Apolipoprotein H Levels in Systemic Lupus Erythematosus Are Not Influenced by Antiphospholipid Serum

Antibodies YUKINOBU ICHIKAWA*, KAZUNAGA TAKAMATSU**, HIROAKI SHIMIZU*, MITSUAKI UCHIYAMA*, JUNKO MORIUCHI*, MASATOSHI TAKAYA*, NOBUMASA KOBAYASHI t, TSUTOMU KAWADA‡ and SHIGERU ARIMORI* of Internal Medicine, Tokai University School of Medicine; **the Second Department of Internal Medicine, Kochi Medical *Department School; †Blood Transfusion Service Center and ‡Central Clinical Laboratory, Tokai University Hospital, Japan

Anticardiolipin antibodies (aCL) were recently discovered to recognize a complex consisting of phospholipids and apolipoprotein H (apo H). In this study, we determined the serum apo H levels in 36 systemic lupus erythematosus (SLE) patients with or without antiphospholipid antibodies (aPL), including aCL and lupus anticoagulants, to clarify the possible effects of aPL on apo H levels in vivo. The apo H levels were low in SLE patients as compared with 22 healthy controls. However, no associations were found between apo H levels and circulating aPL or clinical features of the antiphospholipid antibody syndrome. A secondary hyperlipidemic state, which probably related to lupus nephritis (proteinuria) and/or prednisolone treatment, increased apo H levels in SLE patients.

Key Words: Apolipoprotein H

-β 2 Glycoprotein I Antiphospholipid antibodies Systemic

lupus erythematosus


(aCL) to phospholipids has been shown to require a plasma/serum cofactor, identified as apo H6. Furthermore, some of the aCL might be directed to the cofactor itself’. The interaction between apo H and aCL may relate to the thrombotic diathesis in patients with aPL,

H (apo H), also known as I in human serum, binds to lipoproteins, anionic phospholipids, platelets and other molecules. Although the biological functions of apo H are largely unknown, apo H has been shown to inhibit

Apolipoprotein 0,-glycoprotein


inhibitory functions of apo H in blood coagulation

might be interfered with by aPL. At the present time,

coagulation pathways or platelet aggregation by neutralizing negatively charged molecules that activate blood

we have little information about serum apo H levels in patients with SLE’, aPL or APS. In this study, therefore, we determined serum apo H levels in SLE patients. In addition, the results were compared with those of aPL or with symptoms relating to APS to clarify whether aPL influence serum apo H levels in vivo.


coagulation’ B Antiphospholipid antibodies (aPL), including anticardiolipin antibodies (aCL) and lupus anticoagulants (LA), have been detected in patients with systemic lupus erythematosus (SLE) and other autoimmune diseases. The association of aPL with a unique syndrome consisting of recurrent thrombosis, spontaneous abortions and/or thrombocytopenia has been of great interest, and the syndrome has been named the antiphospholipid antibody syndrome (APS)’. The precise role of aPL in the development of APS is still unknown, while in vitro

Materials and methods Patients

studies have shown that aPL react in many stages of blood coagulation through their interactions with phospholipids5. More recently, the binding of aPL

Fasting sera were obtained from 36 patients with SLE (four males and 32 females) who fulfilled the American Rheumatism Association criteria for the disease9. The patients were receiving prednisolone treatment (11.1 =b 7.8 mg/day, mean ± 1 SD, range 0-30) at the time of this study. Twenty-two healthy adults ( 11 males and 11 females)

Yukinobu Ichikawa, M.D., Department of Internal Medicine, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa, 259-11, Japan.



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served as controls. The mean age of the controls (50.0 ~ 7.4 years, range 34-64) was significantly higher than that of SLE patients (39.2 ± 14.0 years, range 19-76). Determination

of serum

detected in 13 of the 36 patients at the time of this study. In eight of them, proteinuria was greater than 1 g/day. Circulating anti-DNA antibodies (Crithidia method), complement component (C3; nephelometry, normal: ~ 55 mg/dl), immune complexes containing both Clq and IgG (ELISA, Immunomedics, Warren, NJ, USA; normal: < 34.5 p.g/ml), total cholesterol and triglyceride levels were determined simultaneously in the patients. For the statistical analysis, mean values were compared by Wilcoxon’s rank sum test. Prevalence was estimated by chi-square test and Fisher’s exact test. Linear regression analysis was used for the evaluation of correlation coefficients.

