Amegakaryocytic thrombocytopenia (AMT) associated with systemic sclerosis (SSc) has been described in several case reports, but the underlying mechanisms have not been identified. Here we describe a rare case of SSc accompanied by thrombocytopenia and megakaryocytic hypoplasia, in which autoantibody against thrombopoietin receptor (c-Mpl) was detected. A 61-year-old woman with limited SSc was admitted to our hospital because of severe thrombocytopenia (platelet count 0.2 × 104/mm3) with gingival bleeding. Her bone marrow was hypocellular with absent megakaryocytes, consistent with AMT. Treatment with corticosteroids and intravenous immunoglobulin infusions resulted in an increased platelet count, and she sustained a remission over a 1-year period, with a platelet count averaging 10.0 × 104/mm3. Her serum was strongly positive for anti–c-Mpl antibody, and IgG fraction purified from her serum inhibited thrombopoietin-dependent cell proliferation in vitro. Our case report suggests that AMT in patients with SSc could be mediated by the anti–c-Mpl antibody, which functionally blocks an interaction between thrombopoietin and c-Mpl.
Systemic sclerosis (SSc) is a multiorgan disorder that involves not only the skin, but also several of the internal organs. Although thrombocytopenia rarely develops in patients with SSc, the following mechanisms of induction are proposed: microangiopathy, overlapping systemic lupus erythematosus (SLE), aplastic anemia, autoimmune thrombocytopenic purpura (ATP), antiphospholipid syndrome, and hemophagocytic syndrome. In addition, thrombocytopenia may be drug-induced.
We encountered a case of SSc complicated by severe thrombocytopenia with megakaryocytic hypoplasia, which is an extremely rare phenomenon in patients with SSc (only 4 cases have been reported in 25 years). In the search for a mechanism for thrombocytopenia, an autoantibody to c-Mpl, the thrombopoietin (TPO) receptor, was detected in the patient's serum. Recently, one of us (MK) reported the presence of autoantibody to c-Mpl in a subset of patients with SLE who had thrombocytopenia with megakaryocytic hypoplasia (1). In those patients, anti–c-Mpl antibodies functionally blocked an interaction between TPO and c-Mpl and, thus, were thought to be responsible for the impaired thrombopoiesis. The IgG fraction purified from our patient's serum also inhibited TPO-dependent cell proliferation, suggesting that thrombocytopenia in our patient is also mediated by c-Mpl antibodies. This is the first reported case of a patient with SSc who had amegakaryocytic thrombocytopenia (AMT) associated with c-Mpl antibodies.
The patient, a 61-year-old woman, was admitted to our hospital in July 2001 because of gingival bleeding. She first noticed symptoms of Raynaud's phenomenon in 1974. In 1978, a diagnosis of SSc was made, based on the presence of proximal scleroderma in accordance with the American College of Rheumatology (formerly, the American Rheumatism Association) preliminary criteria for SSc (2). The presence of bibasilar pulmonary fibrosis, digital pitting scars, and Raynaud's phenomenon supported the diagnosis. She had limited cutaneous SSc with a stable course and received no medication. In 1996, her platelet count was noted to be low, in the range of 6.0 × 104/mm3, and it decreased gradually thereafter.
Her physical examination showed the presence of palatal petechiae, thickening and shortening of the lingual frenulum, sclerodactyly, atrophy of the soft tissue of her fingers, scattered petechiae over the extremities, and crepitant rales at the base of both lung fields. She had no esophageal involvement, erythema, oral ulcer, lymphadenopathy, or hepatosplenomegaly.
The laboratory examination revealed marked thrombocytopenia (platelet count 0.2 × 104/mm3), leukopenia (white blood cell count 2.8 × 103/mm3) with a normal differential count, and anemia (red blood cell count 3.45 × 106/mm3, hemoglobin level 9.4 gm/dl, reticulocyte count 6.0 × 104/mm3). The erythrocyte sedimentation rate was 57 mm/hour. Electrophoresis of serum proteins showed a polyclonal gammopathy with a total gamma globulin concentration of 2.5 gm/dl. The levels of serum iron, haptoglobin, and complement were normal. An indirect Coombs' test gave a negative result. Antinuclear antibodies were positive at 1:1,280, with a nucleolar pattern. However, anti–Scl-70, anticentromere, anti-RNA polymerase I/III, anti–U1 RNP, anti–U3 RNP, anti-Th/To, anti–PM-Scl, anti–double-stranded DNA, anti-SSA/Ro, anti-SSB/La, and anticardiolipin antibodies were all negative. The prothrombin time, activated partial thromboplastin time, fibrinogen level, and fibrin degradation product level were all normal. The platelet-associated IgG (PAIgG) level was elevated at 3,406 ng/107 platelets (normal 9–25 ng/107 platelets). Renal function and urinalysis were normal.
