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Heavy chain subclass of human anti-platelet antibodies
CLINICAL
IMMUNOLOGY
AND
2, 1-8 (1973)
IMMUNOPATHOLOGY
Heavy Chain Subclass of Human Anti-Platelet Antibodies] SIMON KARPATKIN,~ NATHAN
STRICK,
AND
PETER H. GREGORY
SCHUR,
W. SISKIND~
Department of Medicine, New York University Medical School; Department of Medicine, Harvard Medical School; and Division of Allergy and Immunology, Department of Medicine, Cornell University Medical College, New York, New York 10021 Received
January
5, 1973
The IgG subclass distribution of the antiplatelet antibodies present in several clinical conditions was determined. Studies were carried out on antiplatelet antibodies present in patients with autoimmune thrombocytopenic purpura, systemic lupus erythematosis, drug-induced immunologic thrombocytopenic purpura, and after multiple transfusions. The antiplatelet antibodies in autoimmune thrombocytopenic purpura appeared to be restricted to the rG3 subclass in every one of 15 patients studied. In contrast, nine patients with systemic lupus erythematosis who had antiplatelet antibodies were found to have antiplatelet antibodies detectable in three out of four or in all four subclasses. Different patterns of subclass disdibution were observed in the other clinical conditions studied. The subclass distribution of antiplatelet antibodies may be of assistance in distinguishing systemic lupus erythematosis from autoimmune thrombocytopenic purpura.
Previous communications from this laboratory have pointed out the usefulness of the platelet factor 3 (PF-3) immunoinjury technique for the detection of antiplatelet antibody in various clinical disorders (l-4)j. Employing this method, antiplatelet antibody could be detected in the serum of a high percentage of patients with chronic idiopathic autoimmune thrombocytopenic purpura (ATP), systemic lupus erythematosis (SLE), and drug-induced immunologic thrombocytopenic purpura. The relative nonspecificity of this immunoinjury technique required stringent proof that the antiplatelet factor demonstrated was indeed an autoantibody with platelet specificity. This was established by the following observations: 1. The antiplatelet activity was removed by addition of rabbit anti-human immunoglobulin G (RAHIgG) (l-3)
’ Supported Health Service ford Foundation. ? Career
by grants from (HE 1336, AM
Scientist
the New York Heart 11414, AM 05577, AM
of the Health
Research
Council
Association, from the United States Public 12051, FR 05669) and from the John A. Hartof the City
of New
York
(I-453).
R Career Scientist of the Health Research Council of the City of New York (I-593). ’ Confirmed in several laboratories: H. Horowitz, New York; S. Murphy, Philadelphia; R. Aster, Milwaukee; A. Tartaglia, Albany; J. Dodds, Albany; B. Baer, Kansas City; D. H. Cowan, Cleveland; R. Hirschman, N. I. H.; B. Firkin, Prahran, Australia, J. Caen, Paris; (quoted with permission of investigators). Copyright All rights
@ 1973 by Academic Press, Inc. of reproduction in any form reserved.
I
2
KARPATKIN
ET
AL.
but not by addition of rabbit antibodies to human immunoglobulins A, M, or D. 2. Antiplatelet activity could be absorbed with washed human platelets (l-3) but not with washed human granulocytes or red blood cells. 3. The antiplatelet factor could be eluted from platelets to which it had been adsorbed (l-3) and the antiplatelet activity of the eluted material removed with RAHIgG (l-3). 4. The antiplatelet factor was stable at 56°C (1). 5. It was shown that the serum antiplatelet factor and the antiplatelet activity eluted from platelets to which it had been adsorbed both have a molecular weight indistinguishable from that of IgG (1, 5). Human IgG has been shown to consist of four subclasses, rG1, rG2, yG3, and yG4, which differ not only in structure and relative serum concentrations (yG1: 64-70%, yG2: 23-28%, yG3: 4-7%, yG4: 3-4%), but also in their biological properties (6). The heavy-chain subclass of the antiplatelet antibodies in various clinical disorders was, therefore, studied to ascertain whether there was any relation between yG subclass and diagnosis. This has been examined in patients with ATP, SLE, drug-induced immunologic thrombocytopenic purpura, and a history of numerous blood or platelet transfusions. A unique subclass distribution of the antiplatelet antibodies was found for ATP patients. These patients appear to have their antiplatelet antibodies restricted to the yG3 subclass. This characteristic appears to distinguish ATP patients from other patients with antiplatelet antibodies. MATERIALS
AND
METHODS
