Natural history of platelet antibody formation against αIIbβ3 in a French cohort of Glanzmann thrombasthenia patients

Treatment of the bleeding syndrome in Glanzmann thrombasthenia (GT) is often complicated by naturally occurring isoantibodies directed against the αIIbβ3 integrin that cause the removal of or render ineffective transfused donor platelets. Such antibodies are produced after transfusion or pregnancy when the patient's immune system comes into contact with normal platelets. Despite many reports of anti-αIIbβ3 antibodies in GT patients, there is no consensus pertaining to their frequency, their long-term evolution in the circulation, or their formation in relation to either (i) the extent of the αIIbβ3 deficiency in the patient's platelets or (ii) the nature of the genetic defect (ITGA2B or ITGB3 genes). Antibody screening was performed on a large series of 24 GT patients in South-West France dividing the patients into two cohorts: (i) 16 patients with the French gypsy mutation (c.1544 + 1G>A) within ITGA2B that gives platelets totally lacking αIIbβ3 and (ii) 8 patients carrying other defects of ITGA2B or ITGB3 with different expression levels of αIIbβ3. Our results confirm that patients with premature termination mutations resulting in platelets lacking αIIbβ3 are the most susceptible to form isoantibodies, a finding that may be useful in deciding the choice of therapy between platelet transfusion and the use of recombinant factor VIIa (FVIIa).     

Identification of the integrin β3 L718P mutation in a pedigree with autosomal dominant thrombocytopenia with anisocytosis

αIIbβ3 integrin mutations that result in the complete loss of expression of this molecule on the platelet surface cause Glanzmann thrombasthenia. This is usually autosomal recessive, while other mutations are known to cause dominantly inherited macrothrombocytopenia (although such cases are rare). Here, we report a 4‐generation pedigree including 10 individuals affected by dominantly inherited thrombocytopenia with anisocytosis. Six individuals, whose detailed clinical and laboratory data were available, carried a non‐synonymous ITGB3 gene alteration resulting in mutated integrin β3 (ITGB3)‐L718P. This mutation causes partial activation of the αIIbβ3 complex, which promotes the generation of abnormal pro‐platelet‐like protrusions through downregulating RhoA (RHOA) activity in transfected Chinese Hamster Ovary cells. These findings suggest a model whereby the integrin β3‐L718P mutation contributes to thrombocytopenia through gain‐of‐function mechanisms.     

Castleman disease: delineating the spectrum

Glanzmann thrombasthenia (GT) is caused by inherited defects of the α_IIbβ₃ platelet glycoprotein. This bleeding disorder can be treated with platelet transfusion therapy, but some patients will be immunized and begin to form anti‐human leucocyte antigen (HLA) and/or anti‐α_IIbβ₃ antibodies. These antibodies can bind and interfere with the function of the transfused platelets, rendering treatment ineffective. However, platelet transfusion refractoriness attributable to HLA antibodies may be managed by the selection of compatible donors, although they are not always readily available, particularly in an emergency. Thus, anti‐α_IIbβ₃ antibodies represent one of the most severe complications in GT. Both genetic and environmental factors may contribute to the risk of anti‐α_IIbβ₃ development, but the underlying pathogenic mechanisms are still unknown. This review will summarize the current knowledge of the risk factors for development of anti‐α_IIbβ₃ antibodies in patients with GT and discuss how these findings may influence the clinical management of patients.     

WASP plays a novel role in regulating platelet responses dependent on αIIbβ3 integrin outside‐in signalling

The most consistent feature of Wiskott Aldrich syndrome (WAS) is profound thrombocytopenia with small platelets. The responsible gene encodes WAS protein (WASP), which functions in leucocytes as an actin filament nucleating agent –yet– actin filament nucleation proceeds normally in patient platelets regarding shape change, filopodia and lamellipodia generation. Because WASP localizes in the platelet membrane skeleton and is mobilized by αIIbβ3 integrin outside‐in signalling, we questioned whether its function might be linked to integrin. Agonist‐induced αIIbβ3 activation (PAC‐1 binding) was normal for patient platelets, indicating normal integrin inside‐out signalling. Inside‐out signalling (fibrinogen, JON/A binding) was also normal for wasp‐deficient murine platelets. However, adherence/spreading on immobilized fibrinogen was decreased for patient platelets and wasp‐deficient murine platelets, indicating decreased integrin outside‐in responses. Another integrin outside‐in dependent response, fibrin clot retraction, involving contraction of the post‐aggregation actin cytoskeleton, was also decreased for patient platelets and wasp‐deficient murine platelets. Rebleeding from tail cuts was more frequent for wasp‐deficient mice, suggesting decreased stabilisation of the primary platelet plug. In contrast, phosphatidylserine exposure, a pro‐coagulant response, was enhanced for WASP‐deficient patient and murine platelets. The collective results reveal a novel function for WASP in regulating pro‐aggregatory and pro‐coagulant responses downstream of integrin outside‐in signalling.     

