechnology). The purity of the IgG was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The concentrations of anti-CL/ß2-GPI and anti-PS/PT antibodies in each IgG fraction were confirmed with specific ELISA systems. The final concentration of IgG fractions used in our study was 2 g/L. A pool of a commercially available human IgG (Immuno AG) was used as a control.

effects of IGG fractions with anti-cl/ß2-gpi or anti-ps/pt antibodies on the anticoagulant activity of the apc pathway
The APC-R assay was performed in duplicate with an activated partial thromboplastin time (APTT)-based assay and a KC-10 coagulometer (Amelung). Briefly, 80 µL of normal pooled plasma from 20 healthy donors was incubated with 20 µL of IgG fraction purified from plasma of SLE patients with or without anti-CL/ß2-GPI or anti-PS/PT antibodies. After 5 min of incubation at 37 °C, APTT was measured in the presence and absence of APC with a Coatest Activated Protein C Resistance Kit (Chromogenix). The results are expressed as the ratio of APTT in the presence of APC to the APTT in the absence of APC [APC sensitivity ratio; (APTT with APC)/(APTT without APC)].

adsorption of anti-cl/ß2-gpi or anti-ps/pt from IGG fractions
Cardiolipin-coated wells (Yamasa Shoyu Co. Ltd.) were incubated for 120 min at 37 °C with 20 µL/well of purified human ß2-GPI (50 mg/L) in 10 mmol/L HEPES, 150 mmol/L NaCl, pH 7.4. After this initial incubation, 100 µL of IgG fraction containing anti-CL/ß2-GPI and anti-PS/PT antibodies was added to each well and incubated at 37 °C for 180 min. The supernatant was then collected from each well, and the concentration of anti-CL/ß2-GPI antibodies in this supernatant was measured by ELISA as described above. This procedure was repeated until the anti-CL/ß2-GPI antibody concentrations were below the detection limit of each ELISA.

PL-coated wells were incubated for 120 min at 37 °C with 20 µL/well of purified human PT. After this initial incubation, 100 µL of IgG fraction containing anti-PS/PT antibodies was added to each well and incubated for 180 min at 37 °C. The supernatant was then collected from each well, and the concentration of anti-PS/PT antibodies in this supernatant was measured by ELISA as described above. This procedure was repeated until no anti-PS/PT antibodies were detected.

measurement of anti-thrombin, protein c, and protein s
Anti-thrombin III activity was measured with a chromogenic substrate (cat. no. S-2238; Kabi Vitrium) used in an assay (Testzym; Daiichi Pure Chemicals). Anti-thrombin antigen was assayed by single radial immunodiffusion using M-Partigen plates (Behring-Werke). Protein C activity was measured by use of a chromogenic substrate assay incorporating Pyro-Glu-Pro-Arg-Methoxynitroanilide (Berichrom; Behring-Werke). Protein C antigen was measured by a homogeneous enzyme immunoassay, and protein S activity was measured by a clotting assay (Staclot Protein S; Diagnostica Stago). Plasma concentrations of total and free protein S antigens were measured by ELISAs (Asserachrom Total Protein S and Asserachrom Free Protein S; Diagnostica Stago).

statistical analysis
We used the nonparametric Mann–Whitney test to compare the concentrations of anti-CL/ß2-GPI and anti-PS/PT antibodies between patients with or without VTE and the Fisher exact probability test to evaluate the association between the presence of each antibody and the prevalence of LA activity and/or VTE. As an approximation of the relative risk, we calculated the odds ratios (ORs) and 95% confidence intervals (CIs) for several putative risk factors by multivariate logistic regression analysis with the statistical program Stat Flex (Ver. 4.2; Arthch Inc.). We considered an OR statistically significant when the lower limit of the 95% CI was >1.0. In the multivariate logistic regression analysis of risk factors, we considered P <0.05 statistically significant. We used the Kruskal–Wallis test and the nonparametric Mann–Whitney test to compare the APC sensitivity ratio among groups.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
prevalence of anti-cl/ß2-gpi and anti-ps/pt antibodies
The concentrations of anti-CL/ß2-GPI and anti-PS/PT antibodies in 80 healthy controls, detected by ELISA, were log-transformed to approximate a gaussian distribution by the Stat Flex program before statistical analysis was performed. We chose the mean + 3 SD of each antibody concentration in the 80 controls as the cutoff point. The cutoff values [milliabsorbance units (mAU)] for anti-CL/ß2-GPI and anti-PS/PT antibodies were 398.8 and 403.2 mAU, respectively. We regarded a result as positive when the absorbance exceeded each cutoff value. Anti-CL/ß2-GPI and anti-PS/PT antibodies were detected in 25 (28.7%) and 28 (32.2%) of the 87 SLE patients, respectively. Fourteen patients had both anti-CL/ß2-GPI and anti-PS/PT antibodies, 11 had only anti-CL/ß2-GPI antibodies, and 14 had only anti-PS/PT antibodies.

