Different Antiplatelet Antibody Specificities in Immune Thrombocytopenic Purpura R. L. DONNELL,* R. MCMILLAN, R. J. YELENOSKY, R. L. LONGMIRE AND A. L. LIGHTSEY* L. C.](ICO~)SOIZ Blood Ccriter, Scriyys C h i c arid Rcscarch Forrndatiori, LaJolia, Cal$mia, and * U.S. N a v a l Hospital, Sari Diego, California, U.S.A. (Rcceivcd 37 October 1975; accepted for publicatiorz 15 December 1975) SUMMARY. It is generally accepted that patients with immune thrombocytopenic purpura (ITP) produce antibody against platelet-associated antigens; however, it is not known if these antiplatelet antibodies are directed towards the same or different antigenic sites. In the present studies, quantities ofantiplatelet antibody from different ITP patients, sufficient to saturate platelet antigenic sites, were simultaneously incubated with normal platelets and the quantity of platelet-binding IgG (PBIgG) was determined. In each of the five comparisons made, the amount of PBIgG bound after incubation of normal platelets with saturating quantities of two ITP antibodies approximated to thc sum of the PBIgG bound after incubation with the antibodies separately. These data suggest that the antiplatelet antibody from thesc ITP patients differed in antigenic specificity. Immune thrombocytopenic purpura (ITP) is a syndrome of unknown cause nianifested by thrombocytopenia due to platelet sensitization with an IgG antiplatelet antibody and subsequeiit destruction by the reticuloendothelial system (Baldini, 1966; Harrington ct a / , 1953; Karpatkin, 1971 ; McMillaii et al, 1974a, b; Shulman et a], 1965). The presence of a plateletassociated antigen in this disease seems clear (Handin et a!, 1973; Piessens et a], 1970; Wybran & Fudenberg, 1972) but it is not known if the antigenic sites differ from patient to patient. hi the present studies, the inability of antigen-saturating amounts of splenic produced antiplatelet antibody from one patient with ITP to block binding by antibody from a second patient suggests that the platelet antigenic determinants in the patients studied were different.
METHODS
We have previously shown that IgG produced in culture by splenic cells from ITP patients binds to normal platelets and that under conditions of excess platelet-binding IgG (PBIgG), saturation of platelet-associated antigenic sites occurs (McMillan et al, 1974b). The methods of tissue handling and culture and the quantitation of PBIgG have been previously described in detail (McMillan ct ul, 1972, 1974b). Multiple cultures from six patients were individually pooled and the globulins precipitated with an equal volume of saturated ammonium sulphate. Correspondence: Dr R. McMillaii, Department of Hematology-Oncology, Scripps Clinic and Research Foundation, 476 Prospect Strcct, La Jolla, California 92037, U.S.A.
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R. L.Donne11 et a1
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After dialysis against distilled water and tlien phosphate-buffered saline (pH 7.4, the IgG was concentrated to approximately 30 pg/ml.
Determiiiatioii ojthe ‘Satitrating Quantity’ of PBIgG Duplicate aliquots containing graded amounts of splenic produced IgG from ITP patients (3-60 pg IgG) were incubated in separate tubes with 5 x 108 washed normal platelets (three washes with 0.05 M citrate buffer, pH 6.2; two washes with saline, 25’C) in 0.5 ml saline. After overnight incubation at 5’C, 3.5 ml of cold saline were added and the mixture was centrifuged for 15 niin at 36 ooo g (5OC).The cell button and tube were washed twice with cold saline without disturbing the cells and recentrifuged. After washing, the platelets were suspended in exactly 4 ml of saline and lysed by freezing and thawing five times. Appropriate controls were performed simultaneously. The platelet-associated IgG was determined using the Fab-anti-Fab IgG assay system (McMillan et al, 1972, 1g74b).The anti-Fab antibody was standardized so that 0.5 ml precipitated 50% of the radioactivity in 0.5 ml of an antigen solution containing 20.0 ng of nitrogen 1251Fab. The ability of the test materials to inhibit this reaction was compared to known amounts of pooled, normal human IgG. Platelet-binding IgG was calculated by the difference between ITP and control values and expressed as ng IgG per 109 platelets. The variation (& SD) of the method in replicate experiments was 8.3 k 4.8%. The PBIgG results were plotted against the quantity of IgG in each incubation and a curve .was constructed for each patient (Fig I). The antigen ‘saturating quantity’ of each patient’s PBIgG was estimated from the plateau portion of the curve.
