Autoimmunity, 1992, Vol. 13, pp. 133-140 Reprints available directly from the publisher Photocopying permitted by license only
0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
ANTI-GLYCOPROTEIN IIb/IIIa AUTOANTIBODIES ARE REVERSIBLY INTERNALIZED INTO PLATELETS IN IDIOPATHIC (AUTOIMMUNE) THROMBOCYTOPENIC PURPURA
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SHOSAKU NOMURA*, MUTSUMASA YANABU, TSUTOMU FUKUROI, HIROFUMI KIDO, TOSHIHIRO KAWAKATSU, KAZUYUKI YAMAGUCHI, MASAHIKO SUZUKI, TERUTOSHI KOKAWA and KOJIRO YASUNAGA The First Department ojlnternal Medii,ine, Kansai Medical University, I-Funiizonocho. Moriguchi. Osaka 570, Japan (Received November. S, 1991; in final form April 2 2 , 1 9 9 1 )
We used flow cytometry to investigate the binding of platelet-binding IgG (PBIgG) to unfixed platelets in idiopathic thrombocytopenic purpura (ITP), including that of anti-glycoprotein (CP) IIb/lIIa antibodies. AntiGPIIb/IIIa antibodies were detected in 13/64 ITP patients using antigen-capture ELISA and immunoblotting. When unfixed platelets were incubated with ITP plasma, the PBIgG level was significantly higher than after incubation with normal plasma. When I pM ADP was added to unfixed platelets, which were incubated with ITP plasma and washed, the PBIgC level increased additively. CMP- 140 is a constituent of platelet a-granules, and a monoclonal antibody directed against this protein showed weak binding to platelets after I pM ADP stimulation. The increase of PBIgG produced by ADP was significantly greater when ITP plasma positive for anti-GPlIb/IlIa antibody was used compared with that obtained using antibody-negative ITP plasma. This increase of PBIgC was markedly inhibited by the removal of extracellular calcium with EDTA or the dissociation of the GPIIb/IIIa complex by EDTA treatment at 37°C. These results suggest that anti-GPIIb/IIIa autoantibodies are internalized by unfixed ITP platelets and stored somewhere other than the a-granules. This stored antibody pool can be reversibly redistributed on the platelet surface by weak stimulants such as ADP and a functional GPIlb/llla complex appears to be necessary for this to occur. KEY WORDS: Idiopathic thrombocytopenic purpura, glycoprotein IIb/IIIa autoantibody, platelet binding IgG, internalization, ADP stimulation, unfixed platelets.
gation and GPIIb/IIIa is the most abundant of the platelet integrins". In unstimulated platelets, Idiopathic thrombocytopenic purpura (ITP) is a syn- GPIIb/IIIa is localized on the platelet plasma memdrome caused by circulating antibodies that react with brane, the alpha granule membranes, and the intralumthe platelet membrane'~2.Many assays to detect anti- inal aspects of the intracellular vacuolar structures'*.". platelet antibodies have been developed since the Following platelet stimulation, however, GPIIb/IIIa report of Dixon et Such antibodies can be detected becomes a receptor for several soluble adhesive proby the platelet suspension immunofluorescence test teins, the binding of which is mediated in part by the using a direct method' with ITP platelets or an Arg-Gly-Asp (RGD) recognition sequence, and forms indirect method with ITP plasma. The former detects patches on the platelet surface". Human platelets also platelet-associated IgG (PAIgC) and the latter detects exhibit several molecular trafficking pathways, platelet-binding IgG (PBIgC). PAIgG appears to be including those involved in receptor-mediated patchimportant in the mechanism of ITP because its pres- ing and capping, phagocytosis, and exocytosis"~". ence is closely correlated with t h r o m b ~ c y t o p e n i a ~ ~ " ~For ~ ' , example, Santoso et a/.'' have reported that only but the significance of PBIgG currently remains antibodies directed against GPIIb/IIIa epitopes caused obscure. However, it has been reported that antiglyco- patching and capping, and that they were internalized protein (GP) Ib and anti-GPIIb/IIIa autoantibodies are in a time-dependent fashion. In this study, we detected anti-GPIIb/IIIa autoantipresent in the plasma of patients with chronic ITP,"~"', suggesting that such specific antiplatelet antibodies bodies in ITP plasma by antigen-capture ELISA, and used flow cytometry to investigate the binding of may have a role in this syndrome. The GPIIb/IIIa complex mediates platelet aggre- PBIgG (including anti-GPIIb/IIIa antibodies) to unfixed platelets, in an attempt to further elucidate the *Author for correspondence. Tel: 06-992- 1001. role of these autoantibodies in ITP. INTRODUCTION
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S. NOMURA ET AL
MATERIALS AND METHODS ITP patients
Autoimmunity Downloaded from informahealthcare.com by Chinese University of Hong Kong on 02/04/15 For personal use only.