apo H levels

Serum apo H levels were determined by single radial immunodiffusion with anti-human apo H rabbit serum as reported previously ...... The antiserum was obtained by immunization with apo H isolated from human serum. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the purified apo H demonstrated a single band (50 kD). One precipitation line was observed between apo H and the antiserum used in the present study or rabbit anti-apo H serum purchased from Behringwerke AG Diagnostica (Marburg, Germany) by the Ouchterlony method. The two precipitation lines completely fused with each other. Diluted human sera containing known levels of apo H were employed to produce standard curves. Determination

APS, since they had both aPL (aCL and/or LA) and at least one of the symptoms. Proteinuria (> 30 mg/dl) was



H levels in SLE patients and controls

Serum apo H levels

of aPL

significantly lower in 36 SLE 4.9 mg/dl) than in 22 controls (21.9 ± 0.05). In control subjects, however, no were

patients (20.1 2.4 mg/dl, P < significant difference ±

Serum IgG aCL levels were measured by enzyme-linked immunosorbent assay (ELISA) established by use of standard sera purchased from the Rayne Institute, St Thomas’ Hospital (London, UK). The procedure was almost identical to that described by Gharavi et al.’2. Briefly, wells of a polyvinyl chloride microtiter plate (ICN Biomedicals, Costa Mesa, CA, USA) were coated with cardiolipin (CL; Sigma, St Louis, MO, USA). Ten percent fetal bovine serum (FBS) in phosphate buffered saline (PBS) was used as a blocking solution. Test samples and alkaline phosphatase-conjugated goat antihuman IgG (Sigma) were also diluted in the FBS-PBS. P-nitrophenyl phosphate (Wako Chemical Company, Osaka, Japan) in diethanolamine buffer was used as a substrate. The cut-off level for IgG aCL was 5 IgG anti-

was found in the apo H levels 11 between females (21.2 ± 2.6 mg/dl) and 11males ± 2.1 (22.7 mg/dl), or between nine controls below 50 of years age (mean age: 42.6 ± 4.1 years, apo H: 21.2 ± 1.9 mg/dl) and 13 controls above 50 years of age (mean age: 55.2 ± 3.8 years, apo H: 22.5 ± 2.7 mg/dl), although we could not obtain completely sex- and agematched controls.

Correlations of serum apo H levels with other laboratory results in SLE patients The apo H levels (Table I) were not significantly different between aCL-positive patients (IgG aCL levels: 14.3 ± 12.3 GPL units) and aCL-negative patients (2.5 ± 1.4 GPL units), and between patients with definitely prolonged aPTT (75.2 ± 21.6 s) and those without (34.6 ± 5.8 s). The apo H levels were significantly lower in SLE patients with hypocomplementemia or those without proteinuria (P < 0.05). Compared with those in healthy controls, the apo H levels were also significantly lower in the 23 SLE patients without proteinuria (P < 0.01). The levels were relatively higher in SLE patients with hyperlipidemia, while the other laboratory abnormalities were not associated with the apo H levels. Furthermore, a significant positive correlation was observed between apo H levels (y) and C3 levels (x) (y = 13.0 + 0.13x, r = 0.373, P < 0.05) or total cholesterol levels (x) (y = 15.6 + 0.02x, r = 0.373, P < 0.05) in SLE patients. Total cholesterol levels (y) also correlated with triglyceride levels (x) (y 134 + = r in P < 0.44x, 0.489, 0.05) patients.

phospholipid (GPL) units’3. Activated partial thromboplastin time (aPTT; normal : 31.8-33.8 s) was simultaneously determined in the SLE patients. Of the 12 patients with definitely prolonged aPTT (~ 50 s), 10 were considered as LA positive, since at least two of the following tests were also positive in previous studies: standard mixing test of aPTT, mixing test of kaolin clotting time, dilute tissue thromboplastin test and aPTT-mixing test with dilute phospholipid reagent, as described previously’3. Another two patients with slightly prolonged aPTT (40-49 s) were positive for LA in the previous studies. Other clinical and laboratory parameters Clinical histories of thrombosis, spontaneous abortions or thrombocytopenia were reviewed in each patient. Thirteen of the 36 SLE patients were considered to have



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7.2 t 6.7; and 9.2 t 9.4 vs 7.8 =i= 11.6 GPL units, respectively). No differences were found in apo H levels between patients with and those without each clinical