Specific antisera directed against human heavy-chain subclasses and lightchain types were developed in monkeys and rabbits (6-8). The antisera were absorbed as necessary to make them specific with proteins coupled to Sepharose (9) or to bromacetylcellulose (BAC) (10). Specificity was determined by immunoelectrophoresis, double diffusion in agar, and radial immunodiffusion (11, 12). Antisera were then chromatographed on Sephadex G200 and the second, 7s, peak isolated and concentrated. These fractions were then tested by their ability to specifically agglutinate cells coated with myeloma proteins of the appropriate subclass (8). The antisera were equally potent in their ability to agglutinate specifically sensitized red blood cells (B), but the anti-yG3 was somewhat more potent when employed in radial immunodiffusion (7). The PF-3 immunoinjury technique was performed with globulin fractions of control and test sera on normal platelet-rich plasma in the presence of contact product as described previously (l-3). A lo-set or greater shortening of the fibrin clotting time was considered significant. Antiplatelet activity was titered using normal human globulin as diluent. Based upon our previous experience with this assay (l-3), a titer of greater than 1:4 is regarded as positive. Globulin fractions of sera to be tested and of rabbit antisera to human IgG were prepared by precipitation at 50% cold saturated (NHJ2S04. The globulin fractions were collected by centrifugation, dissolved in and extensively dialyzed against phosphate-buffered saline (PBS: 0.15 NaCI, 0.01 M potas-
ANTI-PLATELET
ANTIBODY
3
sium phosphate buffer, pH 7.4). All globulin fractions were reconstituted to their original serum volume with PBS. The final protein concentration was roughly 15 mglml as determined by the 280:260-rnp absorbancy (13). The immunoglobulin subclass of the patient’s antiplatelet antibody was determined by assaying the ability of subclass-specific antibodies to remove PF-3 activity from the globulin fraction of the patient’s serum. One tenth milliliter of test or control globulin was incubated with lo-40 ~1 of a globulin fraction of rabbit antiserum specific for one of the IgG subclasses or with normal rabbit globulin for 60 min at 37°C and then held at 4°C overnight. Any precipitate formed was removed by centrifugation at 2000g for 15 min at 4°C and the supernate was tested for PF-3 activity. The results are reported as the reciprocal of the highest dilution of the sample giving a positive result (shortening of the fibrin clotting time by 10 set or more) in the PF-3 test. Five samples of ATP sera and five samples of SLE sera were tested with 40 ~1 of anti-subclass antisera. In no case did 40 ~1 of anti-immunoglobulin serum depress the PF-3 titer to a greater extent than did 30~1. Therefore, use of 30~1 was adopted as a standard procedure. One cannot be certain that all IgG of the particular subclass is removed by this procedure. Addition of greater amounts of anti-subclass antisera did not cause any further decrease in antiplatelet antibody activity. The anti-subclass antisera used were prepared in rabbits or monkeys and are of the “precipitating” type which do not tend to give an inhibition zone in antibody excess. The results reported here are based upon addition of 30 ~1 of globulin fraction of rabbit anti-human immunoglobulin antiserum to 0.1 ml of the globulin fraction of the patient’s serum being studied. The results were found to be highly reproducible even in experiments in which samples were handled under “coded” conditions. It should be recognized that antiplatelet antibody titers are only semiquantitative measurements and, therefore, are not linear functions of antibody concentration. Accordingly, we would not expect summation of titer changes to be quantitative. Fifteen patients were studied who had classical clinical histories and hematological findings of chronic ATP (14, 15) with two or more episodes of thrombocytopenia and purpura over a period greater than 6 months. Some had undergone splenectomy (DeM, Bad, Tur) and some were in “clinical remission” (DeM, Bad, Tur, Soy, Min) at the time when serum samples used in this study were obtained. Nine patients with typical clinical and laboratory findings of SLE who were being followed in the Bellevue Hospital SLE clinic were studied. All of these patients had positive LE cell preparations and ANA tests on one or more occasion. None was thrombocytopenic at the time of study. Five patients with drug-induced immunologic thrombocytopenic purpura had both clinical and laboratory evidence for drug-induced thrombocytopenia and have been described in detail elsewhere (4). Laboratory confirmation of the diagnosis was obtained by the addition of the suspected drug to the PF-3 test with a consequent increase in the titer of antiplatelet antibody activity or a change from a negative to a positive PF-3 test. Two patients were studied who had had numerous blood or platelet transfusions. One of these
4
KAFWATKIN
CHnoNIc Patient DeM Qui Bad Han Ben Mar Kil Cor Tur Pal McG SOY Min McM Ale
+NRG 24 40 40 48 32 24 20 20 20 40 16 12 28 32 20
+anti-yG1 24 40 40 48 32 20 20 20 20 - 1’
AUTCXMMUNE tanti-yG2
ET
AL.
TABLE 1 THROMBOCYTOPENIC +anti-yG3 0 0 0 0 0 0 0 0 0 4 0 0 0 0 4
24 36 40 48 32 20 20 20 20 40 16 12 28 32 20
PUHPunA” +anti-yG4
+anti-K
+anti-A
24 40 40 48 32 20 20 20 20
4
4 20 20 8 8 -
40 16 12 28 32 20
16 16 16 8 -
-
” Antiplatelet antibody titer following treatment with anti-heavy chain subclass antibody or anti-light-chain antibody. One-tenth milliliter of globulin fraction of patient’s serum was incubated with 30 ~1 of normal rabbit globulin (NRG) or anti-heavy-chain subclass antibody or anti-light-chain-type antibody. Any precipitate formed was removed and the supernate titered for PF-3 activity. The results are reported as the reciprocal of the highest dilution of the sample giving a positive test. ’ Six of the 15 samples studied were not evaluated with anti-yG1 because no anti-yG1 antisera was available at the time these patients were studied.
