Letters to the editor
Platelet activation by antiplatelet autoantibodies in immune thrombocytopenic purpura To the editor: Immune thrombocytopenic purpura (ITP) is a syndrome caused by circulating antibodies that react with the platelet membrane. Antiplatelet autoantibodies, alloantibodies, and monoclonal antibodies (mAb) have all been reported to initiate platelet activation as measured by aggregation and the release reaction (1). Furthermore, some recent studies have shown that platelet activation by anti-glycoprdtein (GP)IIb/IIIa, anti-CD9, and anti-GPIV mAb is mediated by the FcyII receptor (2-5). We used ITP plasma to investigate IgG-mediated platelet activation and particularly aggregation mediated by the FcyII receptor. Between June 1988 and December 1991, we obtained plasma from 45 chronic ITP patients (12 men and 33 women) and 20 normal subjects. The ITP patients had thrombocytopenia with normal or increased numbers of megakaryocytes and no evidence of secondary immune thrombocytopenia. Platelet aggregation was studied using an NKK Hematracer 1. Blood from healthy volunteers was collected into tubes containing 3.8 % sodium citrate (9: 1, vol:vol), and platelet-rich plasma (PRP) was obtained after centrifugation at 200 g for 10 min. ITP or control plasma (50p1) was added to the PRP (200 pl) and platelet aggregation was monitored for 15 min. Then a goat anti-human IgG antibody (20 pg/ml) was added. In place of this goat antibody, 10 pmol/l ADP or 1.5 mg/ml ristocetin was used for control studies. PBIgG was detected as reported previously (6, 7). In brief, normal washed platelets were prepared and fixed in 1% paraformaldehyde. Then the fixed platelets were incubated with ITP plasma (50 pl) for 30 min at room temperature, followed by incubation with FITC-labeled goat anti-human IgG for 30 min at room temperature. After further washing, the samples were subjected to FACS analysis using operating conditions that have been published previously, (6,7). Detection of plasma autoantibodies directed against GPIIb/IIIa or GPIb was performed by an antigen-capture ELISA, the details of which have also been reported previously '(6, 7).
PBIgG was positive in 34 of the 45 ITP patients. Anti-GPIIb/IIIa autoantibodies were detected in 18 patients and anti-GPIb autoantibodies were found in 7 patients. None of the ITP plasma samples induced the aggregation of normal PRP. Furthermore, the addition of goat anti-human IgG also failed to induce aggregation. ADP-induced aggregation of normal platelets was inhibited by autoantibodies in 10/45 patients, and 8 of them had anti-GPIIb/IIIa antibodies. Ristocetin-induced agglutination was also inhibited in 2 of these patients with anti-GPIb antibodies. It is thought that ITP can occur as a result of several mechanisms (8). These include platelet activation by circulating immune complexes with crosslink platelets via the FcyII receptor. Recent investigators have shown that this can also occur with mAb directed against platelets CD9 (2) or platelets GPIIb/ IIIa (3-5). We investigated whether IgG in ITP plasma could induce platelet aggregation mediated by the FcyII receptor and found that none of the plasma samples used in this study could do so. It was previously reported that autoantibodies could affect platelet function in ITP patients (9, 10). We also found that some anti-GPIIb/IIIa antibodies inhibited ADP-induced platelet aggregation and that PBIgG was positive in these patients. Thus, the autoantibodies found in ITP plasma can bind to normal platelets. Some mAb directed against GPIIb/IIIa appear to cause platelet activation and some do not (1). The reason for this difference is presently unknown, but it may be related to epitope specificity. For ITP autoantibodies to induce platelet aggregation via the FcyII receptor would require the appropriate antigen specificity, e.g., antigen mobility as well as proximity between the antigen and the FcyII receptors, which comprise the majority of the IgG binding sites. If immune complexes of platelet-specific autoantibodies can induce aggregation via the FcyII receptor, this event may participate in the induction of thrombocytopenia in ITP. However, since the autoantibodies found in ITP patients are heterogeneous, this mechanism may not be so common. 109
Letters to the editor References 1. HORSEWOOD P, HAYWARD CPM, WARKENTIN TE, KELTON JG. Investigation of the mechanisms of monoclonal antibody-induced platelet activation. Blood 1991: 78: 10191026. RE, CARROLL RC, BOUCHEIX C. Platelet 2. WORTHINGTON activation by C D 9 monoclonal antibodies is mediated by the FcyII receptor. Br J Haematol 1990: 74: 216-222. 3. RUBINSTEIN E, KOUNSWC, JENNINGS LK, BOUCHEIX C, CARROLLRC. Interaction of two GPIIb/IIIa monoclonal antibodies with platelet Fc receptor (FcyRII). Br J Haematol 1991: 77: 80-86. 4. ANDERSON GP, VAN DE WINKELJGJ, ANDERSONCL. Anti-GPI Ib/llIa (CD41) monoclonal antibody-induced platelet activation requires Fc receptor-dependent cell-cell interaction. Br J Haematol 1991: 79: 75-84. 5. HORNBYEJ, BROWNS, WILKINSON JM, MATTOCKC, AUTHIKS. Activation of human platelets by exposure to monoclonal antibody, PM6/248, to glycoprotein IIb-11Ia. Br J Haematol 1991: 79: 277-285. 6. NOMURAS, YANABUM, KIDO H, et al. Antiplatelet autoantibody-related microparticles in patients with idiopathic (auto-immune) thrombocytopenic purpura. Ann Hematol 1991: 62: 103-107.