Table I Comparison of serum apo H levels in SLE patients with positive laboratory results and in those with negative results.


history. Since the presence of proteinuria significantly influenced the apo H levels in SLE patients, laboratory variables were further compared between patients with and those without proteinuria (Table III). aPL, antiDNA antibodies and C3 levels were not different between the two groups. However, proteinuria-positive patients had high total cholesterol (P < 0.01) and triglyceride levels (P < 0.05), and were receiving high daily doses of prednisolone (P < 0.05) as compared with those without proteinuria. A positive correlation was found between total cholesterol levels (y) and daily doses Table III Comparison of laboratory results and daily doses of prednisolone (PSL) in SLE patients with proteinuria and in

those without. ’The P value between SLE patients with positive results and those with




less than 0.05.

Correlations of apo H levels with clinical histories Table II shows that

IgG aCL levels and aPTT were prolonged in patients with APS (11.5 ~ 9.7 GPL units; 68.7 ~ 26.0 s) than in those without APS (6.7 ± 10.8 GPL units, P < 0.05; 36.6 ± 11.3 s, P < 0.01). The apo H levels, however, were not different between patients with APS and those without. When each symptom of APS was compared, patients with previous thrombosis, spontaneous abortion or thrombocytopenia had more prolonged aPTT (76.1 f 33.9 s, 88.8 ± 25.6 s, and 56.8 ± 25.7 s, respectively) than those without the history (42.8 t 16.4 s,

significantly higher

or more

The P values between the two groups

P < 0.05; 39.1 ~ 9.8 s, P < 0.01; and 41.6 t 19.6 s, P < 0.05), whereas IgG aCL levels were not different between the groups (9.6 ~ 10.5 vs 8.2 ~ 10.7; 9.8 f 5.7

Table IV

less than ao.05 and


Comparison of serum apo H levels in proteinurianegative patients with aPL or related symptoms and in those without the antibodies


Table II Comparison of serum apo H levels in SLE patients with APS or related symptoms and in those without such clinical features.

a Male patients and patients without previous history of pregnancy were




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controls (P < 0.01). Both aPL and apo H have been shown to react with negatively charged phospholipids3. 12. These phospholipids are important for blood coagulation, but they are known to be present within the inner part of the resting platelet membrane&dquo;. If the phospholipids are expressed on the outer surface of platelets that were activated during blood coagulation, and if aPL can react with the platelets in the presence of apo H, both aPL and apo H levels in sera from aCL-positive patients should be decreased through a consumption mechanism. aCL and LA might be different antibody subgroups’6, but both aPL are shown to be neutralized by frozen and thawed platelets’3~’S~ &dquo;. We also reported that activated blood cells, including platelets, can inhibit serum aCL activity in vitro&dquo;. However, aPL activities were still detected in serum that was separated after blood coagulation. In addition, our previous studies showed that aCL activities and apo H levels were similar in sera and plasma simultaneously obtained from the same individuals 10,18. Taken together, these results also indicate that the blood coagulation process in glass tubes at the time of serum separation does not make platelets reactive with aPL or

prednisolone (x)(y 137 + 5.6x, r 0.538, P < 0.05) in all SLE patients. In the 23 proteinuria-negative patients, a possible corof



relation of apo H levels with the presence of aPL or APS also analysed, but no significant differences were found (Table IV).


Discussion were recently discovered to recognize a complex consisting of phospholipids and apo H, or apo H itself in vitro6,’. We considered the possibility that serum apo H levels may be decreased in patients with aPL because of consumption of the protein through in vivo immune complex formations. Our present study, however, demonstrated that circulating aPL do not influence


apo H levels in vivo. Furthermore, apo H levels did not associate with any clinical features of APS. One previous study reported slightly increased apo H serum

levels in 10 SLE patients, although

no further analysis In the present study, we demonstrated that, although apo H levels were low in all SLE patients as compared with healthy controls, a secondary hyperlipidemic state, probably caused by lupus nephritis (proteinuria) and/or prednisolone treatment, can increase apo H levels in SLE patients. The present results were was


apo H . The present results do not support the recently proposed assumption that the interaction of apo H with aPL may relate to thrombotic diathesis in patients with aPL. To clarify further the possibility, however, apo H levels

compatible with previous studies, including ours, which showed increased levels of cholesterol and triglyceride in SLE patients undergoing corticosteroid therapy’4 and high apo H levels in patients with hyperlipidemic states’°. In addition, apo H levels correlated with C3 levels in SLE patients, but there was no difference in C3 levels between patients with and without proteinuria. Liver dysfunctions have been shown to decrease apo H levels, since apo H is probably synthesized in the