Sixty-four patients (21 men and 43 women aged 1.5-85 years) with ITP were studied. All these patients satisfied the usual clinical criteria for ITP and had not received any transfusions; three patients had already been given prednisolone therapy. In addition, 30 patients with a variety of diseases causing nonimmune thrombocytopenia (14 with leukemia, 7 with aplastic anemia, and 9 with liver cirrhosis) were used as disease controls. Preparation of paraformaldehyde-treated platelets Whole blood anticoagulated with 3.8% sodium citrate (9 vol. blood to 1 vol. Na citrate) was centrifuged at 200 g for 10 min at room temperature (RT). After centrifugation, the supernatant was collected without disturbing the other fractions to provide diluted plateletrich plasma (PRP). The PRP was transferred to a second plastic tube containing a cation-free solution of ethylenediaminetetraacetic acid with phosphatebuffered saline (EDTA-PBS, pH 7.2) and 200pg/ml apyrase. Then platelets were pelleted from the PRP by centrifugation for 10 min at 1,400 g and were washed twice at room temperature in EDTA-PBS. After further, the platelets were resuspended in 1 % paraformaldehyde and incubated at RT for 15 min. They were then pelleted and resuspended in EDTA-PBS twice more under the same conditions. After the final centrifugation, the platelets were resuspended in EDTAPBS containing 2% fetal bovine serum and stored at 4°C until analysis. Measurement of PAIgC and PBlgG using fixed platelets Assays were conducted using the platelet suspension immunofluorescence test of Borne et ~ 1 . ~ 1 ) PAIgC PAIgG was measured by a direct immunofluorescence test. Paraformaldehyde-fixed platelets obtained from ITP patients were diluted to 2x1O6/1O0p1 in EDTAPBS, incubated with fluorescein-labeled goat antihuman IgG (Cappel Products, Westchester, PA, USA) for 30min at RT, and washed 3 times by centrifugation. After the final wash, the platelets were resuspended in S00pl of EDTA-PBS and transferred to a plastic tube (Falcon 20.52) for the fluorescence intensity to be determined using a FACS analyzer (Becton Dickinson & Co., Mountain View, CA, USA).
2) PBIgG PBIgG was measured by an indirect immunofluoresc-
ence test using platelets from a selected group of type 0 blood donors. In brief, healthy washed platelets were incubated with ITP plasma (100 pl) for 30 min at RT, washed 3 times by centrifugation (1,400g for 10min at RT), and resuspended in EDTA-PBS. The treated platelets were then incubated with fluoresceinlabeled goat anti-human IgG for 30min at RT, and processed in the same way as for the PAIgC assay. Measurement of PBIgC using unfixed platelets Normal PRP was diluted to 2x106 platelets/100 pl with platelet-poor plasma (PPP), and then incubated with ITP plasma (100 pl) for 30 min at RT. Platelets were resuspended in 1% paraformaldehyde without centrifugation and incubated again at RT for 15 min. The paraformaldehyde-treated platelets were then processed in the same way as the fixed platelets. PBlgG binding to unfixed platelets after various treatments Unfixed platelets were incubated with ITP plasma in the presence of various inhibitors of platelet function. The following inhibitors were used; aspirin (500 pM; Wako Pure Chemical Industries, Osaka, Japan), diltiazem (SO0 pM; Research Biochemical Inc., Tokyo, Japan), prostaglandin E, ( 5 pglml; Sigma Chemical Co., St Louis, MO), EDTA (10 mM; Wako), staurosporine ( 1 0 p M ; Kyowa Medex Co., Ltd., Tokyo, Japan), Arg-Gly-Asp-Ser (RGDS; 500 p M ; Peninsula Laboratories Ltd., Tokyo, Japan, and y-400-411 (HHLGGAKQ AGDV; 500 p M ; Peninsula Laboratories). Detection of plasma autoantihodies against GPllhlllla by microtiter well assay
1 ) Preparation of Platelet Lysate A suspension of washed platelets (lxlO’/pl) in 100 pglml leupeptin and 10 mM EDTA was sonicated on ice and centrifuged at 12,500 g for 30 min. The supernatant was solubilized in 2% Triton X-100 and centrifuged at 100,000 g for 30 min to remove the Triton X100-insoluble fraction. After centrifugation, the supernatant was used as the platelet lysate. 2) Microtiter Well Assay Autoantibodies were assayed by a modification of the method of Woods et al.”“’ In brief, microtiter wells were coated with a monoclonal anti-GPIIb/IIIa antibody (NNKY2- 18)18~1s) by overnight incubation. The platelet lysate was then added to microtiter wells and incubated. After washing, appropriate dilutions of plasma from ITP patients (n=64), disease controls (n= 30), or normal (n=20) were added and incubated.