Neither splenomegaly nor hepatomegaly was observed on abdominal ultrasound examination. Radiography of the chest revealed mild reticulonodular shadows in the bilateral lower lung fields. Bone marrow aspiration and trephine biopsy showed hypocellularity with absent megakaryocytes and decreased myeloid and erythroid cells, and marked fatty infiltration, but dysplasia was not observed (Figure 1). A cytogenetic analysis of the marrow showed a normal female karyotype of 46 XX.
We initiated treatment with methylprednisolone pulse therapy (1.0 gm/day administered intravenously for 3 successive days) followed by prednisolone in an oral dosage of 50 mg/day. The leukopenia and anemia responded quite well to corticosteroid therapy, but the platelet count increased only briefly and then began to decline. Intravenous immunoglobulin (0.4 gm/kg body weight/day for 5 successive days) was given without any effect. The prednisolone dosage was tapered over a 1-month period to 10 mg/day, and then was maintained at 10 mg/day. One month after initiation of corticosteroid therapy, the platelet count increased to 5.3 × 104/mm3, and the PAIgG decreased to 107 ng/107 platelets. Since then, the patient's platelet count has remained stable, averaging 10.0 × 104/mm3 over a 1-year period. Followup bone marrow aspiration revealed the appearance of megakaryocytes (2 megakaryocytes per high-power field), although the marrow was still considered to be hypocellular.
Because in the case presented the bone marrow was hypocellular, with marked megakaryocytic hypoplasia, it is possible that the thrombocytopenia is attributable to aplastic anemia or AMT. However, highly elevated PAIgG is more likely to be associated with ATP than with AMT or aplastic anemia, although the PAIgG level is considered to be less specific for ATP than are platelet-associated anti–glycoprotein IIb-IIIa (anti–GPIIb-IIIa) antibodies (3). To evaluate mechanisms for thrombocytopenia in our patient, we used several newly reported methods, including percentage of reticulated platelets (4), serum autoantibodies to c-Mpl by enzyme-linked immunosorbent assay (ELISA) (the recombinant c-Mpl fragment was provided by Kirin Brewery, Takasaki, Japan) (5), B cells secreting IgG anti–GPIIb-IIIa antibodies in peripheral blood by enzyme-linked immunospot assay (5), and platelet-associated IgG anti–GPIIb-IIIa antibodies by ELISA (5). All of these measurements were carried out after the platelet count had increased to 10.3 × 104/mm3, after treatment with corticosteroids. The percentage of reticulated platelets, which increases in a subset of patients with ATP (4), was 0.2% (normal 0.5–1.7%). The serum anti–c-Mpl antibody level was strongly positive at 77 units (normal <18 units), and it decreased to 24 units 2 months later. In contrast, the frequency of B cells secreting IgG anti–GPIIb-IIIa antibodies in peripheral blood was close to the normal range at 2.5/105 peripheral blood mononuclear cells (normal <2.0/105), and the level of platelet-associated IgG anti–GPIIb-IIIa antibodies was normal at 2.6 units (normal <3.3 units).
To further examine a pathogenic role of anti–c-Mpl antibodies, the IgG fraction was purified from our patient's serum and examined for its capacity to inhibit TPO-induced cell proliferation. The TPO-dependent mouse myeloid leukemia cell line factor-dependent cell/P2 transfected with full-length human c-Mpl complementary DNA (provided by Kirin Brewery) was cultured in the presence of human TPO with the IgG fractions isolated from our patient, 3 patients with SLE (2 with and 1 without anti–c-Mpl antibodies), and 2 healthy controls (1). The IgG fraction from our patient's serum inhibited TPO-induced cell proliferation, as observed in rabbit anti–c-Mpl polyclonal antibodies and the IgG fractions from anti–c-Mpl–positive SLE patients (Figure 2). The inhibitory effect produced by the IgG fraction from our patient was dose-dependent (data not shown).
