CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
57, 125-136 (1990)
Thrombotic Thrombocytopenic Purpura in Two Patients with Systemic Lupus Erythematosus: Clinical Significance of Anti-platelet Antibodies YOSHIHIRO ITOH, HIROMI SEKINE, OSAMU HOSONO, TSUTOMU TAKEUCHI, JUN KOIDE, MAKOTO TAKANO, AND TOHRU ABE The 2nd Department
of Internal Medicine, Saitama Medical Center, Tsujido-machi, Kamoda Kawagoe-shi, Saitama
Saitama Medical 350, Japan
School,
1981
Thrombotic thrombocytopenic purpura (TTP) is a clinical syndrome of unknown etiology and has a high mortality rate due to disseminated platelet thrombi. However, the mechanism of platelet agglutination is not understood. Although an immunological mechanism has been suggested as the basis for the pathogenesis of TTP, any possible immunemediated etiology remains unclear. The association of TTP with systemic lupus erythematosus (SLE) atfords a unique opportunity to study such possibilities, because SLE is a prototype of autoimmune disease. This report describes two patients with SLE who developed TTP. The development of anti-platelet antibodies is one possible immunological mechanism for platelet agglutination in patients with SLE complicated by TTP. More importantly, patient J.Y., who had anti-platelet antibodies, responded dramatically to high doses of prednisolone. o 1990 Academic press, I~C.
INTRODUCTION
Thrombotic thrombocytopenic purpura (TTP) is a clinical syndrome of unknown etiology characterized by microangiopathic hemolytic anemia, thrombocytopenic purpura, neurologic abnormalities, fever, and renal dysfunction (1). Although TTP has a high mortality rate, often due to disseminated platelet thrombi within the arterial circulation, the mechanism of platelet aggregation is unknown (2). Several hypotheses have been proposed to explain the pathogenetic mechanism of this disease. These include (i) the presence of circulating antibody, or immune complexes, which promote microthrombus formation, (ii) injuries to small vessels which trigger platelet adhesion and aggregation, (iii) a defect in the fibrinolytic control mechanism, and (iv) a deficiency of PGI2-stimulating factor or stabilizing factor (3). Although convincing cases of systemic lupus erythematosus (SLE) associated with TTP are rare, there is a small number of cases in which the coexistence of SLE and TTP can be considered well established (4). Since SLE is a prototype of autoimmune disease, patients with SLE complicated by TTP may provide a unique opportunity to elucidate the immunological mechanism of intravascular platelet aggregation. This paper consists of two case reports of patients with well-defined SLE who developed TTP at a time when their SLE was inactive. More importantly, a patient with anti-platelet antibody survived as a result of steroid pulse therapy while another patient who was negative with respect to anti-platelet antibodies 125 0090-1229190 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
126
ITOH
ET
AL.
died, despite steroid pulse therapy, transfusion doses of aspirin and dipiridamole. METHODS
of fresh frozen plasma, and low
AND MATERIALS
Diagnosis of Systemic Lupus Erythematosus Thrombocytopenic Purpura
and Thrombotic
The diagnosis of SLE was made in accordance with the 1982 American Rheumatism Association (ARA) criteria for the classification of SLE (5). A diagnosis of TTP was made if the patient exhibited the triad of microangiopathic hemolytic anemia, thrombocytopenic purpura, and neurologic symptoms (1). The diagnosis was confirmed by the presence of diffuse thrombotic occlusions in the case of patient M.M. Collection
of Plasma
and Platelets
Blood was collected into polyethylene tubes containing 3.8% sodium citrate by a two plastic syringe technique. Platelet-poor plasma (PPP) was prepared by centrifugation at 2400g for 20 min at -4°C after which the supernatant was decanted and spun at 10,OOOg for 10 min at 4°C. The PPP was divided into small samples and stored at 4°C until used (6). Platelets were obtained from platelet-rich plasma (PRP), which was prepared by centrifuging plasma at 180g for 10 min at room temperature. The platelets were washed twice and then suspended in Tris-saline, pH 7.4, containing 133 m.iV NaCl, 15 mA4 Tris-HCl, 5 W KCl, and 1 mikf MgCl, (7). The concentration was adjusted to 7.5 X 105/mm3 for platelet aggregation studies. TTP Plasma-Induced
Platelet Aggregation
Platelet aggregation was achieved by a modification of Lian’s method (7). Briefly, the washed platelets were suspended in Tris-saline buffer, pH 7.4, containing 133 mM NaCl, 15 mM Tris-HCl, 5 r&4 KCl, and 1 n&f MgCl, and adjusted to a concentration of 7.5 x 105/mm3. TTP plasma (0.4 ml), either undiluted or diluted with Tris-saline buffer, was incubated at 37°C in a cell of a Chrono-log platelet aggregometer with a 609~nm red filter. To this mixture, 0.1 ml of platelet suspension was added. The percentage decrease in optical density resulting from platelet aggregation was recorded. Enzyme-Linked
Immunoassay
of Anti-Cardiolipin
Antibody
Anti-cardiolipin antibody was measured by a solid-phase enzyme immunoassay (ELISA) (8). Each well of an Immuron plate was first coated with 50 p,l of cardiolipin, 40 mg/ml, by incubation overnight at 37°C. One hundred microliters of 1% BSA in CBB solution was added to each well and the plate was incubated for 1 hr at room temperature to block nonspecific binding of immunoglobulin to the well surface. After five washings in PBS, 50 pl of serum (diluted with 0.02 M potassium phosphate saline, pH 8.O:PBS containing 5% Tween 20) was added to each well, and the preparations were incubated for 1 hr at room temperature.
