scholarly journals Autoantibodies to the presumptive cytoplasmic domain of platelet glycoprotein IIIa in patients with chronic immune thrombocytopenic purpura [published erratum appears in Blood 1991 Dec 1;78(11):3109]

Blood ◽  
1991 ◽  
Vol 77 (10) ◽  
pp. 2207-2213 ◽  
Author(s):  
K Fujisawa ◽  
TE O'Toole ◽  
P Tani ◽  
JC Loftus ◽  
EF Plow ◽  
...  
Keyword(s):  
Amino Acids ◽  
Immune Thrombocytopenic Purpura ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Antigenic Site ◽  
Cytoplasmic Domain ◽  
Platelet Glycoprotein ◽  
Thrombocytopenic Purpura ◽  
Chronic Immune Thrombocytopenic Purpura ◽  
Chronic Itp

Abstract Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder due to autoantibodies against platelets that result in their destruction. In some patients, these autoantibodies bind to platelet glycoprotein (GP) IIIa. With the aim of better defining the antigenic epitopes, plasma from 13 selected patients with chronic ITP known to have anti-GPIIb/IIIa autoantibodies was tested for reactivity with nine synthetic peptides corresponding to different regions of the GP IIIa molecule. Of these plasmas, five bound significantly (P less than .001) with either peptide 8 (amino acids 721–744) or peptide 9 (amino acids 742–762), which together form most of the carboxyterminal region presumed to be the cytoplasmic domain. Three of these positive plasmas, were tested further. In two of these positive plasmas, the anti-peptide antibodies represented greater than 80% of the detectable circulating autoantibody. To further evaluate the importance of the carboxyterminal region as an antigenic site, the chronic ITP plasmas were tested against Chinese hamster ovary cells transfected with GPIIb and either whole GPIIIa or GPIIIa lacking amino acids 728 to 762. Ten of the 13 plasmas required the presence of this region for significant autoantibody binding. We conclude that the carboxyterminal region is an important area for stimulating antiplatelet autoantibody formation in some patients with chronic ITP. It is not known whether these autoantibodies to the presumed cytoplasmic domain play an important role in the pathogenesis of the disease or occur as a secondary phenomenon during the course of platelet destruction.

Autoantibodies to the presumptive cytoplasmic domain of platelet glycoprotein IIIa in patients with chronic immune thrombocytopenic purpura [published erratum appears in Blood 1991 Dec 1;78(11):3109]

Blood ◽  
1991 ◽  
Vol 77 (10) ◽  
pp. 2207-2213 ◽  
Author(s):  
K Fujisawa ◽  
TE O'Toole ◽  
P Tani ◽  
JC Loftus ◽  
EF Plow ◽  
...  
Keyword(s):  
Amino Acids ◽  
Immune Thrombocytopenic Purpura ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Antigenic Site ◽  
Cytoplasmic Domain ◽  
Platelet Glycoprotein ◽  
Thrombocytopenic Purpura ◽  
Chronic Immune Thrombocytopenic Purpura ◽  
Chronic Itp

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder due to autoantibodies against platelets that result in their destruction. In some patients, these autoantibodies bind to platelet glycoprotein (GP) IIIa. With the aim of better defining the antigenic epitopes, plasma from 13 selected patients with chronic ITP known to have anti-GPIIb/IIIa autoantibodies was tested for reactivity with nine synthetic peptides corresponding to different regions of the GP IIIa molecule. Of these plasmas, five bound significantly (P less than .001) with either peptide 8 (amino acids 721–744) or peptide 9 (amino acids 742–762), which together form most of the carboxyterminal region presumed to be the cytoplasmic domain. Three of these positive plasmas, were tested further. In two of these positive plasmas, the anti-peptide antibodies represented greater than 80% of the detectable circulating autoantibody. To further evaluate the importance of the carboxyterminal region as an antigenic site, the chronic ITP plasmas were tested against Chinese hamster ovary cells transfected with GPIIb and either whole GPIIIa or GPIIIa lacking amino acids 728 to 762. Ten of the 13 plasmas required the presence of this region for significant autoantibody binding. We conclude that the carboxyterminal region is an important area for stimulating antiplatelet autoantibody formation in some patients with chronic ITP. It is not known whether these autoantibodies to the presumed cytoplasmic domain play an important role in the pathogenesis of the disease or occur as a secondary phenomenon during the course of platelet destruction.