was a patient with aplastic anemia (Gar) and the second was a patient who had received multiple whole-blood exchange transfusions and platelet transfusions for infectious hepatitis with hepatic coma (Cap). RESULTS Table 1 gives the results of studies on the immunoglobulin subclass of the antiplatelet antibody present in 15 patients with chronic ATP. In all 15 cases the antiplatelet activity was removed by addition of anti-human yG3. Addition of rabbit antibody specific for either human kappa or lambda light chains resulted in a drop in PF-3 titer suggesting the presence of both light-chain types in the antiplatelet antibody. In Table 2 is presented the results of studies on nine patients with SLE. In every case the antiplatelet antibodies were found to be present in three of the four or in all four IgG subclasses. Antiplatelet antibody was not restricted to a single subclass as it appeared to be in all ATP patients. The antiplatelet antibody titers in SLE patients tended to be higher than in ATP patients. This difference in titers could conceivably make detection of antiplatelet antibody in the yG1, 2, and 4 subclasses more sensitive in the SLE group. However, it
ANTI-PLATELET
Patient __.__-
5
SYSTEMIC
TABLE 2 LUPUS ERYTHEMATOSUS”
+anti-$1
+anti-yG2
+NRG ____
Geo Nun He1 McK sot Cru Bai Wil Hir
ANTIBODY
60 60 48 48 40 20 16 80 48
+anti-yG4
+anti-yC3
20 12 48 48 40 4 16 80 8
32 24 24 8 8
-
20 12 16 8 8 4 8 12 8
fl Antiplatelet antibody titer after treatment with anti-heavy-chain subclass antibody. One tenth milliliter of globulin fraction of patient’s serum was incubated with 30~1 of normal rabbit globulin (NRC) or anti-heavy-chain subclass antibody. Any precipitate formed was removed and the supernate titered for PF-3 activity. The results are reported as the reciprocal of the highest dilution of the sample giving a positive test.
TABLE DRUG-INDUCED Patient Age Vit Zil Lo1 Go1
Drug
IMMUNOLOGIC fanti-yG1
+anti-yG2
+anti-yG3
+anti-yG4 __.
48 16 16 44 32
20 8 16 20 12
4 0 0 0 0
8 0 0 4 4
48 16 16 44 32
antibody titer after treatment with anti-heavy-chain subclass antibody. One of globulin fraction of a patient’s serum was incubated with 30 ~1 of normal (NRG) or anti-heavy-chain subclass antibody. Any precipitate formed was resupernate titered for PF-3 activity in the presence of 0.1 mM of drug. The results the reciprocal of the highest dilution of the sample giving a positive test.
NUMEROUS Patient
Cap Gar
PURPURA”
+NRG
Quinidine Quinidine Quinidine Tegretol Thioguanine
CLAntiplatelet tenth milliliter rabbit globulin moved and the are reported as
3 THROMBOCYTOPENIC
TABLE 4 OR PLATELET
BLOOD
+NRG
+anti-yG
80 32
80 32
1
+anti-yG2 20 4
TRANSFUSIONS~ +anti-yG3 8 0
+anti-yG4 16 4
U Antiplatelet antibody titer after treatment with anti-heavy-chain subclass antibody. One tenth milliliter of globulin fraction of patient’s serum was incubated with 30 4 of normal rabbit globulin (NRG) or anti-heavy chain subclass antibody. Any precipitate formed was removed and the supernate titered for PF-3 activity. The results are reported as the reciprocal of the highest dilution of the sample giving a positive test.
6
KAFWATKIN
ET
AL.