TTP
IN TWO
PATIENTS
WITH
SLE
127
After five washings with PBS, 50 l,~l of alkaline phosphatase (Sigma)-labeled mouse monoclonal anti-human IgG was added to each well and the plate was incubated for 1 hr, then 100 l~,l of p-nitrophenyl phosphate in I M diethanolamine buffer, pH 9.0, was added to each well. After a 30-min incubation, optical absorbance was measured at 405 nm by using a Titertek Multiscan MCC. Anti-platelet
Antibody
Washed platelets (1 x 106) were treated with 100 p,l of patient’s plasma or F(ab’), fragments for 30 min at 4°C with occasional shaking and washed three times with PBS. The platelets were subsequently exposed for 30 min at 4°C to a 150 dilution of FITC-conjugated goat anti-human IgG F(ab) antibody (Cappel Laboratories, Cochransville, PA), washed twice with PBS, then resuspended in 1 ml of PBS. The platelets were examined for staining with a Spectrum III. As a positive control, platelets were treated with CD36 (OKM5), which is known to react with platelets. Absorption
of J. Y. Plasma
by Platelets
from Normal
Donor
The J.Y. plasma was absorbed with varying numbers of platelets from a normal donor. The absorption was carried out with 1 x lo4 or 1 x 10’ platelets/O.5 ml of plasma at 37°C for 45 min. Purification
of ZgG
Five milliliters of patient J.Y.‘s serum was dialyzed overnight against three changes of 100 vol of PBS containing 0.02% NaN,. The dialyzed serum was loaded onto a 1.6 x 6.2-cm DEAE Affi-Gel Blue column and eluted with the same buffer. The flow-through protein fractions were pooled and concentrated to between 1 and 1.5 ml with polyethylene glycol20,OOO and dialyzed overnight against two changes of 500 ml of 0.05 M phosphate buffer, pH 7.0. The purity of the IgG fraction was confirmed by SDS-polyacrylamide electrophoresis under both nonreducing and reducing conditions (9). Preparation
of Human
ZgG F(ab’),
and Fc Fragments
F(ab’), and Fc fragments were prepared by papain digestion using a moditication of the method described by Porter (10). The F(ab’), and Fc fragments were confirmed by 10% SDS-polyacrylamide gel electrophoresis and immunodiffusion studies against commercial anti-F(ab’), (Jackson Immuno-Research Laboratories) and anti-Fc (Wako Pure Chemical Industries, Osaka, Japan) antibodies. Case Histories Patient J. Y. A 47-year-old woman developed fever, polyarthritis, and a butterfly rash in May 1963. Initially the disease affected the proximal interphalangeal joints and knees but additional joints became involved over the next 3 months. During this time there was leukopenia, and the patient was found to be positive for ANA. A diagnosis of SLE was made. Therapy with prednisolone (PSL), 20 mg/ day, relieved her clinical symptoms and the dose was tapered off without recurrence of symptoms. She first experienced Raynaud’s phenomenon during the
128
ITOH
ET AL.