Autoantibodies to αIIbβ3 in patients with chronic immune thrombocytopenic purpura bind primarily to epitopes on αIIb

Blood ◽  
2001 ◽  
Vol 97 (7) ◽  
pp. 2171-2172 ◽  
Author(s):  
Robert McMillan ◽  
Jennifer Lopez-Dee ◽  
Joseph C. Loftus
Keyword(s):  
Immune Thrombocytopenic Purpura ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Surface Proteins ◽  
Platelet Glycoprotein ◽  
Beta Chain ◽  
Platelet Destruction ◽  
Thrombocytopenic Purpura ◽  
Platelet Surface ◽  
Chronic Immune Thrombocytopenic Purpura

Abstract Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disease caused by platelet destruction resulting from autoantibodies against platelet surface proteins, particularly platelet glycoprotein IIb/IIIa (αIIbβ3). To localize the auto-epitopes on platelet αIIbβ3, the binding of autoantibodies to Chinese hamster ovary (CHO) cells expressing either αIIbβ3 or αvβ3was studied. Thirteen of 14 ITP autoantibodies bound only to CHO cells expressing αIIbβ3. Because these 2 integrins have the same beta chain (β3), these results show that most epitopes in chronic ITP are dependent on the presence of glycoprotein αIIb.

scholarly journals Platelet-associated and plasma anti-glycoprotein autoantibodies in chronic ITP

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1040-1045 ◽  
Author(s):  
R McMillan ◽  
P Tani ◽  
F Millard ◽  
P Berchtold ◽  
L Renshaw ◽  
...  
Keyword(s):  
Immune Thrombocytopenic Purpura ◽  
Platelet Glycoprotein ◽  
Antiplatelet Antibody ◽  
Platelet Destruction ◽  
Thrombocytopenic Purpura ◽  
Chronic Immune Thrombocytopenic Purpura ◽  
Chronic Itp ◽  
Negative Results ◽  
Well Assay ◽  
Positive Results

Chronic immune thrombocytopenic purpura (ITP) is due to platelet destruction by circulating antiplatelet antibody. Although autoantibodies against the platelet glycoprotein IIb/IIIa (GPIIb/IIIa) complex and GPIb have been demonstrated using various methods, practical assays for detection of platelet-associated or plasma autoantibodies have not been available. We studied 59 patients with chronic immune thrombocytopenic purpura in whom platelet-associated and plasma autoantibodies against the GPIIb/IIIa complex and GPIb were measured using a newly developed immunobead assay and a previously reported microtiter-well assay. Platelet-associated autoantibody was detected using the immunobead assay in 21 of 28 patients (75.0%; 13 with anti-GPIIb/IIIa, 8 with anti-GPIb). Plasma autoantibodies were noted in 34 of 59 patients (57.6%; 21 with anti-GPIIb/IIIa, 11 with anti-GPIb, and 2 with both). Positive results were noted in 30 of 59 patients using the immunobead assay and in only 14 of 59 using the microtiter-well assay, suggesting that solubilization of the platelets prior to antibody addition, as in the microtiter-well assay, alters epitope stability. Of the 31 thrombocytopenic control patients studied, all gave negative results using both assays. We conclude that these clinically adaptable assays allow detection of autoantibodies in most patients with chronic ITP, confirming the presence of an autoimmune process.

scholarly journals Platelet-associated and plasma anti-glycoprotein autoantibodies in chronic ITP

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1040-1045 ◽  
Author(s):  
R McMillan ◽  
P Tani ◽  
F Millard ◽  
P Berchtold ◽  
L Renshaw ◽  
...  
Keyword(s):  
Immune Thrombocytopenic Purpura ◽  
Platelet Glycoprotein ◽  
Antiplatelet Antibody ◽  
Platelet Destruction ◽  
Thrombocytopenic Purpura ◽  
Chronic Immune Thrombocytopenic Purpura ◽  
Chronic Itp ◽  
Negative Results ◽  
Well Assay ◽  
Positive Results