INTERNALIZATION OF ANTI-GPIIb/IIIa AUTOANTIBODIES IN ITP
After washing again, horseradish peroxidase (HRP)conjugated rabbit anti-human IgG was added, and the amount of IgG which bound to the platelets was determined by measuring the peroxidase activity using a Micro-plate Reader (MPR-A4, Tosoh Inc., Tokyo, Japan). Assay results were expressed in terms of the percent change in peroxidase activity above or below the level in control (normal serum) wells, using the following formula:
Autoimmunity Downloaded from informahealthcare.com by Chinese University of Hong Kong on 02/04/15 For personal use only.
Percent chunge =
OD platelet extract wells-OD control wells OD control wells x 100
Control (normal serum) was obtained from 5 healthy male blood donors (aged 28-37 years; blood types: A,1; B,1; AB,l; and 0,2). Samples with a percentage increase greater than 3 SD above the mean determined in 20 plasma samples were considered to be positive.
135
Operating conditions for FACS analysis The details have been published In brief, the volume gates were set at channels 10 and 255, while F L l markers were set at channels 0 and 255. One negative control (autofluorescence, using unstained platelets) was set at channels 0 and 50. The fluorescence-positive rate was then calculated from the total platelet count above channel 51. The other negative control (platelets incubated with fluoresceinlabeled goat anti-mouse IgG, Kirkegaard & Perry Laboratories) was set so that the fluorescence-positive rate was infinitely close to 1%. To quantify fluorescence, SIGMA was defined as follows: SIGMA=[Mean channel of the positive regionxtotal platelet count of the positive region]/103 Ten thousand events were analyzed in the one-parameter mode.
SDS-PAGE and irnrnunohlotting
Agonist stimulation and changes of the platelet surface membrane Incubation of washed platelets was performed at RT with thrombin or ADP at various concentrations, and the reaction was stopped after 5 min by the addition of 1 % of paraformaldehyde (final concentration). After fixation for 30 min at RT, the samples were diluted in EDTA-PBS. Changes of the platelet surface membrane in response to agonist stimulation were investigated using an anti-GMP- 140 monoclonal antibody (CLB-thromb/6; Immunotech, Marseille)21.Paraformaldehyde-fixed platelets were incubated with 2 pg/ml CLB-thromb/6 for 30min at RT and then washed twice. After washing, the platelets were incubated with fluorescein-labeled goat anti-mouse IgG (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, USA) for 30 min at RT and then washed 3 times by centrifugation. After the final wash, the platelets were resuspended in EDTA-PBS and the fluorescence intensity was determined with a FACS analyzer.