winter of 1973. Her illness had been controlled with PSL, 5-10 mg/day, until September 1988 when she was hospitalized with fever, headache, confusion, purpura, and anemia in the absence of active SLE. Urinalysis revealed no proteinuria, hemoglobin was 6.5 g/d& platelet count was 13,000/mm3, and sedimentation rate was 166 mm/hr. The peripheral blood smear showed fragmentation with approximately 40% schistocytes. Prothrombin time and partial thromboplastin time were normal. Biochemical tests revealed lactic dehydrogenase (LDH), 1623 III/liter, and bilirubin, 4.7 mg/dl. Coom’s test was negative. The ANA test was positive at a titer of 1:1280 with a speckled pattern. Anti-ds DNA antibody was within the normal range. The anti-ss DNA antibody titer was 100 U, anticardiolipin antibody was 1:4, anti-RNP antibody was 1:256, and anti-SSA antibody was 1:256, but anti-Sm, anti-SSB antibody, BFP, and rheumatoid factor were negative. Total hemolytic complement, C3, and C4 were all depressed. The patient’s PA-IgG titer was 1872 ng (normal: 9.0-25.0 r&O’ cells), immune complexes were 9.5 pg (normal: ~20 t&ml), IgG 4850 mg/dl, IgA 579, and IgM 144. A diagnosis of TPP was made clinically, although no gingival biopsy was performed. Treatment with methyl-PSL pulse therapy was initiated. The patient responded dramatically to therapy without anti-platelet drugs and fresh frozen plasma. Clinical and hematological remission has been maintained for over 10 months. Patient M.M. A 31-year-old woman developed SLE in 1976, manifested by polyarthritis, butterfly rash, Raynaud’s phenomenon, membranous glomerulonephritis, and positive ANA with a speckled pattern. She was treated with high-dose PSL and improved, but over the next 10 years she was hospitalized on several occasions for the nephrotic syndrome. In November 1988, she was hospitalized because of headache and confusion for 3 days followed by coma. Her temperature was 38°C and multiple purpurae were seen on her soft palate and extremities. Although she had the nephrotic syndrome, no other active signs of SLE were noted. Hemoglobin was 6.5 g/d1 and the platelet count was 14,000/mm3. A peripheral blood smear showed microangiopathic changes with about 50% schistocytes. Prothrombin time was slightly prolonged but partial prothrombin time was normal. Total bilirubin was 4.7 mgldl, Coomb’s test was negative, and LDH was 1824 III/liter. The PA-IgG titer was 377 ng (normal: 9.0-25.0 &IO’ cells), immune complexes were 6.9 pg (normal: ~20 pg/ml), IgG 1310 mg/dl, IgA 269, and IgM 204. Hemolytic complement was depressed. The anti-ds DNA antibody was negative. Anti-ss DNA, anti-RNP, and anti-SSA antibodies were positive at a 1:4 dilution. The anti-cardiolipin antibody was positive. BFP and rheumatoid factor were negative. Treatment with 1.O mg/ day methyl-PSL, 1200 mg/day aspirin, 600 mg/day dipyridamol, and multiple courses of fresh frozen plasma was instituted. Despite these treatments the patients expired. The diagnosis of TTP was confirmed by autopsy. RESULTS 1. Diagnostic
Fearures
Table 1 summarizes
of SLE and TTP
the major features leading to the diagnosis of SLE and
TTP IN TWO PATIENTS TABLE DIAGNOSTIC
FEATURES
WITH
129
SLE
1 OF SLE AND TTP Patient
SLE Butterfly rash Discoid rash Photosensitivity Oral ulcer Arthritis Serositis Renal disease Neurological abnormality Hematological abnormality Immunological abnormality Antinuclear antibody Diagnosis ARA criteria (24) TTP Microangiopathic hemolytic anemia Thrombocytopenic purpura Neurologic symptoms Renal disease Fever Diagnosis Triad (anemia, purpura, neurologic) Pentad
J.Y.
M.M.
+ +
+ -
+ + +
+ + + +
+
+
+ + + +
+ + + + +
+ -
+ +
TTP. Patients J.Y. and M.M. met the criteria for SLE. At the time TTP was diagnosed, J.Y. exhibited the triad and M.M. the pentad of clinical features of TTP. TTP developed in both cases after the diagnosis of SLE had been established. Figure 1 shows vascular occlusion with hyaline-like, acidophilic, PASpositive material, which blocks the small arterioles in the liver. 2. Platelet Aggregation Induced by TTP Plasma
The TTP plasma from M.M. and J.Y. at the onset of TTP was examined for aggregation of washed platelets from a normal donor. Platelet aggregation was recorded for 14 min. As shown in Fig. 2, the plasma from J.Y. on September 4, 1988, induced aggregation. In contrast, the plasma from M.M. on November 15, 1988, failed to induce aggregation. The results indicate that the plasma from J.Y. on September 4 contained platelet-aggregating factor. 3. Platelet Aggregation Induced by J.Y.‘s Plasma
Since plasma from J.Y. on September 4 induced aggregation of platelets normal donor, the effect of varying the concentration of the plasma on aggregation was determined. As shown in Fig. 3A, the magnitude of aggregation depended on the plasma concentration. Next, specimens of
from a platelet platelet serially
130
ITOH
ET AL.
FIG. 1. Liver of patient M.M. Arterioles within Glisson’s sheath display features of TTP (intraluminal and subendothelial hyahne thrombi; hematoxyhn and eosin, 160x ).
obtained plasma from J.Y. were examined for their ability to aggregate washed platelets. Platelet aggregation was induced by a 50% concentration of her plasma. The results are shown in Fig. 3B. Although the plasma obtained on September 4 induced platelet aggregation, the specimen taken on November 15, when J.Y. had completely recovered from TTP, did not. The same results were obtained by F(ab’), fragments from J.Y.‘s plasma (data not shown). 4. Anti-platelet
Antibody
In a further examination
for platelet-aggregating
factor, we examined
J.Y.‘s
FIG. 2. Platelet aggregation induced by plasma from patients J.Y. and MM. at onset of ‘ITP. The platelet aggregation was induced by TIP plasma in a volume of 0.4 ml, to which 0.1 ml of platelet suspension (7.5 x 105/mm3) was added. The platelet aggregation was recorded for 14 min.
TTP IN TWO PATIENTS
WITH
131
SLE TTP serum 0%
100%
6
8
Minutes
B
serum -
on*
15-11-1988 l-lo-1988
P
.’