relationship between la activity and anti-cl/ß2-gpi or anti-ps/pt antibodies
LA activity was detected in 30 (34.5%) of the 87 SLE patients with or without VTE. Eighteen (72.0%) of 25 anti-CL/ß2-GPI-positive cases had LA activity, and 23 (82.1%) of 28 anti-PS/PT-positive cases had LA activity. Multivariate logistic regression analysis revealed that both anti-CL/ß2-GPI and anti-PS/PT antibodies strongly correlated with the presence of LA activity (ORs = 12.6 and 38.6, respectively; 95% CIs, 2.78–57.6 and 8.74–170, respectively; P <0.002 and P <0.0001, respectively). Because the OR for the presence of anti-PS/PT antibodies was much higher than that for anti-CL/ß2-GPI antibodies, the correlation between LA and anti-PS/PT antibodies was much stronger than that between LA and anti-CL/ß2-GPI antibodies.

relationship between the prevalence of vte and the presence of anti-cl/ß2-gpi or anti-ps/pt antibodies
Among the 87 SLE patients with or without VTE, the concentrations of anti-CL/ß2-GPI and anti-PS/PT antibodies were significantly higher in the patients with VTE than in those without thrombosis: mean (SE) anti-CL/ß2-GPI antibody concentration, 1388 (248) vs 352 (47) mAU (P <0.0001); mean (SE) anti-PS/PT antibody concentration, 883 (154) vs 303 (58) mAU (P <0.0001; Fig. 1 ).



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  		Figure 1. Concentrations of anti-CL/ß2-GPI and anti-PS/PT antibodies in SLE patients with or without VTE.

•, patients with VTE (n = 21); {circ}, patients without VTE (n = 66). The differences in anti-CL/ß2-GPI and anti-PS/PT antibody concentrations in patients with or without VTE were assessed by the nonparametric Mann–Whitney test. The horizontal bars indicate the means. The values in parentheses are the SE.

The prevalences of anti-CL/ß2-GPI and anti-PS/PT antibodies were significantly higher in patients with VTE [anti-CL/ß2-GPI antibodies, 13 of 21 cases (61.9%; P <0.001); anti-PS/PT antibodies, 15 of 21 cases (71.4%; P <0.0001)] than in those without thrombosis [anti-CL/ß2-GPI antibodies, 12 of 66 cases (18.2%); anti-PS/PT antibodies, 13 of 66 cases (19.7%)]. Multivariate logistic analysis of the risk for VTE confirmed that both anti-CL/ß2-GPI and anti-PS/PT antibodies were significant independent risk factors for VTE (ORs = 4.98 and 7.54, respectively; 95% CIs, 1.51–16.4 and 2.30–24.7, respectively).

effect of IGG fractions from plasma with or without anti-cl/ß2-gpi or anti-ps/pt antibodies on the anticoagulant activity of the apc pathway
Because the plasma samples might have contained components other than anti-CL/ß2-GPI and anti-PS/PT antibodies, we purified IgG fractions from plasma from each of the 70 SLE patients. After preincubation of normal pooled plasma with several kinds of IgG fractions, we measured the APC sensitivity ratio [(APTT with APC)/(APTT without APC); Fig. 2 ]. Treatment of normal pooled plasma with IgG fractions without anti-CL/ß2-GPI and anti-PS/PT antibodies did not cause significant changes in the APC sensitivity ratio [n = 30; mean (SD) APC sensitivity ratio, 2.49 (0.24); Fig. 2 ]. In contrast, treatment of normal pooled plasma with IgG fractions containing both anti-CL/ß2-GPI and anti-PS/PT antibodies caused an apparent decrease in the APC sensitivity ratio [n = 15; APC sensitivity ratio, 1.80 (0.43); P <0.0001]. Furthermore, treatment of normal pooled plasma with purified IgG containing either anti-CL/ß2-GPI or anti-PS/PT antibodies also caused decreases in the APC sensitivity ratio [1.88 (0.35); P <0.0001 (n = 11) and 1.81 (0.23); P <0.0001 (n = 14), respectively]. These results raise the possibility that anti-CL/ß2-GPI and anti-PS/PT antibodies cause APC-R independently.