SDS-PAGE was performed by the method of Laemmli22 using 7.5% polyacrylamide gel. Immunoblotting was performed using a modification of the method of Beardsly et al.23 Proteins obtained from a platelet lysate which had been subjected to SDSPAGE were electrophoretically transferred to nitrocellulose paper in 25 mM Tris buffer (pH 8.3). Nitrocellulose strips containing a single lane of platelet proteins were first incubated with 3% bovine serum albumin in 0.85% Tween 20-PBS for 1 hr at RT, and then with test plasma (diluted 1 5 0 in 0.05% Tween 20-PBS). The strips were then washed three times with 0.05% Tween 20-PBS, and antiplatelet antibodies bound to the strips were detected by the avidinbiotin-peroxidase complex method using biotinylated anti-human IgG (Vector Laboratories, Burlingame, Calif.). Molecular weight standards (Bio-Rad, Richmond, Calif.) were subjected to simultaneous electrophoresis with the samples.
Crossed imrnunoelectrophoresis This was performed according to the procedure of Kunicki et al.24 Briefly, first-dimension electroHealthy unfixed platelets (3x 10*/ml) were incubated phoresis was performed at 10 V/cm for 1 hr in 1% with patient plasma for 30 min at RT, after which they (wt/vol) agarose containing 0.5% (v/v) Triton X-100 were washed and incubated with ADP and thrombin in Tris-glycine. Second-dimension electrophoresis for 5 min at RT in the presence of EDTA-PBS. After was then performed at 2 V/cm for 18 hr in a biphasic incubation, the platelets were resuspended in 1% par- gel system, the upper gel of which contained a precipaformaldehyde without centrifugation and incubated itating concentration of a polyspecific rabbit antiagain at RT for 15 min. Paraformaldehyde-treated human platelet antibody. platelets were then incubated with fluorescein-labeled goat anti-human IgG and processed in the same man- Statistical methods ner as for the measurement of PBIgG using unfixed Student's t-test was used in statistical comparisons. platelets.
Changes of PBIgC after agonist stimulation
S. NOMURA ET AL
136
Autoimmunity Downloaded from informahealthcare.com by Chinese University of Hong Kong on 02/04/15 For personal use only.
RESULTS Table 1 shows the platelet counts and the levels of PAIgG and PBIgG in the 30 controls (nonimmune thrombocytopenia) and the 64 ITP patients. In the ITP patients, the platelet count correlated with the PAIgG level (r=-0.48, P
0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
ANTI-GLYCOPROTEIN IIb/IIIa AUTOANTIBODIES ARE REVERSIBLY INTERNALIZED INTO PLATELETS IN IDIOPATHIC (AUTOIMMUNE) THROMBOCYTOPENIC PURPURA
Autoimmunity Downloaded from informahealthcare.com by Chinese University of Hong Kong on 02/04/15 For personal use only.
SHOSAKU NOMURA*, MUTSUMASA YANABU, TSUTOMU FUKUROI, HIROFUMI KIDO, TOSHIHIRO KAWAKATSU, KAZUYUKI YAMAGUCHI, MASAHIKO SUZUKI, TERUTOSHI KOKAWA and KOJIRO YASUNAGA The First Department ojlnternal Medii,ine, Kansai Medical University, I-Funiizonocho. Moriguchi. Osaka 570, Japan (Received November. S, 1991; in final form April 2 2 , 1 9 9 1 )
We used flow cytometry to investigate the binding of platelet-binding IgG (PBIgG) to unfixed platelets in idiopathic thrombocytopenic purpura (ITP), including that of anti-glycoprotein (CP) IIb/lIIa antibodies. AntiGPIIb/IIIa antibodies were detected in 13/64 ITP patients using antigen-capture ELISA and immunoblotting. When unfixed platelets were incubated with ITP plasma, the PBIgG level was significantly higher than after incubation with normal plasma. When I pM ADP was added to unfixed platelets, which were incubated with ITP plasma and washed, the PBIgC level increased additively. CMP- 140 is a constituent of platelet a-granules, and a monoclonal antibody directed against this protein showed weak binding to platelets after I pM ADP stimulation. The increase of PBIgG produced by ADP was significantly greater when ITP plasma positive for anti-GPlIb/IlIa antibody was used compared with that obtained using antibody-negative ITP plasma. This increase of PBIgC was markedly inhibited by the removal of extracellular calcium with EDTA or the dissociation of the GPIIb/IIIa complex by EDTA treatment at 37°C. These results suggest that anti-GPIIb/IIIa autoantibodies are internalized by unfixed ITP platelets and stored somewhere other than the a-granules. This stored antibody pool can be reversibly redistributed on the platelet surface by weak stimulants such as ADP and a functional GPIlb/llla complex appears to be necessary for this to occur. KEY WORDS: Idiopathic thrombocytopenic purpura, glycoprotein IIb/IIIa autoantibody, platelet binding IgG, internalization, ADP stimulation, unfixed platelets.