200 120 Quontity of IgG incuboted (pg/109 plofelefs)
FIGI. Determination of the quantity of platelet-binding IgG (PBIgG) required to saturate the plateletassociated antigen sites. Graded amounts (3-60 fig) of IgG produced in culture by splenic cells from ITP patients were incubated separately with 5 x 10’platelets overnight at 5°C. After ultracentrifugation and washing, the PBIgG of each sample (ng Ig/109 platelets) was determined and plotted against the quantity of IgG incubated (pg/109 platelets). The ‘saturating quantity’ of PBIgG was estimated from the plateau portion of the curve.
Antibody Specijicities in ITP (a) Diffcrenl plateletossociad omtipens
I49
(b) Identical or closely opproximoted plotelet-associotedaMqcns
n
Antibody
B
Antiplotelet antibody incubated
FIG 2. Schematic representation of the predicted results which would be obtained if ‘saturating quantities’ of PBIgG were incubated with target platelets. If the antigenic determinants are different, PBIgG results of incubations containing ‘saturating quantities’ of antiplatelet antibody from two ITP patients should equal the s u m of the individual incubations (Fig 2 a ) ; on the other hand, if the antigenic sites were identical or closely approximated no additive effect would be seen (Fig zb).
Hypothesis and Experimental Design If the platelet-associated antigenic sites in patients with ITP varies, then incubation of normal platelets with saturating quantities of splenic PBIgG from two different ITP patients should result in PBIgG values equal to the sum of the results obtained from incubations containing the individual samples (Fig 2a); conversely, if a common antigen is present, no additive effect would be expected (Fig 2b). Six ITP splenic IgG concentrates were compared. In each comparison, washed platelets from the same O+ donor (3 x 10’) were incubated in solutions containing one of the following: (I) 1.5 times the saturating quantity of PBIgG from patient A, (2) 1.5 times the saturating quantity ofPBIgG from patient B, or (3) 1.5 times the saturating quantities of PBIgG from both patients A and B. Incubation conditions, processing and determination of PBIgG values were performed as described above.
RESULTS The results of five comparisons between splenic IgG concentrates from six ITP patients are shown in Table I. Columns A and B give the PBIgG results after incubation with saturating quantities of IgG from the sera of the individual patients. The middle column is the sum of columns A and B arid is the result predicted ifthe platelet antigenic sites are different. Column 4 shows the results from incubations containing saturating quantities of PBIgG from both patients being compared. In every case, PBIgG values derived from incubations containing both ITP concentrates (column 4) approximated the sum of the individual values (colun~n3). The mean per cent of the predicted additive effect was 82% (range 74-100).
R.L. Donne11 et a1 TABLE I. Comparative antiplatelet antibody specificities of platelet-binding IgG (ng IgG/109 platelets)
A*
B*
Dredictrdt A+B
Observed$ A+B
Per cent predicted
2767 3194 2163 1688 I688
3038 1688 1688 907 982
5805 4882 3851 2595 2670
4306 4882 2782
I00
2332
2544
74 72 90 9s
* Platelet-binding IgG after incubation of target platelets with 1.5 x the saturating amount of the individual antiplatelet antibodies. t Calculated by adding the results of columns I and 2. $ Platelet-bindingIgG after incubation of target platelets with both antibodies simultaneously. DISCUSSION These studies provide evidence that the splenic-produced antiplatclet antibody from these ITP patients is directed towards different platelet-associated antigen sites. If the antigenic sites had been identical or closely approximated, blocking of antibody binding should have occurred. The fact that the PBIgG values after the simultaneous incubation of platelets with antigen saturating quantities of antibody from two ITP patients approximated to the sum of individual incubations provides evidence that the antigenic sites were not only different but were also so spaced that steric hindrance was minimal. A similar conclusion was reached by other investigators (Karpatkin & Siskind, 1975) who noted that the platelets from different donors exposed to ITP sera released differing amounts of platelet factor 3 . Whether these platelet antigenic sites are on different molecules or on remote positions of the same molecule cannot be determined from our studies. ACKNOWLEDGMENTS
These studies were supported by grants AM-16125, AM-16994 and CA-13465 from the U.S. Public Health Service. We are indebted to Mrs Sherry Ryan, Miss Pam Gordon, Mrs Barbara Clark and Miss Paige Gilmaii for their assistance. REFERENCES