should be noted that three SLE samples with titers of 40 or less showed a pattern similar to that in higher titered SLE patients. These samples were in the same titer range as were many of our ATP subjects. One patient(Con) was not included in either Table 1 or 2 because of uncertainty as to the correct diagnosis. The patient was a woman with no history of thrombocytopenia or purpura who gave birth to a thrombocytopenic infant. She had a normal platelet count and her antinuclear antibody titer was nondiagnostic. She was found to have a strongly positive PF-3 test and her antiplatelet antibody titer could be reduced by addition of antibody to each of the four IgG subclasses. Thus, the IgG subclass pattern of her antiplatelet antibody was similar to that usually seen in patients with SLE. Patients with drug-induced thrombocytopenic purpura have antiplatelet antibody present in two or three of the IgG subclasses as illustrated in Table 3. In no case was antiplatelet activity removed by addition of anti-yG4. In Table 4 are results obtained in studies on two patients who had antiplatelet antibody presumably as a consequence of multiple blood or platelet transfusions. In contrast to any of the above conditions antibody activity was detected in all IgG subclasses except yG1. DISCUSSION The results reported here indicate a rather striking restriction of the antiplatelet antibody response of ATP patients to the yG3 immunoglobulin subclass. It is of note that this subclass normally comprises only 4-7% of the total IgG (6,7). This is in marked contrast to the situation in patients with SLE who have antiplatelet antibodies detectable in at least three of the four IgG subclasses and often in all four subclasses. The antiplatelet antibody in ATP did not appear to be monoclonal since both antikappa and antilambda antisera partially removed the antiplatelet antibody activity. The possible diagnostic usefulness of these observations in distinguishing patients with ATP from those with SLE is obvious. It will be of interest to determine ultimately whether this distinction in the subclass distribution of antiplatelet antibodies will permit identification of those patients with chronic ATP who will eventually develop SLE. It should be emphasized that study of larger numbers of patients with chronic ATP who will eventually develop SLE. It should be emphasized that study of larger numbers of patients may reveal exceptions to the simple patterns observed here. Furthermore, the failure to detect antibodies in certain immunoglobulin subclasses may reflect not their absolute absence but merely the relative insensitivity of our detection methods. In any case there is certainly a quantitative if not a qualitative difference in the IgG subclass distribution in patients with ATP as compared to those with SLE. The data obtained regarding the antiplatelet antibody from patients with drug-induced immunologic thrombocytopenic purpura and patients with numerous blood or platelet transfusions are too limited to permit any de&itive conclusions. However, even the limited data do suggest that these conditions may also be associated with predictable patterns of IgG subclasses in the
ANTI-PLATELET
ANTIBODY
7
antiplatelet antibody response. Incomplete removal of IgG subclasses, or formation of antigen antibody complexes, could potentially produce errors in the assay system. Errors introduced in these ways would tend to result in release of PF-3 from platelets: that is, an apparent failure to remove antiplatelet activity. Since the basic observations reported here represent a depletion of PF-3 releasing activity, errors due to the above possibilities could not account for the data presented. It should be noted in addition, that significant depletion of PF-3 activity was observed upon treatment of sera from SLE patients with antisera to IgGl, rG2, and rG4. This clearly indicates that these antisera and the procedures used are capable of removing PF-3 releasing activity when the antiplatelet antibodies are present in these subclasses. The failure of these antisera to decrease antiplatelet activity of sera from ATP patients must be considered together with the complete elimination of antiplatelet activity from these sera with anti-IgG3. These observations can be explained only by assuming that there is at least a quantitative difference in the subclass distribution of the antiplatelet antibodies present in patients with ATP as compared with patients with SLE. While major attention has focused on structural and antigenic differences between the IgG subclasses, using myeloma proteins as models (16), more is being learned about the subclass distribution of IgG antibodies. Some antibody activities appear to be distributed within the yG subclasses in a manner proportional to the distribution of the subclasses in normal sera. These include antibodies directed against thyroglobulin (17) and the antinuclear antibodies (ANA) in patients with SLE, rheumatoid arthritis, and drug-induced SLE (18). 0th er antibodies are present in a more selective manner. Antibodies to nucleoprotein tend to be in the yG1 and rG3 subclasses (which fix complement most efficiently) in those SLE patients with nephritis but in the yG2 and yG4 subclasses in patients without nephritis (19). Antibodies to DNA are primarily of the yG1 and yG3 subclasses (20). The circulating “anticoagulant” antibodies to coagulation factor VIII (found in hemophilia, SLE, and postpartum patients) are found primarily in the subclass of lowest concentration (3-4%) in normal serum, yG4 (21 and 22). Anti-A red blood cell isoagglutinins are primarily of the yG1 subclass (23) while anti-Rh antibodies are mainly yG1 and rG3 (24,25). Antibodies to diphtheria and tetanus toxoid are primarily yG1 (23), whereas antibodies to dextran, levan, and teichoic acid appear predominantly in the yG2 subclass (23). A number of other antibodies have been shown to exhibit some subclass restriction. The details have been reviewed elsewhere (64). Thus, studies on a number of antibodies indicate that many of them are found predominantly in one or in a restricted number of subclasses. On the basis of the normal serum distribution of these immunoglobulin subclasses, one would expect antibody to be present predominantly in the rG1 and in a somewhat lower concentration in the yG2 subclasses. However, when antibodies are present in only one or two subclasses, or if rG2, yG3, or yG4 are overrepresented proportional to their serum concentration, a restriction either
8
KARI’ATMN
ET
AL.
on a genetic or other basis of the antibody response to the particular antigens is suggested. The mechanism for control of this aspect of the immune response is not at present understood. It was shown in this study that the antiplatelet antibodies synthesized by patients with ATP tend to be restricted to the yG3 subclass. Gamma G3 comprises only 4-7% of the total IgG and has the shortest half-life and highest catabolic rate of the four yG subclasses (26,27). It also aggregates readily (28) and fixes complement avidly. The relationship between these properties of yG3 and the biologic potential of antiplatelet antibodies remains uncertain. However, it is conceivable that this propensity for aggregation and complement fixation may play a significant role in the clearance of platelets from the circulation of patients with ATP. ACKNOWLEDGMENTS We greatly appreciate and Miss L. Wolfson.