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7. NOMURAS, YANABUM, SOCAT, et al. Analysis of idiopathic thrombocytopenic purpura patients with antiglycoprotein. IIb/IIIa or Ib autoantibodies. Acta Haematol 1991: 86: 25-30. 8. KARPATKINS. Autoimmune thrombocytopenic purpura. Semin Hematol 1985: 22: 260-288. 9. YANABUM, SUZUKIM, SOGAT, et al. Influences of antiplatelet autoantibodies on platelet function in immune thrombocytopenic purpura. Eur J Haematol 1991: 46: 101-106. 10. YANABUM, NOMURA S, FUKUROI T, et al. Synergistic action in platelet activation induced by an antiplatelet autoantibody in ITP. Br J Haematol 1991: 78: 87-93.
Correspondence: S. Nomura, H. Kido, K. Yamaguchi, T. Fukuroi, M. Yanabu, T. Kawakatsu, M. Suzuki, T. Kokawa, K. Yasunaga First Department of Internal Medicine, Kansai Medical University, 1-Fumizonocho, Moriguchi, Osaka 570, Japan
Platelet activation by antiplatelet autoantibodies in immune thrombocytopenic purpura To the editor: Immune thrombocytopenic purpura (ITP) is a syndrome caused by circulating antibodies that react with the platelet membrane. Antiplatelet autoantibodies, alloantibodies, and monoclonal antibodies (mAb) have all been reported to initiate platelet activation as measured by aggregation and the release reaction (1). Furthermore, some recent studies have shown that platelet activation by anti-glycoprdtein (GP)IIb/IIIa, anti-CD9, and anti-GPIV mAb is mediated by the FcyII receptor (2-5). We used ITP plasma to investigate IgG-mediated platelet activation and particularly aggregation mediated by the FcyII receptor. Between June 1988 and December 1991, we obtained plasma from 45 chronic ITP patients (12 men and 33 women) and 20 normal subjects. The ITP patients had thrombocytopenia with normal or increased numbers of megakaryocytes and no evidence of secondary immune thrombocytopenia. Platelet aggregation was studied using an NKK Hematracer 1. Blood from healthy volunteers was collected into tubes containing 3.8 % sodium citrate (9: 1, vol:vol), and platelet-rich plasma (PRP) was obtained after centrifugation at 200 g for 10 min. ITP or control plasma (50p1) was added to the PRP (200 pl) and platelet aggregation was monitored for 15 min. Then a goat anti-human IgG antibody (20 pg/ml) was added. In place of this goat antibody, 10 pmol/l ADP or 1.5 mg/ml ristocetin was used for control studies. PBIgG was detected as reported previously (6, 7). In brief, normal washed platelets were prepared and fixed in 1% paraformaldehyde. Then the fixed platelets were incubated with ITP plasma (50 pl) for 30 min at room temperature, followed by incubation with FITC-labeled goat anti-human IgG for 30 min at room temperature. After further washing, the samples were subjected to FACS analysis using operating conditions that have been published previously, (6,7). Detection of plasma autoantibodies directed against GPIIb/IIIa or GPIb was performed by an antigen-capture ELISA, the details of which have also been reported previously '(6, 7).