M. (1966) Idiopathic thronibocytopenic BALDINI, purpura. Neiv England Jonrrial of Medicine, 274, 1245, 1301, 1360.
HANDIN,R.L, PIESSENS, W.F. & MOLONEY, W.C. (1973) Stimulation of noninimunizcd lymphocytes by platelet-antibody complexes in idiopathic thrombocytopenic purpura. New England]orrrna\ of’ Medicine, 289, 714.
HARRINGTON, W.J., SPRAGUE, C.C., MINNICH,V.* MOORE, C.V., AULVIN,R.C. & DUBACH, R. (1953) Iminunologic mechanisms in idiopathic and neonatal thrombocytopenic purpura. Annals of Internal Medicine, 38, 433. KARPATKIN, S. (1971)Autoimmune throinbocytopenic purpura. Anierican journal of the Medical Sciences, 261, 127.
Antibody Specificities in ITP KARPATKIN. S. & SISKIND, G.W. (1975) Studies on the specificity of anti-platelet autoantjbodies. Proceedings of the Society j b r Experimental Biology and Medicine, 147. 71.5. MCMILLAN,R., LONGMIRE, R.L., YELENOSKY, R., LANG,J.E., HEATH,V. & CRADDOCK, C.G. (1972) Immunoglobulin synthesis by human lymphoid tissues: normal bone marrow as a major site of IgG production. Journal oflrnrnrmology, 10g. 1386. MCMILLAN,R., LONGMIRE, R.L., TAVASSOLI, M., ARMSTRONG, S. & YELENOSKY, R. (1g74a) In vitro phagocytosis by splenic leukocytes in idiopathic thrombocytopenic purpura. N e w England Journal of Medicine, 290, 249. MCMILLAN,R., LONGMIRE, R.L., YELENOSKY, R., S. (1974b) QuantitaDONNELL, R.L. & ARMSTRONG, tion of platelet-binding IgG produced in vitro by
spleens from patients with idiopathic thrombocytopenic purpura. N e w England Journal qfMedicine, 291, 812. PIFSSENS, W.F., WYBRAN, J., MANASTER, J. & STRIJCKMANS, P.A. (1970) Lymphocyte transformation induced by autologous platelets in a case of thrombocytopenic purpura. Blood, 36, 421. SHULMAN,N.R., MARDER,V.J. & WEINRACH, R.S. (1965) Similarities between known antiplatelet antibodies and the factor responsible for thrornbocytopenia in idiopathic purpura. Physiologic, serologic and isotopic studies. Annals of the New York Academy of Sciences, 124, 499. WYBRAN, J. & FUDENBERG, H.H. (1972) Cellular iininunity to platelets in idiopathic thrombocytopenic purpura. Blood, 40, 856.
METHODS
We have previously shown that IgG produced in culture by splenic cells from ITP patients binds to normal platelets and that under conditions of excess platelet-binding IgG (PBIgG), saturation of platelet-associated antigenic sites occurs (McMillan et al, 1974b). The methods of tissue handling and culture and the quantitation of PBIgG have been previously described in detail (McMillan ct ul, 1972, 1974b). Multiple cultures from six patients were individually pooled and the globulins precipitated with an equal volume of saturated ammonium sulphate. Correspondence: Dr R. McMillaii, Department of Hematology-Oncology, Scripps Clinic and Research Foundation, 476 Prospect Strcct, La Jolla, California 92037, U.S.A.
I47
R. L.Donne11 et a1
148
After dialysis against distilled water and tlien phosphate-buffered saline (pH 7.4, the IgG was concentrated to approximately 30 pg/ml.
