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Rheumatology Advance Access originally published online on January 17, 2006
Rheumatology 2006 45(7):851-854; doi:10.1093/rheumatology/kel010
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Two types of autoantibody-mediated thrombocytopenia in patients with systemic lupus erythematosus

M. Kuwana, J. Kaburaki2, Y. Okazaki, H. Miyazaki3 and Y. Ikeda1

Division of Rheumatology and 1 Division of Haematology, Department of Internal Medicine, Keio University School of Medicine, 2 Department of Internal Medicine, Tokyo Electric Power Company Hospital, Tokyo and 3 Kirin Brewery Company Ltd., Takasaki, Japan.

Correspondence to: Masataka Kuwana, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: kuwanam{at}sc.itc.keio.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. To determine whether autoantibodies to two platelet-specific antigens, glycoprotein IIb/IIIa (GPIIb/IIIa) and thrombopoietin receptor (TPOR), contribute to thrombocytopenia in patients with systemic lupus erythematosus (SLE).

Methods. Circulating B cells producing anti-GPIIb/IIIa antibodies and serum anti-TPOR antibodies were measured in 32 SLE patients with thrombocytopenia, 30 SLE patients without thrombocytopenia, 92 patients with idiopathic thrombocytopenia and 60 healthy controls. The megakaryocyte density in bone-marrow smears from all the patients with thrombocytopenia was evaluated.

Results. Anti-GPIIb/IIIa and anti-TPOR antibody responses were more frequent in SLE patients with thrombocytopenia than in those without thrombocytopenia (88 vs 17%, P<0.0001; and 22% vs 0%, P=0.01, respectively). The frequencies of these platelet-related antibodies were comparable between SLE patients with thrombocytopenia and patients with idiopathic thrombocytopenia. Twenty-nine (91%) SLE patients with thrombocytopenia had either anti-GPIIb/IIIa or anti-TPOR antibody, and six had both. In SLE patients with thrombocytopenia, the anti-TPOR-positive patients had significantly higher frequencies of megakaryocytic hypoplasia and poorer therapeutic responses to corticosteroids and intravenous immunoglobulin than did the anti-TPOR-negative patients, most of whom had the anti-GPIIb/IIIa antibody alone.

Conclusions. Anti-GPIIb/IIIa and anti-TPOR antibodies are major factors contributing to SLE-associated thrombocytopenia, but the clinical presentations associated with these autoantibodies are different.

KEY WORDS: Autoantibodies, Glycoprotein IIb/IIIa, Systemic lupus erythematosus, Thrombocytopenia, Thrombopoietin receptor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Thrombocytopenia is a major haematological complication in patients with systemic lupus erythematosus (SLE) [1]. The pathogenesis of thrombocytopenia in SLE patients is heterogeneous, but the most common mechanism is believed to be increased platelet clearance mediated by anti-platelet autoantibodies, which is analogous to the mechanism seen in patients with idiopathic thrombocytopenic purpura (ITP) [1]. Other potential mechanisms include thrombotic thrombocytopenic purpura, disseminated intravascular coagulation, haemophagocytic syndrome, antiphospholipid syndrome and impaired thrombopoiesis. Anti-platelet autoantibodies in ITP patients preferentially recognize platelet surface glycoproteins (GP), and the most common target is GPIIb/IIIa [2]. A recent study by Michel et al. [3] showed that anti-GPIIb/IIIa antibodies also play a primary role in SLE-associated thrombocytopenia. On the other hand, we recently identified autoantibodies to thrombopoietin receptor (TPOR), also called c-Mpl, which is clinically associated with thrombocytopenia in SLE patients and inhibits thrombopoietin (TPO)-dependent megakaryogenesis in vitro [4]. In this study, the roles of these two types of autoantibody responses in SLE-associated thrombocytopenia were evaluated.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Patients and controls
We studied 32 patients with SLE who had thrombocytopenia (mean platelet count 23x109/l, range 5–57x109/l) and were followed at Keio University Hospital between 1997 and 2004. The inclusion criteria were as follows: (i) requirement for treatment because of a significant bleeding tendency; (ii) pretreatment bone marrow films were available; and (iii) exclusion of clinically apparent conditions that can cause thrombocytopenia, i.e. disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, haemophagocytic syndrome and drug-induced thrombocytopenia. The control subjects were 30 SLE patients who had never been thrombocytopenic and 60 healthy individuals. All the SLE patients satisfied the American College of Rheumatology preliminary criteria [5], and three with thrombocytopenia and four without thrombocytopenia additionally satisfied the Sapporo criteria for antiphospholipid syndrome [6]. We also examined 92 patients with idiopathic thrombocytopenia, defined as thrombocytopenia (<100x109/l) that is not accompanied by morphological evidence of dysplasia in the bone marrow and cannot be attributed to other primary diseases or conditions. Blood samples were obtained after the patients and control subjects had given their written informed consent, as approved by the Keio University Institutional Review Board.