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  		Figure 2. Effect of IgG fractions from plasma of patients with or without anti-CL/ß2-GPI or anti-PS/PT antibodies on the anticoagulant activity of the APC pathway.

IgG fractions were purified by protein G Sepharose chromatography from plasma of the patient groups indicated: anti-CL/ß2-GPI+ · anti-PS/PT+, n = 15; anti-CL/ß2-GPI+ · anti-PS/PT–, n = 11; anti-CL/ß2-GPI– · anti-PS/PT+, n = 14; anti-CL/ß2-GPI– · anti-PS/PT–, n = 30. After preincubation of normal pooled plasma with several kinds of IgG fractions, we measured the APC sensitivity ratio [(APTT with APC)/(APTT without APC)]. The Kruskal–Wallis test and the nonparametric Mann–Whitney test were used to compare the APC sensitivity ratios between groups. D, anti-CL/ß2-GPI– · anti-PS/PT– group. •, values for individual patients.

To clarify this point, we studied the ability of IgG fractions to impede the anticoagulant activity of APC before and after complete removal of anti-CL/ß2-GPI or anti-PS/PT antibodies by adsorption. The change in the APC sensitivity ratio after removal of anti-CL/ß2-GPI or anti-PS/PT antibodies from five IgG fractions is shown in Fig. 3 . Removal of anti-CL/ß2-GPI or anti-PS/PT antibodies from positive IgG samples by adsorption clearly decreased the inhibitory effect of those samples on the anticoagulant activity of APC.



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  		Figure 3. Comparison of the APC sensitivity ratios before and after complete removal of anti-CL/ß2-GPI or anti-PS/PT antibodies from each positive IgG sample by adsorption.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Venous thromboembolic events such as DVT and PE are common manifestations in SLE patients with aPL antibodies (6)(30)(32). Although much effort has been made to clarify the mechanism underlying thromboembolic events in SLE patients with aPL antibodies, the precise mechanism responsible for VTE in these patients remains unclear. The present study showed that acquired APC-R might reflect functional interference of the APC anticoagulant system by anti-CL/ß2-GPI or anti-PS/PT antibodies, which may represent an important mechanism responsible for the development of VTE in patients with SLE.

Recent reports indicated that the presence of LA activity is the strongest risk factor for thromboembolic events in patients with SLE (33)(34). However, LA activity detected by a PL-dependent coagulation assay is heterogeneous, and some investigations showed that LA activity depends on the ability of autoantibodies to bind to PL-bound PT or ß2-GPI (1)(25)(27)(35)(36). We therefore used specific ELISAs to examine the concentrations of anti-CL/ß2-GPI and anti-PS/PT antibodies in 87 SLE patients with or without VTE. Multivariate logistic analysis of risk factors for VTE confirmed that both anti-CL/ß2-GPI and anti-PS/PT antibodies were significant independent risk factors for VTE. The detection of anti-CL/ß2-GPI antibodies and LA activity has been standardized for the diagnosis of antiphospholipid syndrome (37). However, our findings suggest that not only the anti-CL/ß2-GPI ELISA but also the anti-PS/PT ELISA should be performed, regardless of the definition of antiphospholipid syndrome, when patients are suspected of having venous thromboembolic complications.

The protein C pathway is one of the most important anticoagulant systems (17). Protein C is activated on endothelial cells by thrombin bound to thrombomodulin. APC exerts its anticoagulant function by proteolytic cleavage of the procoagulant protein factors Va and VIIIa (17). APC-R is defined as a decreased anticoagulant response to the protein C pathway (38). Hereditary APC-R caused by the factor V Leiden mutation (39)(40)(41) is strongly associated with an increased risk of VTE (42)(43), but the factor V Leiden mutation occurs rarely in the Japanese population. Acquired APC-R, a phenotypic APC-R that occurs in the absence of the factor V Leiden mutation, has been reported in patients with aPL antibodies (18)(19)(20)(21)(22), but the mechanism by which APC-R is acquired in these conditions has not been elucidated.