-
5 a1 8 7-
4-9-1988
*Platelet aggregation by 50% concentration I 2
4
6
8
10
was Induced of TTP serum I 12
I
I
I
14
Minutes
FIG. 3. Effect of varying concentrations of J.Y.‘s plasma and serially obtained plasma on the platelet aggregation. (A) The tracings for platelet aggregation induced by different concentrations of J.Y.‘s plasma. (B) Platelet aggregation was examined by using serially obtained J.Y. plasma.
plasma for anti-platelet antibodies by using Spectrum III. As shown in the left half of Fig. 4, washed platelets reacted with CD36(OKM5), while they failed to bind FITC-conjugated goat anti-human IgG Fab antibody, indicating that the washed platelets did not have IgG on their surface. J.Y .‘s plasma and F(ab’), fragments on September 4 reacted with washed platelets (Fig. 4, top right). In contrast, her plasma and F(ab’), fragment on November 15 failed to bind platelets. The results correlated well with the platelet aggregation determined with a Chrono-log aggregometer. 5. Effect of Absorption on Platelet Aggregation and Anti-Platelet
Antibody
To confirm that the platelet aggregation resulted from anti-platelet antibody, we performed an absorption study. The J.Y. plasma on September 4, 1988 was used because the plasma induced platelet aggregation and contained anti-platelet antibody. As shown in Table 2, the plasma induced 27% platelet aggregation and bound 64% of normal platelets. After absorption with 1 x lo4 cells/O.5 ml of plasma, the percentages were 20 and 46%, respectively. Absorption with an in-
132
ITOH ET AL.
.? ‘0
widwdpbt~t‘a stainodwithOKM5
washsdplatfM +
J.Y. plasmaon 15/11/1988
:: I
‘Jl’et.,,
F I llCl,
*? err,
F I llr,,.
FIG. 4. Graphs of J.Y. and M.M. plasma-treated normal-washed platelets stained with FITCconjugated goat anti-human IgG F(ab) antibody. The washed platelets (1 x 106) were treated with 100 ~1 of J.Y. or M.M. plasma. The platelets were subsequently mixed with FITC-conjugated goat antihuman IgG F(ab) antibody. As a positive control, platelets were treated with CD36 (OKM 5) which is known to stain platelets.
creased number of platelets platelet antibody.
(1 x 10’) eliminated
aggregation
as well as anti-
6. Immunological Features
Immunological findings in patients J.Y. and M.M. are shown in Fig. 5. Pa-IgG and immune complexes were positive in both cases. Anti-Sm and anti-%% antibodies were not detected. Although BFP and rheumatoid factor were negative, anti-cardiolipin antibody was positive at a 1:4 dilution. As is clear from the figure, immunological profiles were similar in the two cases, but the magnitude of immunological abnormalities was greater in J.Y. than in M.M. 7. Clinical Course
The clinical course of both cases is illustrated
in Fig. 6. Patient J.Y. was treated
TTP IN TWO PATIENTS TABLE
WITH
133
SLE
2
EFFECTOFABSORFTIONOFPLATELETAGGREGATION
Number of platelets used for absorption
% platelet aggregationb
None 1 x lo4 1 x 10’
27 20 3
% binding to platelets’ 64 46 5
L1One-half milliliter of J.Y. plasma on April 9, 1988 was absorbed with varying numbers of platelets from a normal donor at 37°C for 45 min. b Platelet aggregation was induced by a 50% concentration of the plasma and recorded by a platelet aggregometer at 10 min. ’ Anti-platelet antibody was determined by percentage binding to normal platelets.
with methyl-PSL pulse therapy. Although the immunological profiles were similar in the two cases, anti-platelet antibody was detected only in patient J.Y. Her clinical findings and laboratory test values responded dramatically to treatment. Clinical and hematological remission has been maintained over 10 months. Treatment of patient M.M. with a high dose of steroids was also started. Unlike patient J.Y., she received anti-platelet drugs and multiple courses of fresh frozen plasma were given (not shown in Fig. 6). Despite this intensive treatment, patient M.M. died 7 days after the onset of TTP. DISCUSSION
In this report we described two patients with preexisting SLE whose course was complicated by the development of TTP. Although the presence of TTP in patients with SLE is frequently mentioned, documentation of the relationship is not solid and the combination is rare (1 l-l 3). J.Y.
47~10
?
(onset, ~3)
MM.
5ly/o
-p (onset, 1974)
13lOmg 269mg 204mg r
256X 256X
Anti-ds
DNA
Anti-so Anti-S-n Anti-RNP Anti-SSA Anti-SSB
DNA antibody antibody antibody antibody antibody
+
Anti-cardiolipin BFP Rheumatoid
antibody
Ab.