Determiiiatioii ojthe ‘Satitrating Quantity’ of PBIgG Duplicate aliquots containing graded amounts of splenic produced IgG from ITP patients (3-60 pg IgG) were incubated in separate tubes with 5 x 108 washed normal platelets (three washes with 0.05 M citrate buffer, pH 6.2; two washes with saline, 25’C) in 0.5 ml saline. After overnight incubation at 5’C, 3.5 ml of cold saline were added and the mixture was centrifuged for 15 niin at 36 ooo g (5OC).The cell button and tube were washed twice with cold saline without disturbing the cells and recentrifuged. After washing, the platelets were suspended in exactly 4 ml of saline and lysed by freezing and thawing five times. Appropriate controls were performed simultaneously. The platelet-associated IgG was determined using the Fab-anti-Fab IgG assay system (McMillan et al, 1972, 1g74b).The anti-Fab antibody was standardized so that 0.5 ml precipitated 50% of the radioactivity in 0.5 ml of an antigen solution containing 20.0 ng of nitrogen 1251Fab. The ability of the test materials to inhibit this reaction was compared to known amounts of pooled, normal human IgG. Platelet-binding IgG was calculated by the difference between ITP and control values and expressed as ng IgG per 109 platelets. The variation (& SD) of the method in replicate experiments was 8.3 k 4.8%. The PBIgG results were plotted against the quantity of IgG in each incubation and a curve .was constructed for each patient (Fig I). The antigen ‘saturating quantity’ of each patient’s PBIgG was estimated from the plateau portion of the curve.
200 120 Quontity of IgG incuboted (pg/109 plofelefs)
FIGI. Determination of the quantity of platelet-binding IgG (PBIgG) required to saturate the plateletassociated antigen sites. Graded amounts (3-60 fig) of IgG produced in culture by splenic cells from ITP patients were incubated separately with 5 x 10’platelets overnight at 5°C. After ultracentrifugation and washing, the PBIgG of each sample (ng Ig/109 platelets) was determined and plotted against the quantity of IgG incubated (pg/109 platelets). The ‘saturating quantity’ of PBIgG was estimated from the plateau portion of the curve.
Antibody Specijicities in ITP (a) Diffcrenl plateletossociad omtipens
I49
(b) Identical or closely opproximoted plotelet-associotedaMqcns
n
Antibody
B
Antiplotelet antibody incubated
FIG 2. Schematic representation of the predicted results which would be obtained if ‘saturating quantities’ of PBIgG were incubated with target platelets. If the antigenic determinants are different, PBIgG results of incubations containing ‘saturating quantities’ of antiplatelet antibody from two ITP patients should equal the s u m of the individual incubations (Fig 2 a ) ; on the other hand, if the antigenic sites were identical or closely approximated no additive effect would be seen (Fig zb).
Hypothesis and Experimental Design If the platelet-associated antigenic sites in patients with ITP varies, then incubation of normal platelets with saturating quantities of splenic PBIgG from two different ITP patients should result in PBIgG values equal to the sum of the results obtained from incubations containing the individual samples (Fig 2a); conversely, if a common antigen is present, no additive effect would be expected (Fig 2b). Six ITP splenic IgG concentrates were compared. In each comparison, washed platelets from the same O+ donor (3 x 10’) were incubated in solutions containing one of the following: (I) 1.5 times the saturating quantity of PBIgG from patient A, (2) 1.5 times the saturating quantity ofPBIgG from patient B, or (3) 1.5 times the saturating quantities of PBIgG from both patients A and B. Incubation conditions, processing and determination of PBIgG values were performed as described above.
RESULTS The results of five comparisons between splenic IgG concentrates from six ITP patients are shown in Table I. Columns A and B give the PBIgG results after incubation with saturating quantities of IgG from the sera of the individual patients. The middle column is the sum of columns A and B arid is the result predicted ifthe platelet antigenic sites are different. Column 4 shows the results from incubations containing saturating quantities of PBIgG from both patients being compared. In every case, PBIgG values derived from incubations containing both ITP concentrates (column 4) approximated the sum of the individual values (colun~n3). The mean per cent of the predicted additive effect was 82% (range 74-100).