the excellent
technical
assistance
of Miss M. Monroe
REFERENCES 1. KARPATKIN, S., AND SISKIND, G. W., Blood 33, 795, 1969. 2. KARPATKIN, S., STRICK, N., KARPATKIN, M. H., AND SISUND, G. W., Amer. J. Aled. 52, 776, 1972. S., STRICX, N., AND SISI
IMMUNOLOGY
AND
2, 1-8 (1973)
IMMUNOPATHOLOGY
Heavy Chain Subclass of Human Anti-Platelet Antibodies] SIMON KARPATKIN,~ NATHAN
STRICK,
AND
PETER H. GREGORY
SCHUR,
W. SISKIND~
Department of Medicine, New York University Medical School; Department of Medicine, Harvard Medical School; and Division of Allergy and Immunology, Department of Medicine, Cornell University Medical College, New York, New York 10021 Received
January
5, 1973
The IgG subclass distribution of the antiplatelet antibodies present in several clinical conditions was determined. Studies were carried out on antiplatelet antibodies present in patients with autoimmune thrombocytopenic purpura, systemic lupus erythematosis, drug-induced immunologic thrombocytopenic purpura, and after multiple transfusions. The antiplatelet antibodies in autoimmune thrombocytopenic purpura appeared to be restricted to the rG3 subclass in every one of 15 patients studied. In contrast, nine patients with systemic lupus erythematosis who had antiplatelet antibodies were found to have antiplatelet antibodies detectable in three out of four or in all four subclasses. Different patterns of subclass disdibution were observed in the other clinical conditions studied. The subclass distribution of antiplatelet antibodies may be of assistance in distinguishing systemic lupus erythematosis from autoimmune thrombocytopenic purpura.
Previous communications from this laboratory have pointed out the usefulness of the platelet factor 3 (PF-3) immunoinjury technique for the detection of antiplatelet antibody in various clinical disorders (l-4)j. Employing this method, antiplatelet antibody could be detected in the serum of a high percentage of patients with chronic idiopathic autoimmune thrombocytopenic purpura (ATP), systemic lupus erythematosis (SLE), and drug-induced immunologic thrombocytopenic purpura. The relative nonspecificity of this immunoinjury technique required stringent proof that the antiplatelet factor demonstrated was indeed an autoantibody with platelet specificity. This was established by the following observations: 1. The antiplatelet activity was removed by addition of rabbit anti-human immunoglobulin G (RAHIgG) (l-3)
’ Supported Health Service ford Foundation. ? Career
by grants from (HE 1336, AM
Scientist
the New York Heart 11414, AM 05577, AM
of the Health
Research
Council
Association, from the United States Public 12051, FR 05669) and from the John A. Hartof the City
of New
York
(I-453).
R Career Scientist of the Health Research Council of the City of New York (I-593). ’ Confirmed in several laboratories: H. Horowitz, New York; S. Murphy, Philadelphia; R. Aster, Milwaukee; A. Tartaglia, Albany; J. Dodds, Albany; B. Baer, Kansas City; D. H. Cowan, Cleveland; R. Hirschman, N. I. H.; B. Firkin, Prahran, Australia, J. Caen, Paris; (quoted with permission of investigators). Copyright All rights
@ 1973 by Academic Press, Inc. of reproduction in any form reserved.
I
2
KARPATKIN
ET
AL.
but not by addition of rabbit antibodies to human immunoglobulins A, M, or D. 2. Antiplatelet activity could be absorbed with washed human platelets (l-3) but not with washed human granulocytes or red blood cells. 3. The antiplatelet factor could be eluted from platelets to which it had been adsorbed (l-3) and the antiplatelet activity of the eluted material removed with RAHIgG (l-3). 4. The antiplatelet factor was stable at 56°C (1). 5. It was shown that the serum antiplatelet factor and the antiplatelet activity eluted from platelets to which it had been adsorbed both have a molecular weight indistinguishable from that of IgG (1, 5). Human IgG has been shown to consist of four subclasses, rG1, rG2, yG3, and yG4, which differ not only in structure and relative serum concentrations (yG1: 64-70%, yG2: 23-28%, yG3: 4-7%, yG4: 3-4%), but also in their biological properties (6). The heavy-chain subclass of the antiplatelet antibodies in various clinical disorders was, therefore, studied to ascertain whether there was any relation between yG subclass and diagnosis. This has been examined in patients with ATP, SLE, drug-induced immunologic thrombocytopenic purpura, and a history of numerous blood or platelet transfusions. A unique subclass distribution of the antiplatelet antibodies was found for ATP patients. These patients appear to have their antiplatelet antibodies restricted to the yG3 subclass. This characteristic appears to distinguish ATP patients from other patients with antiplatelet antibodies. MATERIALS
AND
METHODS