+
factor
FIG. 5. Immunological findings in J.Y. and M.M. The immunological profiles were similar, but the magnitude of the immunological abnormalities was greater in J.Y. than in M.M. PA IgG: normal 9.0-25.0 ng/lO’ cells. IC: normal
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
57, 125-136 (1990)
Thrombotic Thrombocytopenic Purpura in Two Patients with Systemic Lupus Erythematosus: Clinical Significance of Anti-platelet Antibodies YOSHIHIRO ITOH, HIROMI SEKINE, OSAMU HOSONO, TSUTOMU TAKEUCHI, JUN KOIDE, MAKOTO TAKANO, AND TOHRU ABE The 2nd Department
of Internal Medicine, Saitama Medical Center, Tsujido-machi, Kamoda Kawagoe-shi, Saitama
Saitama Medical 350, Japan
School,
1981
Thrombotic thrombocytopenic purpura (TTP) is a clinical syndrome of unknown etiology and has a high mortality rate due to disseminated platelet thrombi. However, the mechanism of platelet agglutination is not understood. Although an immunological mechanism has been suggested as the basis for the pathogenesis of TTP, any possible immunemediated etiology remains unclear. The association of TTP with systemic lupus erythematosus (SLE) atfords a unique opportunity to study such possibilities, because SLE is a prototype of autoimmune disease. This report describes two patients with SLE who developed TTP. The development of anti-platelet antibodies is one possible immunological mechanism for platelet agglutination in patients with SLE complicated by TTP. More importantly, patient J.Y., who had anti-platelet antibodies, responded dramatically to high doses of prednisolone. o 1990 Academic press, I~C.
INTRODUCTION
Thrombotic thrombocytopenic purpura (TTP) is a clinical syndrome of unknown etiology characterized by microangiopathic hemolytic anemia, thrombocytopenic purpura, neurologic abnormalities, fever, and renal dysfunction (1). Although TTP has a high mortality rate, often due to disseminated platelet thrombi within the arterial circulation, the mechanism of platelet aggregation is unknown (2). Several hypotheses have been proposed to explain the pathogenetic mechanism of this disease. These include (i) the presence of circulating antibody, or immune complexes, which promote microthrombus formation, (ii) injuries to small vessels which trigger platelet adhesion and aggregation, (iii) a defect in the fibrinolytic control mechanism, and (iv) a deficiency of PGI2-stimulating factor or stabilizing factor (3). Although convincing cases of systemic lupus erythematosus (SLE) associated with TTP are rare, there is a small number of cases in which the coexistence of SLE and TTP can be considered well established (4). Since SLE is a prototype of autoimmune disease, patients with SLE complicated by TTP may provide a unique opportunity to elucidate the immunological mechanism of intravascular platelet aggregation. This paper consists of two case reports of patients with well-defined SLE who developed TTP at a time when their SLE was inactive. More importantly, a patient with anti-platelet antibody survived as a result of steroid pulse therapy while another patient who was negative with respect to anti-platelet antibodies 125 0090-1229190 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
126
ITOH
ET
AL.
died, despite steroid pulse therapy, transfusion doses of aspirin and dipiridamole. METHODS
of fresh frozen plasma, and low
AND MATERIALS
Diagnosis of Systemic Lupus Erythematosus Thrombocytopenic Purpura
and Thrombotic
The diagnosis of SLE was made in accordance with the 1982 American Rheumatism Association (ARA) criteria for the classification of SLE (5). A diagnosis of TTP was made if the patient exhibited the triad of microangiopathic hemolytic anemia, thrombocytopenic purpura, and neurologic symptoms (1). The diagnosis was confirmed by the presence of diffuse thrombotic occlusions in the case of patient M.M. Collection
of Plasma
and Platelets
Blood was collected into polyethylene tubes containing 3.8% sodium citrate by a two plastic syringe technique. Platelet-poor plasma (PPP) was prepared by centrifugation at 2400g for 20 min at -4°C after which the supernatant was decanted and spun at 10,OOOg for 10 min at 4°C. The PPP was divided into small samples and stored at 4°C until used (6). Platelets were obtained from platelet-rich plasma (PRP), which was prepared by centrifuging plasma at 180g for 10 min at room temperature. The platelets were washed twice and then suspended in Tris-saline, pH 7.4, containing 133 m.iV NaCl, 15 mA4 Tris-HCl, 5 W KCl, and 1 mikf MgCl, (7). The concentration was adjusted to 7.5 X 105/mm3 for platelet aggregation studies. TTP Plasma-Induced
Platelet Aggregation
Platelet aggregation was achieved by a modification of Lian’s method (7). Briefly, the washed platelets were suspended in Tris-saline buffer, pH 7.4, containing 133 mM NaCl, 15 mM Tris-HCl, 5 r&4 KCl, and 1 n&f MgCl, and adjusted to a concentration of 7.5 x 105/mm3. TTP plasma (0.4 ml), either undiluted or diluted with Tris-saline buffer, was incubated at 37°C in a cell of a Chrono-log platelet aggregometer with a 609~nm red filter. To this mixture, 0.1 ml of platelet suspension was added. The percentage decrease in optical density resulting from platelet aggregation was recorded. Enzyme-Linked
Immunoassay
of Anti-Cardiolipin
Antibody
Anti-cardiolipin antibody was measured by a solid-phase enzyme immunoassay (ELISA) (8). Each well of an Immuron plate was first coated with 50 p,l of cardiolipin, 40 mg/ml, by incubation overnight at 37°C. One hundred microliters of 1% BSA in CBB solution was added to each well and the plate was incubated for 1 hr at room temperature to block nonspecific binding of immunoglobulin to the well surface. After five washings in PBS, 50 pl of serum (diluted with 0.02 M potassium phosphate saline, pH 8.O:PBS containing 5% Tween 20) was added to each well, and the preparations were incubated for 1 hr at room temperature.