Abstract Chronic immune thrombocytopenic purpura (ITP) is due to platelet destruction by circulating antiplatelet antibody. Although autoantibodies against the platelet glycoprotein IIb/IIIa (GPIIb/IIIa) complex and GPIb have been demonstrated using various methods, practical assays for detection of platelet-associated or plasma autoantibodies have not been available. We studied 59 patients with chronic immune thrombocytopenic purpura in whom platelet-associated and plasma autoantibodies against the GPIIb/IIIa complex and GPIb were measured using a newly developed immunobead assay and a previously reported microtiter-well assay. Platelet-associated autoantibody was detected using the immunobead assay in 21 of 28 patients (75.0%; 13 with anti-GPIIb/IIIa, 8 with anti-GPIb). Plasma autoantibodies were noted in 34 of 59 patients (57.6%; 21 with anti-GPIIb/IIIa, 11 with anti-GPIb, and 2 with both). Positive results were noted in 30 of 59 patients using the immunobead assay and in only 14 of 59 using the microtiter-well assay, suggesting that solubilization of the platelets prior to antibody addition, as in the microtiter-well assay, alters epitope stability. Of the 31 thrombocytopenic control patients studied, all gave negative results using both assays. We conclude that these clinically adaptable assays allow detection of autoantibodies in most patients with chronic ITP, confirming the presence of an autoimmune process.

scholarly journals Platelet-associated antibody to glycoprotein IIb/IIIa from chronic immune thrombocytopenic purpura patients often binds to divalent cation- dependent antigens

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1284-1289 ◽  
Author(s):  
K Fujisawa ◽  
P Tani ◽  
R McMillan
Keyword(s):  
Divalent Cation ◽  
Immune Thrombocytopenic Purpura ◽  
Platelet Glycoprotein ◽  
Thrombocytopenic Purpura ◽  
Inhibition Ratio ◽  
Chronic Immune Thrombocytopenic Purpura ◽  
Chronic Itp ◽  
Capture Assay ◽  
Antigen Capture ◽  
Posttransfusion Purpura

Chronic immune thrombocytopenic purpura (ITP) is a syndrome of destructive thrombocytopenia due to autoantibodies against platelet- associated antigens. These antigens are most commonly located on the platelet glycoprotein (GP) IIb/IIIa complex. In the present studies, we show that many platelet-associated anti-GPIIb/IIIa autoantibodies from chronic ITP patients depend on conformationally intact GPIIb/IIIa for maximal binding. We studied anti-GPIIb/IIIa autoantibodies from 19 ITP patients (15 platelet-associated, 8 plasma) and alloantibodies from three patients with posttransfusion purpura (anti-PIA1). Antibodies were preincubated with purified intact GPIIb/IIIa, EDTA-dissociated GPIIb/IIIa, GPIIIa, or GPIIb for 2 hours and then residual antibody was measured in an antigen capture assay. The binding results were compared with those obtained using antibody preincubated in buffer. Of the 15 platelet-associated autoantibodies studied, the intact GPIIb/IIIa complex resulted in greater inhibition of antibody binding than the EDTA-dissociated complex, with a mean inhibition ratio (intact/dissociated) of 7.9 (range, 1.4 to 30.3). Little inhibition was noted using either GPIIb or GPIIIa. Conversely, plasma anti-PIA1 alloantibodies or plasma autoantibodies from ITP patients against the c- terminal region of GPIIIa were more efficiently inhibited by the dissociated complex or purified GPIIIa. We conclude that platelet- associated anti-GPIIb/IIIa autoantibodies in chronic ITP are frequently directed to cation-dependent conformational antigens.

scholarly journals Autoantibodies against platelet membrane glycoproteins in children with acute and chronic immune thrombocytopenic purpura

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1600-1602 ◽  
Author(s):  
P Berchtold ◽  
R McMillan ◽  
P Tani ◽  
S Sommerville-Nielsen ◽  
VS Blanchette
Keyword(s):  
Immune Thrombocytopenic Purpura ◽  
Platelet Glycoprotein ◽  
Membrane Glycoproteins ◽  
Pathogenetic Mechanism ◽  
Normal Range ◽  
Thrombocytopenic Purpura ◽  
Chronic Immune Thrombocytopenic Purpura ◽  
Chronic Itp ◽  
Acute Itp ◽  
Platelet Autoantibodies