PBIgG was positive in 34 of the 45 ITP patients. Anti-GPIIb/IIIa autoantibodies were detected in 18 patients and anti-GPIb autoantibodies were found in 7 patients. None of the ITP plasma samples induced the aggregation of normal PRP. Furthermore, the addition of goat anti-human IgG also failed to induce aggregation. ADP-induced aggregation of normal platelets was inhibited by autoantibodies in 10/45 patients, and 8 of them had anti-GPIIb/IIIa antibodies. Ristocetin-induced agglutination was also inhibited in 2 of these patients with anti-GPIb antibodies. It is thought that ITP can occur as a result of several mechanisms (8). These include platelet activation by circulating immune complexes with crosslink platelets via the FcyII receptor. Recent investigators have shown that this can also occur with mAb directed against platelets CD9 (2) or platelets GPIIb/ IIIa (3-5). We investigated whether IgG in ITP plasma could induce platelet aggregation mediated by the FcyII receptor and found that none of the plasma samples used in this study could do so. It was previously reported that autoantibodies could affect platelet function in ITP patients (9, 10). We also found that some anti-GPIIb/IIIa antibodies inhibited ADP-induced platelet aggregation and that PBIgG was positive in these patients. Thus, the autoantibodies found in ITP plasma can bind to normal platelets. Some mAb directed against GPIIb/IIIa appear to cause platelet activation and some do not (1). The reason for this difference is presently unknown, but it may be related to epitope specificity. For ITP autoantibodies to induce platelet aggregation via the FcyII receptor would require the appropriate antigen specificity, e.g., antigen mobility as well as proximity between the antigen and the FcyII receptors, which comprise the majority of the IgG binding sites. If immune complexes of platelet-specific autoantibodies can induce aggregation via the FcyII receptor, this event may participate in the induction of thrombocytopenia in ITP. However, since the autoantibodies found in ITP patients are heterogeneous, this mechanism may not be so common. 109
Letters to the editor References 1. HORSEWOOD P, HAYWARD CPM, WARKENTIN TE, KELTON JG. Investigation of the mechanisms of monoclonal antibody-induced platelet activation. Blood 1991: 78: 10191026. RE, CARROLL RC, BOUCHEIX C. Platelet 2. WORTHINGTON activation by C D 9 monoclonal antibodies is mediated by the FcyII receptor. Br J Haematol 1990: 74: 216-222. 3. RUBINSTEIN E, KOUNSWC, JENNINGS LK, BOUCHEIX C, CARROLLRC. Interaction of two GPIIb/IIIa monoclonal antibodies with platelet Fc receptor (FcyRII). Br J Haematol 1991: 77: 80-86. 4. ANDERSON GP, VAN DE WINKELJGJ, ANDERSONCL. Anti-GPI Ib/llIa (CD41) monoclonal antibody-induced platelet activation requires Fc receptor-dependent cell-cell interaction. Br J Haematol 1991: 79: 75-84. 5. HORNBYEJ, BROWNS, WILKINSON JM, MATTOCKC, AUTHIKS. Activation of human platelets by exposure to monoclonal antibody, PM6/248, to glycoprotein IIb-11Ia. Br J Haematol 1991: 79: 277-285. 6. NOMURAS, YANABUM, KIDO H, et al. Antiplatelet autoantibody-related microparticles in patients with idiopathic (auto-immune) thrombocytopenic purpura. Ann Hematol 1991: 62: 103-107.
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7. NOMURAS, YANABUM, SOCAT, et al. Analysis of idiopathic thrombocytopenic purpura patients with antiglycoprotein. IIb/IIIa or Ib autoantibodies. Acta Haematol 1991: 86: 25-30. 8. KARPATKINS. Autoimmune thrombocytopenic purpura. Semin Hematol 1985: 22: 260-288. 9. YANABUM, SUZUKIM, SOGAT, et al. Influences of antiplatelet autoantibodies on platelet function in immune thrombocytopenic purpura. Eur J Haematol 1991: 46: 101-106. 10. YANABUM, NOMURA S, FUKUROI T, et al. Synergistic action in platelet activation induced by an antiplatelet autoantibody in ITP. Br J Haematol 1991: 78: 87-93.
Correspondence: S. Nomura, H. Kido, K. Yamaguchi, T. Fukuroi, M. Yanabu, T. Kawakatsu, M. Suzuki, T. Kokawa, K. Yasunaga First Department of Internal Medicine, Kansai Medical University, 1-Fumizonocho, Moriguchi, Osaka 570, Japan