may need to be determined at the time of thrombotic in patients with APS. In fact, no patient has been reported who has an impaired blood coagulation that is definitely related to low serum apo H levels, although


several patients with disseminated intravascular coagulation have low apo H levels2. 3. Additionally, FBS used in the present ELISA for aCL contains apo H. This method, therefore, probably detects antibodies that are reactive with CL, CL-apo H complex and/or the cofactor. Antibodies to apo H were not detected in any of our patients’ sera by the Ouchterlony method, but assays that can selectively detect each of the antibodies will be useful for future studies. Finally, the low apo H levels detected in SLE patients may suggest a hypercoagulable state in the disease. Conversely, the increase of apo H levels observed in proteinuria-positive patients may be a compensatory phenomenon for more hypercoagulable conditions in patients with hyperlipidemia and/or nephrotic

liver8.1O. Our results, therefore, may reflect the function of the liver (C3 and apo H synthesis) in the SLE patients studied. Serum apo H levels are regulated by a pair of autosomal codominant alleles, Bg’ and Bg’. In six of the 36 SLE patients, including one with chronic hepatitis, the apo H levels (11.2-14.8 mg/dl) were within the range of healthy subjects who are heterozygous for Bg’ and Bg’. However, the frequency of SLE patients who are possibly homozygous for Bg’ or heterozygous for Bg’ and Bg D was similar between patients with and without aPL or APS, and was not significantly different from that observed in healthy Japanese populations&dquo;. When the six SLE patients who had low apo H levels were excluded, apo H levels were not significantly different between SLE patients and controls. The apo H levels, however, were still low in the 19 proteinuria-negative SLE patients (19.7 f 2.8 mg/dl), excluding one patient with chronic hepatitis and three possible heterozygous patients who did not have proteinuria, as compared with

syndrome. References Nimpf J, Wurm H, Kostner GM. Interaction of β -glycoprotein-I 2 with human blood platelets: influence upon the ADP-induced aggregation. Thrombosis Haemostas 1985; 54: 397-401. 2. Schousboe I. β -Glycoprotein I: a plasma inhibitor of the contact 2 activation of the intrinsic blood coagulation pathway. Blood 1985; 66: 1086-91. 1.


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8. 9.


Japanese population. Jpn J Human Genet 1989; 34: 279-83. 12. Gharavi AE, Harris EN, Ascherson RA, Hughes GRV. Anticardiolipin antibodies: isotype distribution and phospholipid specificity. Ann Rheum Dis 1987; 46: 1-6. et al. Reactivities of 13. Ichikawa Y, Kobayashi N, Kawada T antiphospholipid antibodies to blood cells and their effects on 1990; 8: 461-8. platelet aggregation in vitro. Clin Exp Rheumatol 14. Ettinger WH, Goldberg AP, Applebaum-Bowden D, Hazzard WR. Dyslipoproteinemia in systemic lupus erythematosus. Am J Med 1987; 83: 503-8. et al. Immune mediated 15. Khamashita MA, Harris EN, Gharavi AE mechanism for thrombosis: antiphospholipid antibody binding to platelet membrane. Ann Rheum Dis 1988; 47: 849-54. 16. McNeil HP, Chesterman CN, Krilis SA. Anticardiolipin antibodies and lupus anticoagulants comprise separate antibody subgroups with different phospholipid binding characteristics. Br 1989; J Haematol 73: 506-13. 17. Triplett DA, Brandt JT, Kaczor D, Schaeffer J. Laboratory diagnosis of lupus inhibitors: a comparison of the tissue thromboplastin inhibition procedure with a new platelet neutralization 1983; 79: 678-82. procedure. Am J Clin Pathol 18. Ichikawa Y, Kobayashi N, Kawada T et al. Anticardiolipin antibodies by an enzyme-linked immunosorbent assay: fundamental studies on the conditions for antigen-application and specificity of the assay. Tokai J Exp Clin Med 1989; 14: 103-12.

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Serum apolipoprotein H levels in systemic lupus erythematosus are not influenced by antiphospholipid antibodies.

Anticardiolipin antibodies (aCL) were recently discovered to recognize a complex consisting of phospholipids and apolipoprotein H (apo H). In this stu...
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