Preliminary Investigation about the Expression of G Protein-Coupled Receptors in Platelets from Patients with Chronic Immune Thrombocytopenic Purpura

2021 ◽  
pp. 1-9
Author(s):  
Daming Xu ◽  
Long Xie ◽  
Zewen Zhang ◽  
Duanxu Wang ◽  
Jinfeng Qiu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
G Protein ◽  
Immune Thrombocytopenic Purpura ◽  
Peripheral Blood ◽  
G Protein Coupled Receptors ◽  
Thrombocytopenic Purpura ◽  
Blood Samples ◽  
Chronic Immune Thrombocytopenic Purpura ◽  
Chronic Itp ◽  
G Protein Coupled

<b><i>Objective:</i></b> The objective of this study was to determine the expression of G protein-coupled receptors (GPCRs) in platelets from adult patients with chronic immune thrombocytopenic purpura (ITP). <b><i>Methods:</i></b> Peripheral blood samples were collected from 40 patients with chronic ITP in the Second Affiliated Hospital of Shantou University Medical College, and 40 peripheral blood samples from healthy volunteers were collected; expressions of the adenosine diphosphate receptors (P2Y1 and P2Y12), alpha-2A adrenergic receptor (α2A-AR), and thromboxane A<sub>2</sub> receptor (TP) in platelets were detected by flow cytometry. Gα protein, protease-activated receptor 1 (PAR1), and protease-activated receptor 4 (PAR4) were analyzed by Western blot and analyzed statistically. <b><i>Results:</i></b> Flow cytometry measurements of mean fluorescence intensities showed platelets from patients with chronic ITP, compared to healthy individuals, had significantly higher levels of P2Y1 (31.4 ± 2.2 vs. 7.8 ± 0.8), P2Y12 (29.6 ± 2.1 vs. 7.2 ± 1.3), α2A-AR (25.8 ± 2.9 vs. 9.8 ± 0.9), and TP (39.8 ± 3.1 vs. 4.7 ± 0.6) (all <i>p</i> &#x3c; 0.01). Similarly, integrated optical density analysis of Western blots showed that platelets from patients with chronic ITP had significantly higher levels of Gα (1046.3 ± 159.96 vs. 254.49 ± 39.51), PAR1 (832.98 ± 98.81 vs. 203.92 ± 27.47), and PAR4 (1518.80 ± 272.45 vs. 431.27 ± 41.86) (all <i>p</i> &#x3c; 0.01). <b><i>Conclusion:</i></b> Expression of GPCRs is increased in platelets from patients with chronic ITP, suggesting that platelets of chronic ITP may participate in the complicated biological process by means of GPCR-mediated signaling pathways.

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.

Treatment of Chronic Immune Thrombocytopenic Purpura with Homeopathic Dilutions of Patient Blood

2018 ◽  
Vol 25 (2) ◽  
pp. 114-116
Author(s):  
Heiner Frei
Keyword(s):  
Case Report ◽  
Oral Administration ◽  
Effective Treatment ◽  
Immune Thrombocytopenic Purpura ◽  
Capillary Blood ◽  
Normal Range ◽  
Thrombocytopenic Purpura ◽  
Chronic Immune Thrombocytopenic Purpura ◽  
Chronic Itp ◽  
Treatment Onset

Background: Conventional or homeopathic treatment of chronic immune thrombocytopenic purpura (ITP) is often difficult. The use of homeopathic dilutions of patient blood (HPB) for immunomodulation has been described, which inspired us to try the method in an ITP case. Case Report: A 2-year-old girl with chronic ITP was treated with homeopathic dilutions of her own capillary blood, given orally over 5 months. Immediately after treatment onset there was a rapid normalization of the thrombocyte counts. Within 6 weeks, they rose from 15,000/µl to 254,000/µl. After treatment stop, they decreased to 155,000/µl, increased again spontaneously to 270,000/µl and remained within normal range for over 3 years. Conclusions: Oral administration of homeopathic dilutions of capillary patient blood may possibly be an effective treatment in chronic ITP. If our results can be reproduced, this will revolutionize the treatment of ITP.

Sphingosine 1-Phosphate (S1P) Induces Migration and ERK/MAP-Kinase-Dependent Proliferation in Chronic Lymphocytic Leukemia (B-CLL) Due to Expression of the G Protein-Coupled Receptors S1P1/4.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4996-4996
Author(s):  
Gabriele Seitz ◽  
Sedat Yildirim ◽  
Andreas M. Boehmler ◽  
Lothar Kanz ◽  
Robert Möhle
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
G Protein ◽  
Cell Lines ◽  
Peripheral Blood ◽  
G Protein Coupled Receptors ◽  
Lymphocytic Leukemia ◽  
S1p Receptors ◽  
Sphingosine 1 Phosphate ◽  
G Protein Coupled