Abstract The autoimmune nature of chronic immune thrombocytopenic purpura (ITP) in adults is widely accepted. In contrast, the pathogenetic mechanism in acute and chronic ITP in children is not known. In this report, we studied 39 children with destructive thrombocytopenia, 15 patients with acute ITP and 24 patients with chronic ITP. Platelet autoantibodies to platelet glycoprotein IIb/IIIa were detected in 14 of 24 patients (58.3%) in the chronic ITP group and in four of 15 (26.7%) with acute ITP. Binding ratios (+/- SD) of positive patients were significantly greater (P = .01) in chronic ITP (8.0 +/- 9.1) when compared with those of acute ITP where the binding ratios were only slightly above the normal range (1.9 +/- 0.4). The results show that autoantibodies against platelet glycoproteins are present in the majority of children with chronic ITP confirming the autoimmune nature of this disorder. The minimal elevation seen in the positive children with acute ITP suggests a different pathogenetic mechanism. These data suggest that this approach may be useful in differentiating acute from chronic ITP patients.

scholarly journals Autoantibodies against platelet glycoprotein Ib in patients with chronic immune thrombocytopenic purpura

Blood ◽  
1984 ◽  
Vol 64 (1) ◽  
pp. 156-160 ◽  
Author(s):  
VL Jr Woods ◽  
Y Kurata ◽  
RR Montgomery ◽  
P Tani ◽  
D Mason ◽  
...  
Keyword(s):  
Immune Thrombocytopenic Purpura ◽  
Control Cell ◽  
Normal Subjects ◽  
Cell Extract ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
Thrombocytopenic Purpura ◽  
Chronic Immune Thrombocytopenic Purpura ◽  
Chronic Itp ◽  
Negative Results

The present studies provide direct evidence that some patients with chronic immune thrombocytopenic purpura (ITP) have autoantibodies reactive with platelet glycoprotein Ib ( GPIb ). Microtiter wells coated with a monoclonal antibody that recognized GPIb were reacted with either platelet extract or a control cell extract. After washing and incubating with test plasma, well-bound IgG was quantitated using radioactive anti-IgG. When compared to plasma from normal subjects, plasma from 3 of 106 patients with chronic ITP had significantly increased quantities of IgG bound to microtiter wells reacted with platelet extracts. Negative results were obtained with the remaining 103 patients with chronic ITP and 59 patients with a variety of other platelet disorders. Plasma from two of the three positive patients precipitated a protein from 125I-surface-labeled platelet extract with a molecular weight similar to GPlb . One of the three patients with anti- GPlb antibody also had demonstrable autoantibodies to the platelet glycoprotein llb / llla complex.

scholarly journals Different specificities of platelet-associated and plasma autoantibodies to platelet GPIIb-IIIa in patients with chronic immune thrombocytopenic purpura

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1441-1446 ◽  
Author(s):  
K Fujisawa ◽  
P Tani ◽  
TE O'Toole ◽  
MH Ginsberg ◽  
R McMillan
Keyword(s):  
Immune Thrombocytopenic Purpura ◽  
Severe Disease ◽  
Chinese Hamster ◽  
Autoimmune Disorder ◽  
Antibody Binding ◽  
Carboxy Terminus ◽  
Thrombocytopenic Purpura ◽  
Chronic Immune Thrombocytopenic Purpura ◽  
Chronic Itp ◽  
C Terminus

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder due to antiplatelet autoantibodies, many of which are directed against platelet membrane glycoprotein (GP) IIb-IIIa or GPIb-IX. In a recent study, we described plasma autoantibodies from 13 selected ITP patients, which required the presence of the putative GPIIIa cytoplasmic region for antibody binding. Since this region may not be available for antibody binding under physiologic conditions, we evaluated the frequency of binding to this or other regions of GPIIb- IIIa by platelet-associated and plasma autoantibody from a group of chronic ITP patients. We studied platelet-associated autoantibodies in 27 patients and plasma antibodies in 21 patients; in 15 patients, both were studied. To determine if autoantibodies were directed to the cytoplasmic portion of GPIIIa or to another portion of the GPIIb-IIIa molecule, antibody eluted from patient platelets or plasma antibody was tested in an antigen capture assay for binding to GPIIb-IIIa obtained from Chinese hamster ovary (CHO) cells transfected with GPIIb and either intact GPIIIa or GPIIIa lacking the carboxy terminal 35 residues. Of the 21 plasma autoantibodies tested, 13 bound primarily to the carboxy terminus of GPIIIa and eight to other epitopes. Conversely, all 26 platelet-associated autoantibodies, including eight of the 13 with anti-carboxy terminus antibodies, bound to epitopes in other regions of GPIIb-IIIa. Comparison of the degree of antibody adsorption by intact or lysed platelets indicated that epitopes on the c-terminal region of GPIIIa are relatively inaccessible on the surface of intact washed platelets when compared with other epitopes. We conclude that the importance of plasma autoantibodies in chronic ITP patients should be interpreted cautiously, since their specificity may differ from that of antibodies bound to the platelet. Whether antibodies against the c- terminus of GPIIIa are of pathogenetic importance remains to be determined, although patients with these antibodies have particularly severe disease.