gation and GPIIb/IIIa is the most abundant of the platelet integrins". In unstimulated platelets, Idiopathic thrombocytopenic purpura (ITP) is a syn- GPIIb/IIIa is localized on the platelet plasma memdrome caused by circulating antibodies that react with brane, the alpha granule membranes, and the intralumthe platelet membrane'~2.Many assays to detect anti- inal aspects of the intracellular vacuolar structures'*.". platelet antibodies have been developed since the Following platelet stimulation, however, GPIIb/IIIa report of Dixon et Such antibodies can be detected becomes a receptor for several soluble adhesive proby the platelet suspension immunofluorescence test teins, the binding of which is mediated in part by the using a direct method' with ITP platelets or an Arg-Gly-Asp (RGD) recognition sequence, and forms indirect method with ITP plasma. The former detects patches on the platelet surface". Human platelets also platelet-associated IgG (PAIgC) and the latter detects exhibit several molecular trafficking pathways, platelet-binding IgG (PBIgC). PAIgG appears to be including those involved in receptor-mediated patchimportant in the mechanism of ITP because its pres- ing and capping, phagocytosis, and exocytosis"~". ence is closely correlated with t h r o m b ~ c y t o p e n i a ~ ~ " ~For ~ ' , example, Santoso et a/.'' have reported that only but the significance of PBIgG currently remains antibodies directed against GPIIb/IIIa epitopes caused obscure. However, it has been reported that antiglyco- patching and capping, and that they were internalized protein (GP) Ib and anti-GPIIb/IIIa autoantibodies are in a time-dependent fashion. In this study, we detected anti-GPIIb/IIIa autoantipresent in the plasma of patients with chronic ITP,"~"', suggesting that such specific antiplatelet antibodies bodies in ITP plasma by antigen-capture ELISA, and used flow cytometry to investigate the binding of may have a role in this syndrome. The GPIIb/IIIa complex mediates platelet aggre- PBIgG (including anti-GPIIb/IIIa antibodies) to unfixed platelets, in an attempt to further elucidate the *Author for correspondence. Tel: 06-992- 1001. role of these autoantibodies in ITP. INTRODUCTION
133
134
S. NOMURA ET AL
MATERIALS AND METHODS ITP patients
Autoimmunity Downloaded from informahealthcare.com by Chinese University of Hong Kong on 02/04/15 For personal use only.
Sixty-four patients (21 men and 43 women aged 1.5-85 years) with ITP were studied. All these patients satisfied the usual clinical criteria for ITP and had not received any transfusions; three patients had already been given prednisolone therapy. In addition, 30 patients with a variety of diseases causing nonimmune thrombocytopenia (14 with leukemia, 7 with aplastic anemia, and 9 with liver cirrhosis) were used as disease controls. Preparation of paraformaldehyde-treated platelets Whole blood anticoagulated with 3.8% sodium citrate (9 vol. blood to 1 vol. Na citrate) was centrifuged at 200 g for 10 min at room temperature (RT). After centrifugation, the supernatant was collected without disturbing the other fractions to provide diluted plateletrich plasma (PRP). The PRP was transferred to a second plastic tube containing a cation-free solution of ethylenediaminetetraacetic acid with phosphatebuffered saline (EDTA-PBS, pH 7.2) and 200pg/ml apyrase. Then platelets were pelleted from the PRP by centrifugation for 10 min at 1,400 g and were washed twice at room temperature in EDTA-PBS. After further, the platelets were resuspended in 1 % paraformaldehyde and incubated at RT for 15 min. They were then pelleted and resuspended in EDTA-PBS twice more under the same conditions. After the final centrifugation, the platelets were resuspended in EDTAPBS containing 2% fetal bovine serum and stored at 4°C until analysis. Measurement of PAIgC and PBlgG using fixed platelets Assays were conducted using the platelet suspension immunofluorescence test of Borne et ~ 1 . ~ 1 ) PAIgC PAIgG was measured by a direct immunofluorescence test. Paraformaldehyde-fixed platelets obtained from ITP patients were diluted to 2x1O6/1O0p1 in EDTAPBS, incubated with fluorescein-labeled goat antihuman IgG (Cappel Products, Westchester, PA, USA) for 30min at RT, and washed 3 times by centrifugation. After the final wash, the platelets were resuspended in S00pl of EDTA-PBS and transferred to a plastic tube (Falcon 20.52) for the fluorescence intensity to be determined using a FACS analyzer (Becton Dickinson & Co., Mountain View, CA, USA).