TTP
IN TWO
PATIENTS
WITH
SLE
127
After five washings with PBS, 50 l,~l of alkaline phosphatase (Sigma)-labeled mouse monoclonal anti-human IgG was added to each well and the plate was incubated for 1 hr, then 100 l~,l of p-nitrophenyl phosphate in I M diethanolamine buffer, pH 9.0, was added to each well. After a 30-min incubation, optical absorbance was measured at 405 nm by using a Titertek Multiscan MCC. Anti-platelet
Antibody
Washed platelets (1 x 106) were treated with 100 p,l of patient’s plasma or F(ab’), fragments for 30 min at 4°C with occasional shaking and washed three times with PBS. The platelets were subsequently exposed for 30 min at 4°C to a 150 dilution of FITC-conjugated goat anti-human IgG F(ab) antibody (Cappel Laboratories, Cochransville, PA), washed twice with PBS, then resuspended in 1 ml of PBS. The platelets were examined for staining with a Spectrum III. As a positive control, platelets were treated with CD36 (OKM5), which is known to react with platelets. Absorption
of J. Y. Plasma
by Platelets
from Normal
Donor
The J.Y. plasma was absorbed with varying numbers of platelets from a normal donor. The absorption was carried out with 1 x lo4 or 1 x 10’ platelets/O.5 ml of plasma at 37°C for 45 min. Purification
of ZgG
Five milliliters of patient J.Y.‘s serum was dialyzed overnight against three changes of 100 vol of PBS containing 0.02% NaN,. The dialyzed serum was loaded onto a 1.6 x 6.2-cm DEAE Affi-Gel Blue column and eluted with the same buffer. The flow-through protein fractions were pooled and concentrated to between 1 and 1.5 ml with polyethylene glycol20,OOO and dialyzed overnight against two changes of 500 ml of 0.05 M phosphate buffer, pH 7.0. The purity of the IgG fraction was confirmed by SDS-polyacrylamide electrophoresis under both nonreducing and reducing conditions (9). Preparation
of Human
ZgG F(ab’),
and Fc Fragments
F(ab’), and Fc fragments were prepared by papain digestion using a moditication of the method described by Porter (10). The F(ab’), and Fc fragments were confirmed by 10% SDS-polyacrylamide gel electrophoresis and immunodiffusion studies against commercial anti-F(ab’), (Jackson Immuno-Research Laboratories) and anti-Fc (Wako Pure Chemical Industries, Osaka, Japan) antibodies. Case Histories Patient J. Y. A 47-year-old woman developed fever, polyarthritis, and a butterfly rash in May 1963. Initially the disease affected the proximal interphalangeal joints and knees but additional joints became involved over the next 3 months. During this time there was leukopenia, and the patient was found to be positive for ANA. A diagnosis of SLE was made. Therapy with prednisolone (PSL), 20 mg/ day, relieved her clinical symptoms and the dose was tapered off without recurrence of symptoms. She first experienced Raynaud’s phenomenon during the
128
ITOH
ET AL.