This is the first report describing the presence of autoantibodies to c-Mpl in a patient with SSc. Because overlapping autoimmune disease was not found by detailed and systemic evaluation, it is likely that in our patient AMT is associated with SSc. The present case demonstrates the possibility that AMT associated with SSc can be mediated by anti–c-Mpl antibody.
TPO has been identified as a megakaryocyte colony-stimulating factor and a megakaryocyte potentiator. TPO binds to its receptor, c-Mpl, on the surface of hematopoietic stem cells and megakaryocytes and induces their proliferation and maturation. It was recently demonstrated that anti–c-Mpl antibodies are present in patients with SLE who have thrombocytopenia with decreased bone marrow megakaryocytes (1). Based on the findings that IgG fractions purified from anti–c-Mpl antibody–positive SLE sera bind to c-Mpl expressed on the cell surface and inhibit TPO-dependent cell proliferation as well as megakaryocyte colony formation, the impaired thrombopoiesis in those SLE patients is considered to be mediated by the anti–c-Mpl antibodies, which functionally block an interaction between TPO and c-Mpl. In our patient, who had SSc and thrombocytopenia with megakaryocytic hypoplasia, the level of serum anti–c-Mpl antibodies was significantly elevated, and the IgG fraction inhibited TPO-dependent cell proliferation in vitro. Therefore, it is likely that the mechanism for thrombocytopenia in our SSc patient is similar to that in patients with SLE who present with megakaryocytic hypoplasia.
Leukopenia and anemia were also noted in our patient, and myeloid- and erythroid-lineage cells were also decreased in the bone marrow. However, these changes were relatively mild compared with the observed thrombocytopenia and decrease in megakaryocytes. Because it has been reported that recombinant human TPO acts not only on megakaryocyte progenitors but also on erythroid, myeloid, and multipotential progenitors in vitro (6), the mild leukopenia and anemia observed in our patient may also be explained by the presence of anti–c-Mpl autoantibodies.
GPIIb-IIIa is known to be the main target of pathogenic antiplatelet autoantibodies in patients with ATP (3). In our patient, the level of platelet-associated IgG anti–GPIIb-IIIa antibodies was normal, and the frequency of IgG anti–GPIIb-IIIa antibody–producing B cells was borderline. However, because we were unable to perform these evaluations before initiation of therapy, the possibility that anti–GPIIb-IIIa antibodies had increased before treatment and affected the clinical course cannot be excluded. In this regard, more than half of SLE patients with anti–c-Mpl antibodies had coexistent anti–GPIIb-IIIa antibodies (1), suggesting that impaired thrombopoiesis is not the sole mechanism for thrombocytopenia in those patients.
Corticosteroid therapy appeared to be effective in increasing the platelet count in our patient, but it took a month to detect a significant increase in platelet count, which is unusual for ATP. The precise reason is unclear, but it is possible that additional time for differentiation from impaired hematopoietic stem cells to megakaryocytes is necessary for recovery from impaired thrombopoiesis.
Nine SSc patients with thrombocytopenia, excluding drug-induced thrombocytopenia, microangiopathy, overlapping syndrome with SLE, antiphospholipid syndrome, or hemophagocytic syndrome, were identified in the literature (Table 1). Five of them had an increased or normal number of megakaryocytes in bone marrow, consistent with ATP (7–10). In contrast, the other 4 patients with SSc showed hypocellular marrow with absent megakaryocytes, similar to what was observed in our patient (11–14). It is interesting to note that 3 of these 4 patients also had leukopenia and/or anemia. Antinuclear antibody testing on indirect immunofluorescence was performed in 3 patients. In 2 of them results were negative, and the remaining patient was weakly positive for antinuclear antibodies, with a speckled pattern. The mechanism for thrombocytopenia in these SSc patients with AMT has not been elucidated, but it is possible that impaired thrombopoiesis induced by the anti–c-Mpl antibody might be the mechanism in some of them. However, we must mention that our patient's case is very unusual, because AMT is an extremely rare cause for thrombocytopenia in patients with SSc, and scleroderma-related autoantibodies are generally considered not to cause any specific abnormalities.
Table 1. Complete blood cell count and bone marrow megakaryocytes in SSc patients with thrombocytopenia*
Author, year (ref.)
Complete blood cell count
Bone marrow megakaryocytes
ANA (staining pattern)
Patients with drug-induced thrombocytopenia were excluded. SSc = systemic sclerosis; WBC = white blood cell; ANA = antinuclear antibodies; ND = not determined.