Clinical findings
The demographic and clinical features of each SLE patient were evaluated at the time of blood collection. Thirty-seven clinical and laboratory findings were recorded; these were the individual items included in the American College of Rheumatology preliminary classification criteria [5] and the SLE Disease Activity Index (SLEDAI) [7]. All SLE patients with thrombocytopenia received moderate- to high-dose oral corticosteroids (>40 mg/day; n = 26) or methylprednisolone pulse therapy (1 g/day for 3 days; n = 6), and eight of them simultaneously received intravenous immunoglobulin (IVIG; 0.4 g/day for 3–5 days) and/or platelet transfusion. During the course of the disease, 19 patients who required surgical or invasive procedure received IVIG without increase in the corticosteroid dosage or the initiation of immunosuppressant. A therapeutic response was defined as a platelet count >100x109/l in association with these therapies. The efficacy of the corticosteroid treatment was assessed 3 months afterwards, when the potential influence of IVIG or platelet transfusion could be ignored; the efficacy of IVIG was assessed at 1 week.

Autoantibody analysis
Anti-double-stranded DNA antibody was measured by the Farr assay, and anti-Sm and anti-SSA antibodies were identified using an RNA immunoprecipitation assay [8]. IgG anti-cardiolipin antibodies were measured with an enzyme-linked immunosorbent assay (ELISA) kit (MBL, Nagano, Japan).

Anti-GPIIb/IIIa antibody-producing B cells
The anti-GPIIb/IIIa antibody response was evaluated by detecting peripheral blood B cells secreting IgG anti-GPIIb/IIIa antibodies. For this, we used an enzyme-linked immunospot assay, which is a sensitive and specific method for evaluating the presence or absence of autoantibody-mediated thrombocytopenia [9]. Briefly, peripheral blood mononuclear cells (105/well) were cultured in pentaplicate on GPIIb/IIIa-coated 96-well microplates at 37°C for 4 h, and subsequently incubated with alkaline phosphatase-conjugated goat anti-human IgG. Finally, the anti-GPIIb/IIIa antibodies that bound to the membrane were visualized as spots by incubation with a substrate. The frequency of circulating anti-GPIIb/IIIa antibody-producing B cells was calculated as the number per 105 peripheral blood mononuclear cells. The cut-off value was defined as 2.0 [9].

Anti-TPOR antibody
Serum anti-TPOR antibody was detected by ELISA using a recombinant protein encoding the entire extracellular domain of human TPOR as the antigen, as described before [4]. Antibody units were calculated from the optical density at 450 nm, using a standard curve obtained from serial concentrations of rabbit anti-human TPOR polyclonal antibodies (Kirin Brewery, Takasaki, Japan), and the cut-off value was defined as 18.0 units [4].

Evaluation of bone-marrow megakaryocyte density
Bone-marrow films from all the patients with thrombocytopenia were available. The proportion of megakaryocytes to the total number of nucleated cells was evaluated from Wright–Giemsa-stained bone-marrow smears. At least 1000 nucleated cells were counted for each sample. A proportion of megakaryocytes that was ≤0.2% was regarded as a decrease and one of >1.0% as an increase.