winter of 1973. Her illness had been controlled with PSL, 5-10 mg/day, until September 1988 when she was hospitalized with fever, headache, confusion, purpura, and anemia in the absence of active SLE. Urinalysis revealed no proteinuria, hemoglobin was 6.5 g/d& platelet count was 13,000/mm3, and sedimentation rate was 166 mm/hr. The peripheral blood smear showed fragmentation with approximately 40% schistocytes. Prothrombin time and partial thromboplastin time were normal. Biochemical tests revealed lactic dehydrogenase (LDH), 1623 III/liter, and bilirubin, 4.7 mg/dl. Coom’s test was negative. The ANA test was positive at a titer of 1:1280 with a speckled pattern. Anti-ds DNA antibody was within the normal range. The anti-ss DNA antibody titer was 100 U, anticardiolipin antibody was 1:4, anti-RNP antibody was 1:256, and anti-SSA antibody was 1:256, but anti-Sm, anti-SSB antibody, BFP, and rheumatoid factor were negative. Total hemolytic complement, C3, and C4 were all depressed. The patient’s PA-IgG titer was 1872 ng (normal: 9.0-25.0 r&O’ cells), immune complexes were 9.5 pg (normal: ~20 t&ml), IgG 4850 mg/dl, IgA 579, and IgM 144. A diagnosis of TPP was made clinically, although no gingival biopsy was performed. Treatment with methyl-PSL pulse therapy was initiated. The patient responded dramatically to therapy without anti-platelet drugs and fresh frozen plasma. Clinical and hematological remission has been maintained for over 10 months. Patient M.M. A 31-year-old woman developed SLE in 1976, manifested by polyarthritis, butterfly rash, Raynaud’s phenomenon, membranous glomerulonephritis, and positive ANA with a speckled pattern. She was treated with high-dose PSL and improved, but over the next 10 years she was hospitalized on several occasions for the nephrotic syndrome. In November 1988, she was hospitalized because of headache and confusion for 3 days followed by coma. Her temperature was 38°C and multiple purpurae were seen on her soft palate and extremities. Although she had the nephrotic syndrome, no other active signs of SLE were noted. Hemoglobin was 6.5 g/d1 and the platelet count was 14,000/mm3. A peripheral blood smear showed microangiopathic changes with about 50% schistocytes. Prothrombin time was slightly prolonged but partial prothrombin time was normal. Total bilirubin was 4.7 mgldl, Coomb’s test was negative, and LDH was 1824 III/liter. The PA-IgG titer was 377 ng (normal: 9.0-25.0 &IO’ cells), immune complexes were 6.9 pg (normal: ~20 pg/ml), IgG 1310 mg/dl, IgA 269, and IgM 204. Hemolytic complement was depressed. The anti-ds DNA antibody was negative. Anti-ss DNA, anti-RNP, and anti-SSA antibodies were positive at a 1:4 dilution. The anti-cardiolipin antibody was positive. BFP and rheumatoid factor were negative. Treatment with 1.O mg/ day methyl-PSL, 1200 mg/day aspirin, 600 mg/day dipyridamol, and multiple courses of fresh frozen plasma was instituted. Despite these treatments the patients expired. The diagnosis of TTP was confirmed by autopsy. RESULTS 1. Diagnostic
Fearures
Table 1 summarizes
of SLE and TTP
the major features leading to the diagnosis of SLE and
TTP IN TWO PATIENTS TABLE DIAGNOSTIC
FEATURES
WITH
129
SLE
1 OF SLE AND TTP Patient
SLE Butterfly rash Discoid rash Photosensitivity Oral ulcer Arthritis Serositis Renal disease Neurological abnormality Hematological abnormality Immunological abnormality Antinuclear antibody Diagnosis ARA criteria (24) TTP Microangiopathic hemolytic anemia Thrombocytopenic purpura Neurologic symptoms Renal disease Fever Diagnosis Triad (anemia, purpura, neurologic) Pentad
J.Y.
M.M.
+ +
+ -
+ + +
+ + + +
+
+
+ + + +
+ + + + +
+ -
+ +
TTP. Patients J.Y. and M.M. met the criteria for SLE. At the time TTP was diagnosed, J.Y. exhibited the triad and M.M. the pentad of clinical features of TTP. TTP developed in both cases after the diagnosis of SLE had been established. Figure 1 shows vascular occlusion with hyaline-like, acidophilic, PASpositive material, which blocks the small arterioles in the liver. 2. Platelet Aggregation Induced by TTP Plasma
The TTP plasma from M.M. and J.Y. at the onset of TTP was examined for aggregation of washed platelets from a normal donor. Platelet aggregation was recorded for 14 min. As shown in Fig. 2, the plasma from J.Y. on September 4, 1988, induced aggregation. In contrast, the plasma from M.M. on November 15, 1988, failed to induce aggregation. The results indicate that the plasma from J.Y. on September 4 contained platelet-aggregating factor. 3. Platelet Aggregation Induced by J.Y.‘s Plasma
Since plasma from J.Y. on September 4 induced aggregation of platelets normal donor, the effect of varying the concentration of the plasma on aggregation was determined. As shown in Fig. 3A, the magnitude of aggregation depended on the plasma concentration. Next, specimens of
from a platelet platelet serially
130
ITOH
ET AL.
FIG. 1. Liver of patient M.M. Arterioles within Glisson’s sheath display features of TTP (intraluminal and subendothelial hyahne thrombi; hematoxyhn and eosin, 160x ).
obtained plasma from J.Y. were examined for their ability to aggregate washed platelets. Platelet aggregation was induced by a 50% concentration of her plasma. The results are shown in Fig. 3B. Although the plasma obtained on September 4 induced platelet aggregation, the specimen taken on November 15, when J.Y. had completely recovered from TTP, did not. The same results were obtained by F(ab’), fragments from J.Y.‘s plasma (data not shown). 4. Anti-platelet
Antibody
In a further examination
for platelet-aggregating
factor, we examined
J.Y.‘s
FIG. 2. Platelet aggregation induced by plasma from patients J.Y. and MM. at onset of ‘ITP. The platelet aggregation was induced by TIP plasma in a volume of 0.4 ml, to which 0.1 ml of platelet suspension (7.5 x 105/mm3) was added. The platelet aggregation was recorded for 14 min.
TTP IN TWO PATIENTS
WITH
131
SLE TTP serum 0%
100%
6
8
Minutes
B
serum -
on*
15-11-1988 l-lo-1988
P
.’