Specific antisera directed against human heavy-chain subclasses and lightchain types were developed in monkeys and rabbits (6-8). The antisera were absorbed as necessary to make them specific with proteins coupled to Sepharose (9) or to bromacetylcellulose (BAC) (10). Specificity was determined by immunoelectrophoresis, double diffusion in agar, and radial immunodiffusion (11, 12). Antisera were then chromatographed on Sephadex G200 and the second, 7s, peak isolated and concentrated. These fractions were then tested by their ability to specifically agglutinate cells coated with myeloma proteins of the appropriate subclass (8). The antisera were equally potent in their ability to agglutinate specifically sensitized red blood cells (B), but the anti-yG3 was somewhat more potent when employed in radial immunodiffusion (7). The PF-3 immunoinjury technique was performed with globulin fractions of control and test sera on normal platelet-rich plasma in the presence of contact product as described previously (l-3). A lo-set or greater shortening of the fibrin clotting time was considered significant. Antiplatelet activity was titered using normal human globulin as diluent. Based upon our previous experience with this assay (l-3), a titer of greater than 1:4 is regarded as positive. Globulin fractions of sera to be tested and of rabbit antisera to human IgG were prepared by precipitation at 50% cold saturated (NHJ2S04. The globulin fractions were collected by centrifugation, dissolved in and extensively dialyzed against phosphate-buffered saline (PBS: 0.15 NaCI, 0.01 M potas-
ANTI-PLATELET
ANTIBODY
3
sium phosphate buffer, pH 7.4). All globulin fractions were reconstituted to their original serum volume with PBS. The final protein concentration was roughly 15 mglml as determined by the 280:260-rnp absorbancy (13). The immunoglobulin subclass of the patient’s antiplatelet antibody was determined by assaying the ability of subclass-specific antibodies to remove PF-3 activity from the globulin fraction of the patient’s serum. One tenth milliliter of test or control globulin was incubated with lo-40 ~1 of a globulin fraction of rabbit antiserum specific for one of the IgG subclasses or with normal rabbit globulin for 60 min at 37°C and then held at 4°C overnight. Any precipitate formed was removed by centrifugation at 2000g for 15 min at 4°C and the supernate was tested for PF-3 activity. The results are reported as the reciprocal of the highest dilution of the sample giving a positive result (shortening of the fibrin clotting time by 10 set or more) in the PF-3 test. Five samples of ATP sera and five samples of SLE sera were tested with 40 ~1 of anti-subclass antisera. In no case did 40 ~1 of anti-immunoglobulin serum depress the PF-3 titer to a greater extent than did 30~1. Therefore, use of 30~1 was adopted as a standard procedure. One cannot be certain that all IgG of the particular subclass is removed by this procedure. Addition of greater amounts of anti-subclass antisera did not cause any further decrease in antiplatelet antibody activity. The anti-subclass antisera used were prepared in rabbits or monkeys and are of the “precipitating” type which do not tend to give an inhibition zone in antibody excess. The results reported here are based upon addition of 30 ~1 of globulin fraction of rabbit anti-human immunoglobulin antiserum to 0.1 ml of the globulin fraction of the patient’s serum being studied. The results were found to be highly reproducible even in experiments in which samples were handled under “coded” conditions. It should be recognized that antiplatelet antibody titers are only semiquantitative measurements and, therefore, are not linear functions of antibody concentration. Accordingly, we would not expect summation of titer changes to be quantitative. Fifteen patients were studied who had classical clinical histories and hematological findings of chronic ATP (14, 15) with two or more episodes of thrombocytopenia and purpura over a period greater than 6 months. Some had undergone splenectomy (DeM, Bad, Tur) and some were in “clinical remission” (DeM, Bad, Tur, Soy, Min) at the time when serum samples used in this study were obtained. Nine patients with typical clinical and laboratory findings of SLE who were being followed in the Bellevue Hospital SLE clinic were studied. All of these patients had positive LE cell preparations and ANA tests on one or more occasion. None was thrombocytopenic at the time of study. Five patients with drug-induced immunologic thrombocytopenic purpura had both clinical and laboratory evidence for drug-induced thrombocytopenia and have been described in detail elsewhere (4). Laboratory confirmation of the diagnosis was obtained by the addition of the suspected drug to the PF-3 test with a consequent increase in the titer of antiplatelet antibody activity or a change from a negative to a positive PF-3 test. Two patients were studied who had had numerous blood or platelet transfusions. One of these
4
KAFWATKIN
CHnoNIc Patient DeM Qui Bad Han Ben Mar Kil Cor Tur Pal McG SOY Min McM Ale
+NRG 24 40 40 48 32 24 20 20 20 40 16 12 28 32 20
+anti-yG1 24 40 40 48 32 20 20 20 20 - 1’
AUTCXMMUNE tanti-yG2
ET
AL.
TABLE 1 THROMBOCYTOPENIC +anti-yG3 0 0 0 0 0 0 0 0 0 4 0 0 0 0 4
24 36 40 48 32 20 20 20 20 40 16 12 28 32 20
PUHPunA” +anti-yG4
+anti-K
+anti-A
24 40 40 48 32 20 20 20 20
4
4 20 20 8 8 -
40 16 12 28 32 20
16 16 16 8 -
-
” Antiplatelet antibody titer following treatment with anti-heavy chain subclass antibody or anti-light-chain antibody. One-tenth milliliter of globulin fraction of patient’s serum was incubated with 30 ~1 of normal rabbit globulin (NRG) or anti-heavy-chain subclass antibody or anti-light-chain-type antibody. Any precipitate formed was removed and the supernate titered for PF-3 activity. The results are reported as the reciprocal of the highest dilution of the sample giving a positive test. ’ Six of the 15 samples studied were not evaluated with anti-yG1 because no anti-yG1 antisera was available at the time these patients were studied.