Statistical analysis
All continuous results were expressed as the mean±S.D. Comparisons to determine statistical significance between two groups were performed using Fisher's exact test or unpaired Student's t-test, as appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Anti-GPIIb/IIIa and anti-TPOR antibody responses in SLE patients with thrombocytopenia
Anti-GPIIb/IIIa antibody-producing B cells and anti-TPOR antibody levels in SLE patients with thrombocytopenia were significantly higher than in SLE patients without thrombocytopenia or healthy controls, but were comparable to those in patients with idiopathic thrombocytopenia (Fig. 1). When all the subjects were stratified above or below the cut-off, an anti-GPIIb/IIIa antibody response was detected in 28 (88%) SLE patients with thrombocytopenia, but in five (17%) without thrombocytopenia (P<0.0001). Anti-TPOR antibody was detected exclusively in SLE patients with thrombocytopenia and in those with idiopathic thrombocytopenia, and its frequency was significantly higher in SLE patients with thrombocytopenia than in SLE patients without it (22 vs 0%, P = 0.01). The respective frequencies of anti-GPIIb/IIIa and anti-TPOR antibodies in SLE patients with thrombocytopenia were comparable to those in patients with idiopathic thrombocytopenia (86 and 10%). Finally, 29 (91%) of the SLE patients with thrombocytopenia produced either anti-GPIIb/IIIa or anti-TPOR antibody, and six of these patients produced both.


Figure 1
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  		FIG. 1. Circulating anti-GPIIb/IIIa antibody-producing B cells (A) and serum anti-TPOR antibody (B) in 32 SLE patients with thrombocytopenia (TP), 30 SLE patients without thrombocytopenia, 92 patients with idiopathic thrombocytopenia and 60 healthy controls. A broken line indicates a cut-off level (2.0 for anti-GPIIb/IIIa antibody-producing B cells and 18.0 for anti-TPOR antibody). Levels were compared between SLE patients with thrombocytopenia and other groups using the unpaired t-test. NS, not significant (P>0.05).

 
Megakaryocyte density in association with autoantibody status
Examination of the bone marrow from SLE patients with thrombocytopenia revealed that eight (25%), 17 (53%) and seven (22%) patients had increased, normal and decreased bone megakaryocytes, respectively. A similar distribution was observed in the 92 patients with idiopathic thrombocytopenia; i.e. 20, 66 and 14% had increased, normal and decreased bone megakaryocytes, respectively. The status of anti-GPIIb/IIIa and anti-TPOR antibodies was compared with the bone marrow megakaryocyte density of patients with SLE and thrombocytopenia and those with idiopathic thrombocytopenia. Seven SLE patients who had anti-TPOR antibody had a significantly higher frequency of megakaryocytic hypoplasia than 25 patients who did not (86 vs 4%, P<0.0001; Table 1), and this association appeared to be independent of anti-GPIIb/IIIa antibody production. In contrast, none of the SLE patients who produced anti-GPIIb/IIIa antibody but not anti-TPOR antibody had megakaryocytic hypoplasia. Similarly, in patients with idiopathic thrombocytopenia, megakaryocytic hypoplasia was significantly more frequent in the nine patients with anti-TPOR antibody than in the 83 without this antibody (79 vs 7%, P<0.0001).


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  		TABLE 1. Clinical and laboratory findings for SLE patients with thrombocytopenia who did or did not produce anti-TPOR antibody

 
Clinical associations with anti-TPOR antibody
Additional clinical and laboratory findings for SLE patients with thrombocytopenia were compared based on the presence or absence of anti-TPOR antibody (Table 1). There was no significant difference in sex, age at examination, SLE-related clinical findings, lowest platelet count, autoantibody status, including anti-GPIIb/IIIa antibody-producing B cells, or SLEDAI between these two groups. A poor therapeutic response to corticosteroids was more prevalent in patients with anti-TPOR antibody than in those without, most of whom had anti-GPIIb/IIIa antibody alone (P = 0.0006). Thus, the immunosuppressant use for thrombocytopenia was significantly more frequent in anti-TPOR-positive patients than in anti-TPOR-negative patients (71 vs 8%, P = 0.002). Furthermore, all five anti-TPOR-positive patients who received IVIG were non-responders, while only one patient (10%) without this antibody showed a poor response to IVIG (P = 0.002).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Our findings demonstrate that both anti-GPIIb/IIIa and anti-TPOR antibodies are associated with thrombocytopenia in SLE patients, although the tests used were not necessarily comparable: antibody-secreting peripheral blood B cells were measured to detect the anti-GPIIb/IIIa antibody response while serum samples were used to detect the anti-TPOR antibody response. More than 90% of SLE patients with thrombocytopenia had at least one of these platelet-related autoantibodies, indicating that thrombocytopenia mediated by these two types of autoantibody is a dominant mechanism for SLE-associated thrombocytopenia, as for idiopathic thrombocytopenia.