Recently, Male et al. (24) reported that the presence of LA was significantly associated with the prevalence of acquired APC-R in patients with SLE: the prevalence of acquired APC-R was significantly higher in LA-positive patients (50%) than in LA-negative patients (21%). Furthermore, the authors of some studies have suggested that plasma or purified IgG fractions from LA-positive patients impair APC-mediated factor Va inactivation (23). Acquired APC-R induced by LA has been hypothesized to be a possible mechanism of aPL-associated VTE in patients with SLE, but some investigations showed that LA activity could be caused by anti-CL/ß2-GPI or anti-PS/PT antibodies (27)(44). In the present study, we observed that the antibodies detected by the anti-PS/PT ELISA differed from the antibodies detected by the anti-CL/ß2-GPI ELISA and that anti-PS/PT and anti-CL/ß2-GPI antibodies were independently responsible for the LA activity.

Because it is not clear whether anti-CL/ß2-GPI, anti-PS/PT, or both antibodies are associated with acquired APC-R, we investigated the in vitro effects of these antibodies on the anticoagulant activity of the APC pathway. The IgG fractions purified from plasma of SLE patients with neither anti-CL/ß2-GPI nor anti-PS/PT did not significantly impede the anticoagulant activity of the APC pathway. In contrast, purified IgG containing both anti-CL/ß2-GPI and anti-PS/PT antibodies significantly impeded the anticoagulant activity of APC. However, purified IgG containing either anti-CL/ß2-GPI or anti-PS/PT antibodies also impeded the anticoagulant activity of the APC pathway. These results raise the possibility that anti-CL/ß2-GPI and anti-PS/PT antibodies independently cause APC-R. To test this possibility, we studied the ability of IgG fractions to impede the anticoagulant activity of APC before and after complete removal of anti-CL/ß2-GPI or anti-PS/PT antibodies by adsorption. We confirmed that the ability of IgG fractions to impair the anticoagulant activity of APC was clearly decreased by removal of anti-CL/ß2-GPI or anti-PS/PT activity from each positive IgG sample. These findings suggest that acquired APC-R in SLE patients is attributable to functional interference of the protein C pathway by anti-CL/ß2-GPI or anti-PS/PT antibodies. Several recent studies showed that anti-CL/ß2-GPI/anti-PS/PT antibodies enhance the binding of ß2-GPI/PT to negatively charged PL in the prothrombinase reaction (35)(45)(46)(47)(48). It is possible that these antibodies could indirectly inhibit the PL-dependent reactions of the APC pathway.

In this study, acquired APC-R, which was not found in any patients with the factor V Leiden mutation, was present in 24 (27.6%) of the 87 patients with SLE. Eleven (44.0%) of 25 anti-CL/ß2-GPI-positive cases had acquired APC-R, and 15 (53.6%) of 28 anti-PS/PT-positive cases had acquired APC-R. However, one half of SLE patients with anti-CL/ß2-GPI and anti-PS/PT antibodies had no acquired APC-R. These results suggest that the phenomenon observed in the in vitro experiment using IgG fractions containing anti-CL/ß2-GPI and anti-PS/PT antibodies may not always occur in vivo.

Recently, several authors have suggested that the complex of PL and plasma proteins such as protein C and protein S are also recognized by aPL antibodies and that these aPL antibodies may be involved in acquired APC-R (1)(2)(5). Further studies are currently in progress to elucidate the mechanisms by which these aPL antibodies inhibit the APC pathway.


   Acknowledgments
 
This work was supported by the Charitable Trust Laboratory Medicine Foundation of Japan.


   Footnotes
 
1 Nonstandard abbreviations: aPL, anti-phospholipid; anti-CL/ß2-GPI, anti-cardiolipin/ß2-glycoprotein I; anti-PS/PT, anti-phosphatidylserine/prothrombin; LA, lupus anticoagulant; SLE, systemic lupus erythematosus; PE, pulmonary embolism; VTE, venous thromboembolism; APC, activated protein C; APC-R, activated protein C resistance; DVT, deep vein thrombosis; TBS, Tris-buffered saline; BSA, bovine serum albumin; APTT, activated partial thromboplastin time; OR, odds ratio; CI, confidence interval; and mAU, milliabsorbance unit(s).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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