-
5 a1 8 7-
4-9-1988
*Platelet aggregation by 50% concentration I 2
4
6
8
10
was Induced of TTP serum I 12
I
I
I
14
Minutes
FIG. 3. Effect of varying concentrations of J.Y.‘s plasma and serially obtained plasma on the platelet aggregation. (A) The tracings for platelet aggregation induced by different concentrations of J.Y.‘s plasma. (B) Platelet aggregation was examined by using serially obtained J.Y. plasma.
plasma for anti-platelet antibodies by using Spectrum III. As shown in the left half of Fig. 4, washed platelets reacted with CD36(OKM5), while they failed to bind FITC-conjugated goat anti-human IgG Fab antibody, indicating that the washed platelets did not have IgG on their surface. J.Y .‘s plasma and F(ab’), fragments on September 4 reacted with washed platelets (Fig. 4, top right). In contrast, her plasma and F(ab’), fragment on November 15 failed to bind platelets. The results correlated well with the platelet aggregation determined with a Chrono-log aggregometer. 5. Effect of Absorption on Platelet Aggregation and Anti-Platelet
Antibody
To confirm that the platelet aggregation resulted from anti-platelet antibody, we performed an absorption study. The J.Y. plasma on September 4, 1988 was used because the plasma induced platelet aggregation and contained anti-platelet antibody. As shown in Table 2, the plasma induced 27% platelet aggregation and bound 64% of normal platelets. After absorption with 1 x lo4 cells/O.5 ml of plasma, the percentages were 20 and 46%, respectively. Absorption with an in-
132
ITOH ET AL.
.? ‘0
widwdpbt~t‘a stainodwithOKM5
washsdplatfM +
J.Y. plasmaon 15/11/1988
:: I
‘Jl’et.,,
F I llCl,
*? err,
F I llr,,.
FIG. 4. Graphs of J.Y. and M.M. plasma-treated normal-washed platelets stained with FITCconjugated goat anti-human IgG F(ab) antibody. The washed platelets (1 x 106) were treated with 100 ~1 of J.Y. or M.M. plasma. The platelets were subsequently mixed with FITC-conjugated goat antihuman IgG F(ab) antibody. As a positive control, platelets were treated with CD36 (OKM 5) which is known to stain platelets.
creased number of platelets platelet antibody.
(1 x 10’) eliminated
aggregation
as well as anti-
6. Immunological Features
Immunological findings in patients J.Y. and M.M. are shown in Fig. 5. Pa-IgG and immune complexes were positive in both cases. Anti-Sm and anti-%% antibodies were not detected. Although BFP and rheumatoid factor were negative, anti-cardiolipin antibody was positive at a 1:4 dilution. As is clear from the figure, immunological profiles were similar in the two cases, but the magnitude of immunological abnormalities was greater in J.Y. than in M.M. 7. Clinical Course
The clinical course of both cases is illustrated
in Fig. 6. Patient J.Y. was treated
TTP IN TWO PATIENTS TABLE
WITH
133
SLE
2
EFFECTOFABSORFTIONOFPLATELETAGGREGATION
Number of platelets used for absorption
% platelet aggregationb
None 1 x lo4 1 x 10’
27 20 3
% binding to platelets’ 64 46 5
L1One-half milliliter of J.Y. plasma on April 9, 1988 was absorbed with varying numbers of platelets from a normal donor at 37°C for 45 min. b Platelet aggregation was induced by a 50% concentration of the plasma and recorded by a platelet aggregometer at 10 min. ’ Anti-platelet antibody was determined by percentage binding to normal platelets.
with methyl-PSL pulse therapy. Although the immunological profiles were similar in the two cases, anti-platelet antibody was detected only in patient J.Y. Her clinical findings and laboratory test values responded dramatically to treatment. Clinical and hematological remission has been maintained over 10 months. Treatment of patient M.M. with a high dose of steroids was also started. Unlike patient J.Y., she received anti-platelet drugs and multiple courses of fresh frozen plasma were given (not shown in Fig. 6). Despite this intensive treatment, patient M.M. died 7 days after the onset of TTP. DISCUSSION
In this report we described two patients with preexisting SLE whose course was complicated by the development of TTP. Although the presence of TTP in patients with SLE is frequently mentioned, documentation of the relationship is not solid and the combination is rare (1 l-l 3). J.Y.
47~10
?
(onset, ~3)
MM.
5ly/o
-p (onset, 1974)
13lOmg 269mg 204mg r
256X 256X
Anti-ds
DNA
Anti-so Anti-S-n Anti-RNP Anti-SSA Anti-SSB
DNA antibody antibody antibody antibody antibody
+
Anti-cardiolipin BFP Rheumatoid
antibody
Ab.
+
factor
FIG. 5. Immunological findings in J.Y. and M.M. The immunological profiles were similar, but the magnitude of the immunological abnormalities was greater in J.Y. than in M.M. PA IgG: normal 9.0-25.0 ng/lO’ cells. IC: normal