R.L. Donne11 et a1 TABLE I. Comparative antiplatelet antibody specificities of platelet-binding IgG (ng IgG/109 platelets)
A*
B*
Dredictrdt A+B
Observed$ A+B
Per cent predicted
2767 3194 2163 1688 I688
3038 1688 1688 907 982
5805 4882 3851 2595 2670
4306 4882 2782
I00
2332
2544
74 72 90 9s
* Platelet-binding IgG after incubation of target platelets with 1.5 x the saturating amount of the individual antiplatelet antibodies. t Calculated by adding the results of columns I and 2. $ Platelet-bindingIgG after incubation of target platelets with both antibodies simultaneously. DISCUSSION These studies provide evidence that the splenic-produced antiplatclet antibody from these ITP patients is directed towards different platelet-associated antigen sites. If the antigenic sites had been identical or closely approximated, blocking of antibody binding should have occurred. The fact that the PBIgG values after the simultaneous incubation of platelets with antigen saturating quantities of antibody from two ITP patients approximated to the sum of individual incubations provides evidence that the antigenic sites were not only different but were also so spaced that steric hindrance was minimal. A similar conclusion was reached by other investigators (Karpatkin & Siskind, 1975) who noted that the platelets from different donors exposed to ITP sera released differing amounts of platelet factor 3 . Whether these platelet antigenic sites are on different molecules or on remote positions of the same molecule cannot be determined from our studies. ACKNOWLEDGMENTS
These studies were supported by grants AM-16125, AM-16994 and CA-13465 from the U.S. Public Health Service. We are indebted to Mrs Sherry Ryan, Miss Pam Gordon, Mrs Barbara Clark and Miss Paige Gilmaii for their assistance. REFERENCES
M. (1966) Idiopathic thronibocytopenic BALDINI, purpura. Neiv England Jonrrial of Medicine, 274, 1245, 1301, 1360.
HANDIN,R.L, PIESSENS, W.F. & MOLONEY, W.C. (1973) Stimulation of noninimunizcd lymphocytes by platelet-antibody complexes in idiopathic thrombocytopenic purpura. New England]orrrna\ of’ Medicine, 289, 714.
HARRINGTON, W.J., SPRAGUE, C.C., MINNICH,V.* MOORE, C.V., AULVIN,R.C. & DUBACH, R. (1953) Iminunologic mechanisms in idiopathic and neonatal thrombocytopenic purpura. Annals of Internal Medicine, 38, 433. KARPATKIN, S. (1971)Autoimmune throinbocytopenic purpura. Anierican journal of the Medical Sciences, 261, 127.
Antibody Specificities in ITP KARPATKIN. S. & SISKIND, G.W. (1975) Studies on the specificity of anti-platelet autoantjbodies. Proceedings of the Society j b r Experimental Biology and Medicine, 147. 71.5. MCMILLAN,R., LONGMIRE, R.L., YELENOSKY, R., LANG,J.E., HEATH,V. & CRADDOCK, C.G. (1972) Immunoglobulin synthesis by human lymphoid tissues: normal bone marrow as a major site of IgG production. Journal oflrnrnrmology, 10g. 1386. MCMILLAN,R., LONGMIRE, R.L., TAVASSOLI, M., ARMSTRONG, S. & YELENOSKY, R. (1g74a) In vitro phagocytosis by splenic leukocytes in idiopathic thrombocytopenic purpura. N e w England Journal of Medicine, 290, 249. MCMILLAN,R., LONGMIRE, R.L., YELENOSKY, R., S. (1974b) QuantitaDONNELL, R.L. & ARMSTRONG, tion of platelet-binding IgG produced in vitro by
spleens from patients with idiopathic thrombocytopenic purpura. N e w England Journal qfMedicine, 291, 812. PIFSSENS, W.F., WYBRAN, J., MANASTER, J. & STRIJCKMANS, P.A. (1970) Lymphocyte transformation induced by autologous platelets in a case of thrombocytopenic purpura. Blood, 36, 421. SHULMAN,N.R., MARDER,V.J. & WEINRACH, R.S. (1965) Similarities between known antiplatelet antibodies and the factor responsible for thrornbocytopenia in idiopathic purpura. Physiologic, serologic and isotopic studies. Annals of the New York Academy of Sciences, 124, 499. WYBRAN, J. & FUDENBERG, H.H. (1972) Cellular iininunity to platelets in idiopathic thrombocytopenic purpura. Blood, 40, 856.