2) PBIgG PBIgG was measured by an indirect immunofluoresc-
ence test using platelets from a selected group of type 0 blood donors. In brief, healthy washed platelets were incubated with ITP plasma (100 pl) for 30 min at RT, washed 3 times by centrifugation (1,400g for 10min at RT), and resuspended in EDTA-PBS. The treated platelets were then incubated with fluoresceinlabeled goat anti-human IgG for 30min at RT, and processed in the same way as for the PAIgC assay. Measurement of PBIgC using unfixed platelets Normal PRP was diluted to 2x106 platelets/100 pl with platelet-poor plasma (PPP), and then incubated with ITP plasma (100 pl) for 30 min at RT. Platelets were resuspended in 1% paraformaldehyde without centrifugation and incubated again at RT for 15 min. The paraformaldehyde-treated platelets were then processed in the same way as the fixed platelets. PBlgG binding to unfixed platelets after various treatments Unfixed platelets were incubated with ITP plasma in the presence of various inhibitors of platelet function. The following inhibitors were used; aspirin (500 pM; Wako Pure Chemical Industries, Osaka, Japan), diltiazem (SO0 pM; Research Biochemical Inc., Tokyo, Japan), prostaglandin E, ( 5 pglml; Sigma Chemical Co., St Louis, MO), EDTA (10 mM; Wako), staurosporine ( 1 0 p M ; Kyowa Medex Co., Ltd., Tokyo, Japan), Arg-Gly-Asp-Ser (RGDS; 500 p M ; Peninsula Laboratories Ltd., Tokyo, Japan, and y-400-411 (HHLGGAKQ AGDV; 500 p M ; Peninsula Laboratories). Detection of plasma autoantihodies against GPllhlllla by microtiter well assay
1 ) Preparation of Platelet Lysate A suspension of washed platelets (lxlO’/pl) in 100 pglml leupeptin and 10 mM EDTA was sonicated on ice and centrifuged at 12,500 g for 30 min. The supernatant was solubilized in 2% Triton X-100 and centrifuged at 100,000 g for 30 min to remove the Triton X100-insoluble fraction. After centrifugation, the supernatant was used as the platelet lysate. 2) Microtiter Well Assay Autoantibodies were assayed by a modification of the method of Woods et al.”“’ In brief, microtiter wells were coated with a monoclonal anti-GPIIb/IIIa antibody (NNKY2- 18)18~1s) by overnight incubation. The platelet lysate was then added to microtiter wells and incubated. After washing, appropriate dilutions of plasma from ITP patients (n=64), disease controls (n= 30), or normal (n=20) were added and incubated.
INTERNALIZATION OF ANTI-GPIIb/IIIa AUTOANTIBODIES IN ITP
After washing again, horseradish peroxidase (HRP)conjugated rabbit anti-human IgG was added, and the amount of IgG which bound to the platelets was determined by measuring the peroxidase activity using a Micro-plate Reader (MPR-A4, Tosoh Inc., Tokyo, Japan). Assay results were expressed in terms of the percent change in peroxidase activity above or below the level in control (normal serum) wells, using the following formula:
Autoimmunity Downloaded from informahealthcare.com by Chinese University of Hong Kong on 02/04/15 For personal use only.
Percent chunge =
OD platelet extract wells-OD control wells OD control wells x 100
Control (normal serum) was obtained from 5 healthy male blood donors (aged 28-37 years; blood types: A,1; B,1; AB,l; and 0,2). Samples with a percentage increase greater than 3 SD above the mean determined in 20 plasma samples were considered to be positive.