was a patient with aplastic anemia (Gar) and the second was a patient who had received multiple whole-blood exchange transfusions and platelet transfusions for infectious hepatitis with hepatic coma (Cap). RESULTS Table 1 gives the results of studies on the immunoglobulin subclass of the antiplatelet antibody present in 15 patients with chronic ATP. In all 15 cases the antiplatelet activity was removed by addition of anti-human yG3. Addition of rabbit antibody specific for either human kappa or lambda light chains resulted in a drop in PF-3 titer suggesting the presence of both light-chain types in the antiplatelet antibody. In Table 2 is presented the results of studies on nine patients with SLE. In every case the antiplatelet antibodies were found to be present in three of the four or in all four IgG subclasses. Antiplatelet antibody was not restricted to a single subclass as it appeared to be in all ATP patients. The antiplatelet antibody titers in SLE patients tended to be higher than in ATP patients. This difference in titers could conceivably make detection of antiplatelet antibody in the yG1, 2, and 4 subclasses more sensitive in the SLE group. However, it
ANTI-PLATELET
Patient __.__-
5
SYSTEMIC
TABLE 2 LUPUS ERYTHEMATOSUS”
+anti-$1
+anti-yG2
+NRG ____
Geo Nun He1 McK sot Cru Bai Wil Hir
ANTIBODY
60 60 48 48 40 20 16 80 48
+anti-yG4
+anti-yC3
20 12 48 48 40 4 16 80 8
32 24 24 8 8
-
20 12 16 8 8 4 8 12 8
fl Antiplatelet antibody titer after treatment with anti-heavy-chain subclass antibody. One tenth milliliter of globulin fraction of patient’s serum was incubated with 30~1 of normal rabbit globulin (NRC) or anti-heavy-chain subclass antibody. Any precipitate formed was removed and the supernate titered for PF-3 activity. The results are reported as the reciprocal of the highest dilution of the sample giving a positive test.
TABLE DRUG-INDUCED Patient Age Vit Zil Lo1 Go1
Drug
IMMUNOLOGIC fanti-yG1
+anti-yG2
+anti-yG3
+anti-yG4 __.
48 16 16 44 32
20 8 16 20 12
4 0 0 0 0
8 0 0 4 4
48 16 16 44 32
antibody titer after treatment with anti-heavy-chain subclass antibody. One of globulin fraction of a patient’s serum was incubated with 30 ~1 of normal (NRG) or anti-heavy-chain subclass antibody. Any precipitate formed was resupernate titered for PF-3 activity in the presence of 0.1 mM of drug. The results the reciprocal of the highest dilution of the sample giving a positive test.
NUMEROUS Patient
Cap Gar
PURPURA”
+NRG
Quinidine Quinidine Quinidine Tegretol Thioguanine
CLAntiplatelet tenth milliliter rabbit globulin moved and the are reported as
3 THROMBOCYTOPENIC
TABLE 4 OR PLATELET
BLOOD
+NRG
+anti-yG
80 32
80 32
1
+anti-yG2 20 4
TRANSFUSIONS~ +anti-yG3 8 0
+anti-yG4 16 4
U Antiplatelet antibody titer after treatment with anti-heavy-chain subclass antibody. One tenth milliliter of globulin fraction of patient’s serum was incubated with 30 4 of normal rabbit globulin (NRG) or anti-heavy chain subclass antibody. Any precipitate formed was removed and the supernate titered for PF-3 activity. The results are reported as the reciprocal of the highest dilution of the sample giving a positive test.
6
KAFWATKIN
ET
AL.