Abstract Egress of lymphocytes from lymphoid organs into the circulation has been shown to depend on the presence of the lipid mediator sphingosine 1-phosphate (S1P) in the peripheral blood, and expression of corresponding S1P receptors (i.e., S1P1), that belong to the family of 7-transmembrane G protein-coupled receptors (GPCR). As circulating lymphocytic lymphoma cells are a hallmark of chronic lymphocytic leukemia, we analyzed expression of different S1P receptors and the effects of S1P on B-CLL cells. By qualitative and quantitative (TaqMan) RT-PCR, significant mRNA expression of S1P1 and S1P4 was found in CLL cell lines (EHEB, MEC-1) and in most samples (S1P1 in 88%, S1P4 in 100%) of primary CD19+ cells isolated from the peripheral blood of untreated B-CLL patients. mRNA of other S1P receptors (S1P2, S1P3, S1P5) was less consistently detected. Normal, nonmalignant B cells were strongly positive for S1P1, while other S1P receptors were weakly expressed or negative. S1P induced typical effects of chemotactic GPCR, such as actin polymerization (analyzed by flow cytometry) and chemotaxis (measured in a modified Boyden chamber assay) in CLL cell lines and primary B-CLL cells. After serum deprivation in vitro, S1P induced phosphorylation of ERK/MAP-kinase as analyzed by Western blot, demonstrating that S1P receptors expressed in CLL were able to activate signaling pathways of GPCR not only related to cell migration and chemotaxis, but also to cell proliferation. Of note, the S1P1 ligand FTY720, which induces receptor internalization after prolonged exposure and acts as an antagonist, resulted in apoptosis in CLL cell lines and primary CLL cells in vitro, as measured by MTT-test and staining with Annexin-FITC, respectively. We conclude that sphingosine 1-phosphate, which is present in the peripheral blood in considerable amounts, contributes to the trafficking of B-CLL cells expressing the GPCRs S1P1/4, and to their prolonged survival.

Profiles of Th1, Th2, Th17 and Treg Cells in Patients with Chronic Idiopathic Thrombocytopenic Purpura.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3404-3404
Author(s):  
Rong-Fu Zhou ◽  
Jian Ou-yang ◽  
Da-Yu Chang ◽  
Jing-Yan Xu ◽  
Bing Chen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Idiopathic Thrombocytopenic Purpura ◽  
Peripheral Blood ◽  
Th17 Cells ◽  
Treg Cells ◽  
Chronic Idiopathic Thrombocytopenic Purpura ◽  
Active Stage ◽  
Thrombocytopenic Purpura ◽  
Chronic Itp ◽  
Ifn Γ

Abstract Objective: To explore the profiles of Th1,Th2, Th17 and Treg cells in patients with chronic idiopathic thrombocytopenic purpura. Methods: Samples of peripheral blood were collected from 30 chronic ITP patients ( 9 males and 21 females), aged 41, 21 being in active stage, and 9 in remission stage, and 9 healthy persons in control (3 males and 6 females), aged 36. Peripheral blood was cultured, and activated with PMA/ionomycin when Th1, Th2 and Th17 cells were detected. Flow cytometry was used to measure the intracellular cytokines interferon (IFN)-γ, interleukin (IL)-4 and interleukin (IL)-17 so as to identify the Th1 cells (CD3+ CD8− IFN-γ+ IL-4− cells), Th2 cells (CD3+ CD8− IFN-γ − IL-4+ cells) and IL-17 cells (CD3+ CD8− IL-17+ cells); Treg cells were identified to CD4+ CD25+ Foxp3+ cells and uncultured peripheral blood was used to measured the CD4+ CD25+ Foxp3+ cells by flow cytometry. The ratios of Th1/Th2 were calculated. Results: The Th1/Th2 ratio for patients in active stage was 15.04±9.67, significantly higher than those for patients in remission stage (7.17±5.38, P <0.05) and in control (8.47±3.78, P <0.05); the percentage of Treg cells of the patients in active stage was 0.89±0.58%, significantly decreased than those of patients in remission stage (6.41±1.86%, P <0.001) and in control (6.06±0.85%, P <0.001); the percentage of Th17 cells was 1.94±0.77% for patients in active stage, 2.16±0.52% for patients in remission stage and 1.82±0.58% for patients in control, respectively, and there was no statistic significance between them. Conclusion: Chronic ITP is a Th1 predominant disease; decreased number and function of Treg cells might be one of mechanisms that cause immune regulation dysfunction in chronic ITP; Th17 cells might not play a role in the development of chronic ITP.

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 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.

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.

Rituximab efficacy and safety in adult splenectomy candidates with chronic immune thrombocytopenic purpura: results of a prospective multicenter phase 2 study

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 999-1004 ◽  
Author(s):  
Bertrand Godeau ◽  
Raphael Porcher ◽  
Olivier Fain ◽  
François Lefrère ◽  
Pierre Fenaux ◽  
...  
Keyword(s):  
Immune Thrombocytopenic Purpura ◽  
Phase 2 ◽  
Open Label ◽  
Thrombocytopenic Purpura ◽  
Platelet Counts ◽  
Chronic Immune Thrombocytopenic Purpura ◽  
Chronic Itp ◽  
Phase 2 Trial ◽  
Phase 2 Study

Abstract Whether rituximab could effectively and safely avoid splenectomy for adults with chronic immune thrombocytopenic purpura (ITP) remains unresolved. A multicenter, prospective, open-label, single-arm, phase 2 trial was conducted to assess rituximab safety and efficacy in adult splenectomy candidates with chronic ITP. Sixty patients with chronic (≥ 6 months) ITP and platelet counts less than 30 × 109/L received a weekly intravenous infusion of rituximab (375 mg/m2) for 4 weeks. All other ITP treatments were stopped. A good response was defined as a platelet count 50 × 109/L or more, with at least a doubling of the initial value at 1 and 2 years after the first rituximab infusion. Patients who required another treatment during follow up were considered nonresponders. Sixteen patients experienced transient side effects that necessitated treatment discontinuation for only 1. Good 1-year responses were obtained in 40% of the patients (24/60 [95% confidence interval: 28%-52%]). At 2 years, 33.3% (20/60 patients) had good responses and 6.7% (4/60) had sustained platelet counts of 30 × 109/L or more without treatment. Thirty-six (60%) patients failed to respond; 25 underwent splenectomy. Based on these results, rituximab was an apparently safe and effective splenectomy-avoiding option in some adults with chronic ITP. This trial is registered at http://clinicaltrials.gov as NCT00225875.

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