135
Operating conditions for FACS analysis The details have been published In brief, the volume gates were set at channels 10 and 255, while F L l markers were set at channels 0 and 255. One negative control (autofluorescence, using unstained platelets) was set at channels 0 and 50. The fluorescence-positive rate was then calculated from the total platelet count above channel 51. The other negative control (platelets incubated with fluoresceinlabeled goat anti-mouse IgG, Kirkegaard & Perry Laboratories) was set so that the fluorescence-positive rate was infinitely close to 1%. To quantify fluorescence, SIGMA was defined as follows: SIGMA=[Mean channel of the positive regionxtotal platelet count of the positive region]/103 Ten thousand events were analyzed in the one-parameter mode.
SDS-PAGE and irnrnunohlotting
Agonist stimulation and changes of the platelet surface membrane Incubation of washed platelets was performed at RT with thrombin or ADP at various concentrations, and the reaction was stopped after 5 min by the addition of 1 % of paraformaldehyde (final concentration). After fixation for 30 min at RT, the samples were diluted in EDTA-PBS. Changes of the platelet surface membrane in response to agonist stimulation were investigated using an anti-GMP- 140 monoclonal antibody (CLB-thromb/6; Immunotech, Marseille)21.Paraformaldehyde-fixed platelets were incubated with 2 pg/ml CLB-thromb/6 for 30min at RT and then washed twice. After washing, the platelets were incubated with fluorescein-labeled goat anti-mouse IgG (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, USA) for 30 min at RT and then washed 3 times by centrifugation. After the final wash, the platelets were resuspended in EDTA-PBS and the fluorescence intensity was determined with a FACS analyzer.
SDS-PAGE was performed by the method of Laemmli22 using 7.5% polyacrylamide gel. Immunoblotting was performed using a modification of the method of Beardsly et al.23 Proteins obtained from a platelet lysate which had been subjected to SDSPAGE were electrophoretically transferred to nitrocellulose paper in 25 mM Tris buffer (pH 8.3). Nitrocellulose strips containing a single lane of platelet proteins were first incubated with 3% bovine serum albumin in 0.85% Tween 20-PBS for 1 hr at RT, and then with test plasma (diluted 1 5 0 in 0.05% Tween 20-PBS). The strips were then washed three times with 0.05% Tween 20-PBS, and antiplatelet antibodies bound to the strips were detected by the avidinbiotin-peroxidase complex method using biotinylated anti-human IgG (Vector Laboratories, Burlingame, Calif.). Molecular weight standards (Bio-Rad, Richmond, Calif.) were subjected to simultaneous electrophoresis with the samples.
Crossed imrnunoelectrophoresis This was performed according to the procedure of Kunicki et al.24 Briefly, first-dimension electroHealthy unfixed platelets (3x 10*/ml) were incubated phoresis was performed at 10 V/cm for 1 hr in 1% with patient plasma for 30 min at RT, after which they (wt/vol) agarose containing 0.5% (v/v) Triton X-100 were washed and incubated with ADP and thrombin in Tris-glycine. Second-dimension electrophoresis for 5 min at RT in the presence of EDTA-PBS. After was then performed at 2 V/cm for 18 hr in a biphasic incubation, the platelets were resuspended in 1% par- gel system, the upper gel of which contained a precipaformaldehyde without centrifugation and incubated itating concentration of a polyspecific rabbit antiagain at RT for 15 min. Paraformaldehyde-treated human platelet antibody. platelets were then incubated with fluorescein-labeled goat anti-human IgG and processed in the same man- Statistical methods ner as for the measurement of PBIgG using unfixed Student's t-test was used in statistical comparisons. platelets.
Changes of PBIgC after agonist stimulation
S. NOMURA ET AL
136
Autoimmunity Downloaded from informahealthcare.com by Chinese University of Hong Kong on 02/04/15 For personal use only.
RESULTS Table 1 shows the platelet counts and the levels of PAIgG and PBIgG in the 30 controls (nonimmune thrombocytopenia) and the 64 ITP patients. In the ITP patients, the platelet count correlated with the PAIgG level (r=-0.48, P