should be noted that three SLE samples with titers of 40 or less showed a pattern similar to that in higher titered SLE patients. These samples were in the same titer range as were many of our ATP subjects. One patient(Con) was not included in either Table 1 or 2 because of uncertainty as to the correct diagnosis. The patient was a woman with no history of thrombocytopenia or purpura who gave birth to a thrombocytopenic infant. She had a normal platelet count and her antinuclear antibody titer was nondiagnostic. She was found to have a strongly positive PF-3 test and her antiplatelet antibody titer could be reduced by addition of antibody to each of the four IgG subclasses. Thus, the IgG subclass pattern of her antiplatelet antibody was similar to that usually seen in patients with SLE. Patients with drug-induced thrombocytopenic purpura have antiplatelet antibody present in two or three of the IgG subclasses as illustrated in Table 3. In no case was antiplatelet activity removed by addition of anti-yG4. In Table 4 are results obtained in studies on two patients who had antiplatelet antibody presumably as a consequence of multiple blood or platelet transfusions. In contrast to any of the above conditions antibody activity was detected in all IgG subclasses except yG1. DISCUSSION The results reported here indicate a rather striking restriction of the antiplatelet antibody response of ATP patients to the yG3 immunoglobulin subclass. It is of note that this subclass normally comprises only 4-7% of the total IgG (6,7). This is in marked contrast to the situation in patients with SLE who have antiplatelet antibodies detectable in at least three of the four IgG subclasses and often in all four subclasses. The antiplatelet antibody in ATP did not appear to be monoclonal since both antikappa and antilambda antisera partially removed the antiplatelet antibody activity. The possible diagnostic usefulness of these observations in distinguishing patients with ATP from those with SLE is obvious. It will be of interest to determine ultimately whether this distinction in the subclass distribution of antiplatelet antibodies will permit identification of those patients with chronic ATP who will eventually develop SLE. It should be emphasized that study of larger numbers of patients with chronic ATP who will eventually develop SLE. It should be emphasized that study of larger numbers of patients may reveal exceptions to the simple patterns observed here. Furthermore, the failure to detect antibodies in certain immunoglobulin subclasses may reflect not their absolute absence but merely the relative insensitivity of our detection methods. In any case there is certainly a quantitative if not a qualitative difference in the IgG subclass distribution in patients with ATP as compared to those with SLE. The data obtained regarding the antiplatelet antibody from patients with drug-induced immunologic thrombocytopenic purpura and patients with numerous blood or platelet transfusions are too limited to permit any de&itive conclusions. However, even the limited data do suggest that these conditions may also be associated with predictable patterns of IgG subclasses in the
ANTI-PLATELET
ANTIBODY
7
antiplatelet antibody response. Incomplete removal of IgG subclasses, or formation of antigen antibody complexes, could potentially produce errors in the assay system. Errors introduced in these ways would tend to result in release of PF-3 from platelets: that is, an apparent failure to remove antiplatelet activity. Since the basic observations reported here represent a depletion of PF-3 releasing activity, errors due to the above possibilities could not account for the data presented. It should be noted in addition, that significant depletion of PF-3 activity was observed upon treatment of sera from SLE patients with antisera to IgGl, rG2, and rG4. This clearly indicates that these antisera and the procedures used are capable of removing PF-3 releasing activity when the antiplatelet antibodies are present in these subclasses. The failure of these antisera to decrease antiplatelet activity of sera from ATP patients must be considered together with the complete elimination of antiplatelet activity from these sera with anti-IgG3. These observations can be explained only by assuming that there is at least a quantitative difference in the subclass distribution of the antiplatelet antibodies present in patients with ATP as compared with patients with SLE. While major attention has focused on structural and antigenic differences between the IgG subclasses, using myeloma proteins as models (16), more is being learned about the subclass distribution of IgG antibodies. Some antibody activities appear to be distributed within the yG subclasses in a manner proportional to the distribution of the subclasses in normal sera. These include antibodies directed against thyroglobulin (17) and the antinuclear antibodies (ANA) in patients with SLE, rheumatoid arthritis, and drug-induced SLE (18). 0th er antibodies are present in a more selective manner. Antibodies to nucleoprotein tend to be in the yG1 and rG3 subclasses (which fix complement most efficiently) in those SLE patients with nephritis but in the yG2 and yG4 subclasses in patients without nephritis (19). Antibodies to DNA are primarily of the yG1 and yG3 subclasses (20). The circulating “anticoagulant” antibodies to coagulation factor VIII (found in hemophilia, SLE, and postpartum patients) are found primarily in the subclass of lowest concentration (3-4%) in normal serum, yG4 (21 and 22). Anti-A red blood cell isoagglutinins are primarily of the yG1 subclass (23) while anti-Rh antibodies are mainly yG1 and rG3 (24,25). Antibodies to diphtheria and tetanus toxoid are primarily yG1 (23), whereas antibodies to dextran, levan, and teichoic acid appear predominantly in the yG2 subclass (23). A number of other antibodies have been shown to exhibit some subclass restriction. The details have been reviewed elsewhere (64). Thus, studies on a number of antibodies indicate that many of them are found predominantly in one or in a restricted number of subclasses. On the basis of the normal serum distribution of these immunoglobulin subclasses, one would expect antibody to be present predominantly in the rG1 and in a somewhat lower concentration in the yG2 subclasses. However, when antibodies are present in only one or two subclasses, or if rG2, yG3, or yG4 are overrepresented proportional to their serum concentration, a restriction either
8
KARI’ATMN
ET
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on a genetic or other basis of the antibody response to the particular antigens is suggested. The mechanism for control of this aspect of the immune response is not at present understood. It was shown in this study that the antiplatelet antibodies synthesized by patients with ATP tend to be restricted to the yG3 subclass. Gamma G3 comprises only 4-7% of the total IgG and has the shortest half-life and highest catabolic rate of the four yG subclasses (26,27). It also aggregates readily (28) and fixes complement avidly. The relationship between these properties of yG3 and the biologic potential of antiplatelet antibodies remains uncertain. However, it is conceivable that this propensity for aggregation and complement fixation may play a significant role in the clearance of platelets from the circulation of patients with ATP. ACKNOWLEDGMENTS We greatly appreciate and Miss L. Wolfson.
the excellent
technical
assistance
of Miss M. Monroe
REFERENCES 1. KARPATKIN, S., AND SISKIND, G. W., Blood 33, 795, 1969. 2. KARPATKIN, S., STRICK, N., KARPATKIN, M. H., AND SISUND, G. W., Amer. J. Aled. 52, 776, 1972. S., STRICX, N., AND SISI