scholarly journals Autoantibodies against platelet glycoprotein Ib in patients with chronic immune thrombocytopenic purpura

Blood ◽  
1984 ◽  
Vol 64 (1) ◽  
pp. 156-160 ◽  
Author(s):  
VL Jr Woods ◽  
Y Kurata ◽  
RR Montgomery ◽  
P Tani ◽  
D Mason ◽  
...  
Keyword(s):  
Immune Thrombocytopenic Purpura ◽  
Control Cell ◽  
Normal Subjects ◽  
Cell Extract ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
Thrombocytopenic Purpura ◽  
Chronic Immune Thrombocytopenic Purpura ◽  
Chronic Itp ◽  
Negative Results

Abstract The present studies provide direct evidence that some patients with chronic immune thrombocytopenic purpura (ITP) have autoantibodies reactive with platelet glycoprotein Ib ( GPIb ). Microtiter wells coated with a monoclonal antibody that recognized GPIb were reacted with either platelet extract or a control cell extract. After washing and incubating with test plasma, well-bound IgG was quantitated using radioactive anti-IgG. When compared to plasma from normal subjects, plasma from 3 of 106 patients with chronic ITP had significantly increased quantities of IgG bound to microtiter wells reacted with platelet extracts. Negative results were obtained with the remaining 103 patients with chronic ITP and 59 patients with a variety of other platelet disorders. Plasma from two of the three positive patients precipitated a protein from 125I-surface-labeled platelet extract with a molecular weight similar to GPlb . One of the three patients with anti- GPlb antibody also had demonstrable autoantibodies to the platelet glycoprotein llb / llla complex.

scholarly journals Platelet-associated antibody to glycoprotein IIb/IIIa from chronic immune thrombocytopenic purpura patients often binds to divalent cation- dependent antigens

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1284-1289 ◽  
Author(s):  
K Fujisawa ◽  
P Tani ◽  
R McMillan
Keyword(s):  
Divalent Cation ◽  
Immune Thrombocytopenic Purpura ◽  
Platelet Glycoprotein ◽  
Thrombocytopenic Purpura ◽  
Inhibition Ratio ◽  
Chronic Immune Thrombocytopenic Purpura ◽  
Chronic Itp ◽  
Capture Assay ◽  
Antigen Capture ◽  
Posttransfusion Purpura

Abstract Chronic immune thrombocytopenic purpura (ITP) is a syndrome of destructive thrombocytopenia due to autoantibodies against platelet- associated antigens. These antigens are most commonly located on the platelet glycoprotein (GP) IIb/IIIa complex. In the present studies, we show that many platelet-associated anti-GPIIb/IIIa autoantibodies from chronic ITP patients depend on conformationally intact GPIIb/IIIa for maximal binding. We studied anti-GPIIb/IIIa autoantibodies from 19 ITP patients (15 platelet-associated, 8 plasma) and alloantibodies from three patients with posttransfusion purpura (anti-PIA1). Antibodies were preincubated with purified intact GPIIb/IIIa, EDTA-dissociated GPIIb/IIIa, GPIIIa, or GPIIb for 2 hours and then residual antibody was measured in an antigen capture assay. The binding results were compared with those obtained using antibody preincubated in buffer. Of the 15 platelet-associated autoantibodies studied, the intact GPIIb/IIIa complex resulted in greater inhibition of antibody binding than the EDTA-dissociated complex, with a mean inhibition ratio (intact/dissociated) of 7.9 (range, 1.4 to 30.3). Little inhibition was noted using either GPIIb or GPIIIa. Conversely, plasma anti-PIA1 alloantibodies or plasma autoantibodies from ITP patients against the c- terminal region of GPIIIa were more efficiently inhibited by the dissociated complex or purified GPIIIa. We conclude that platelet- associated anti-GPIIb/IIIa autoantibodies in chronic ITP are frequently directed to cation-dependent conformational antigens.

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