Interestingly, anti-GPIIb/IIIa and anti-TPOR antibodies were associated with different phenotypes of thrombocytopenia, in terms of bone-marrow megakaryocyte density and therapeutic responses to standard treatment regimens for immune thrombocytopenia. All the SLE patients with anti-GPIIb/IIIa antibody alone had normal or increased megakaryocyte density, whereas the anti-TPOR antibody was strongly associated with megakaryocytic hypoplasia. This different phenotype can be explained by the distinct biological effects of these antibodies: anti-GPIIb/IIIa antibody binds circulating platelets and facilitates Fc{gamma} receptor-mediated clearance of opsonized platelets by reticuloendothelial phagocytes [2], whereas anti-TPOR antibody blocks TPO signalling, resulting in inhibition of megakaryogenesis in the bone marrow [4]. This different mode of action may also account for the lack of therapeutic response to IVIG of patients with anti-TPOR antibody. Since interactions between the Fc portion of the infused immunoglobulins and the Fc receptors on target cells are thought to be a primary action of IVIG [10], it is likely that IVIG has little effect on the TPO signal blockade through the variable region of the antibodies.

In summary, measurement of anti-GPIIb/IIIa anti-TPOR antibody responses is useful in distinguishing between subsets of patients with SLE and thrombocytopenia and predicting their therapeutic response.
Figure 2


   Acknowledgments
 
This work was supported by grants from the Japanese Ministry of Health and Welfare.

No conflict of interest has been declared by the authors.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Quismorio FP Jr. Hematologic and lymphoid abnormalities in systemic lupus erythematosus. In: Wallace DJ, Hahn BH, eds. Duboi's lupus erythematosus. Baltimore, MD: Williams & Wilkins, 1997:793–816.
  2. Cines DB, Blanchette VS. Immune thrombocytopenic purpura. N Engl J Med 2002;346:995–1008.[Free Full Text]
  3. Michel M, Lee K, Piette JC et al. Platelet autoantibodies and lupus-associated thrombocytopenia. Br J Haematol 2002;119:354–8.[Medline]
  4. Kuwana M, Okazaki Y, Kajihara M et al. Autoantibody to c-Mpl (thrombopoietin receptor) in systemic lupus erythematosus: relationship to thrombocytopenia with megakaryocytic hypoplasia. Arthritis Rheum 2002;46:2148–59.[Medline]
  5. Tan EM, Cohan AS, Fries JF et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271–7.[Web of Science][Medline]
  6. Wilson WA, Gharavi AE, Koike T et al. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome. Arthritis Rheum 1999;42:1309–11.[CrossRef][Web of Science][Medline]
  7. Bombardier C, Gladman DD, Urowitz MB et al. Derivation of the SLEDAI: a disease activity index for lupus patients. Arthritis Rheum 1992;35:630–40.[Web of Science][Medline]
  8. Forman MS, Nakamura M, Mimori T, Gelpi C, Hardin JA. Detection of antibodies to small nuclear ribonucleoproteins and small cytoplasmic ribonucleoproteins using unlabelled cell extracts. Arthritis Rheum 1985;28:1356–61.[Medline]
  9. Kuwana M, Okazaki Y, Kaburaki J, Ikeda Y. Detection of circulating B cells secreting platelet-specific autoantibody is useful in the diagnosis of autoimmune thrombocytopenia. Am J Med 2003;114:322–5.[CrossRef][Medline]
  10. Kazatchkine MD, Kaveri SV. Immunomodulation of autoimmune and inflammatory diseases with intravenous immune globulin. N Engl J Med 2001;345:747–55.[Free Full Text]
Submitted 17 October 2005; revised version accepted 8 December 2005.
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