Immunotherapy with the trifunctional anti‐CD20 x anti‐CD3 antibody FBTA05 (Lymphomun) in paediatric high‐risk patients with recurrent CD20‐positive B cell malignancies

Children with B cell malignancies refractory to standard therapy are known to have a poor prognosis and very limited treatment options. Here, we report on the treatment and follow‐up of ten patients diagnosed with relapsed or refractory mature B‐cell Non Hodgkin Lymphoma (B‐NHL), Burkitt leukaemia (B‐AL) or pre B‐acute lymphoblastic leukaemia (pre B‐ALL). All children were treated with FBTA05 (now designated Lymphomun), an anti‐CD3 x anti‐CD20 trifunctional bispecific antibody (trAb) in compassionate use. Within individual treatment schedules, Lymphomun was applied (a) after allogeneic stem cell transplantation (allo‐SCT, n = 6) to induce sustained long‐term remission, or (b) stand alone prior to subsequent chemotherapy to eradicate residual disease before allo‐SCT (n = 4). Nine of ten children displayed a clinical response: three stable diseases (SD), one partial remission (PR) and five induced or sustained complete remissions (CR). Five of these nine responders died during follow‐up. The other patients still maintain CR with a current overall survival of 874–1424 days (median: 1150 days). In conclusion, despite the dismal clinical prognosis of children refractory to standard therapy, immunotherapy with Lymphomun resulted in a favourable clinical outcome in this cohort of refractory paediatric patients.     

Platelet gene therapy improves hemostatic function for integrin alphaIIbbeta3-deficient dogs

Activated blood platelets mediate the primary response to vascular injury. Although molecular abnormalities of platelet proteins occur infrequently, taken collectively, an inherited platelet defect accounts for a bleeding diathesis in ≈1:20,000 individuals. One rare example of a platelet disorder, Glanzmann thrombasthenia (GT), is characterized by life-long morbidity and mortality due to molecular abnormalities in a major platelet adhesion receptor, integrin αIIbβ3. Transfusion therapy is frequently inadequate because patients often generate antibodies to αIIbβ3, leading to immune-mediated destruction of healthy platelets. In the most severe cases allogeneic bone marrow transplantation has been used, yet because of the risk of the procedure it has been limited to few patients. Thus, hematopoietic stem cell gene transfer was explored as a strategy to improve platelet function within a canine model for GT. Bleeding complications necessitated the use of a mild pretransplant conditioning regimen; therefore, in vivo drug selection was used to improve engraftment of autologously transplanted cells. Approximately 5,000 αIIbβ3 receptors formed on 10% of platelets. These modest levels allowed platelets to adhere to αIIbβ3's major ligand (fibrinogen), form aggregates, and mediate retraction of a fibrin clot. Remarkably, improved hemostatic function was evident, with ≤135-fold reduced blood loss, and improved buccal bleeding times decreased to 4 min for up to 5 y after transplant. One of four transplanted dogs developed a significant antibody response to αIIbβ3 that was attenuated effectively with transient immune suppression. These results indicate that gene therapy could become a practical approach for treating inherited platelet defects.     

Significance of anti‐α‐fodrin autoantibody in diagnosis of Sjogren's syndrome

Objective: Anti‐α‐fodrin autoantibody has been reported to be a highly specific and sensitive test for the diagnosis of Sjogren's syndrome (SS). The objective of our report is to investigate the sensitivity and specificity of anti‐α‐fodrin antibody in patients with SS and its correlation with clinical manifestations. Methods: Recombinant human α‐fodrin was used as envelope antigen in enzyme‐linked immunosorbent assay (ELISA) to detect the relatively specific autoantibody in sera of 42 primary SS, 24 secondary SS with systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA), and 40 other connective tissue diseases (CTDs) (SLE 17, RA 8, ankylosing spondylitis 5, dermatomyositis 5, systemic schlerosis 3, mixed connective tissue disease 1, Takayasu's disease 1) patients. Results: Antibodies against α‐fodrin were present in 59.5% of primary SS patients, 31.5% of secondary SS patients, 35.0% and 11.3% of other CTD patients and controls, respectively; the specificity of anti‐α‐fodrin antibody was 79.4% in patients with SS. It showed no significant difference between primary and secondary SS (P > 0.05), as well as SS compared with other CTD patients (P > 0.05). The positive rates of antibodies against α‐fodrin in CTD patients were significantly higher than those in non‐CTD patients and normal controls (P < 0.01). The presence of anti‐α‐fodrin antibodies has no significant correlation with clinical manifestations or other autoantibodies, while the levels of sera IgG and erythrocyte sedimentation rate (ESR) are higher in α‐fodrin antibody‐positive patients (IgG: 23.2 vs. 18.6, P < 0.05; ESR: 52.9 vs. 37.1, P < 0.05) than α‐fodrin antibody‐negative patients. Anti‐α‐fodrin antibodies are all negative in anti‐SS antigen A and antinuclear antibody‐negative SS patients. Conclusion: The result showed a lower sensitivity and specificity for anti‐α‐fodrin antibody as a diagnostic marker of SS, compared with previous reports. Anti‐α‐fodrin antibodies had no significant association with clinical manifestations, but might be related to the sera IgG level. Antibodies against α‐fodrin played no important roles in diagnosis of antibody‐negative SS patients.     

The infectious profiles of anti‐tumor necrosis factor agents in a Thai population: a retrospective study a the university‐based hospital

Aims: Anti‐tumour necrosis factor‐α (anti‐TNF) agents represented treatment advances in a number of rheumatologic diseases. However, adverse effects of anti‐TNF agents have been identified through both clinical trials and post‐marketing surveillance, especially an increased risk of serious infections. This study firstly described the infectious profiles of anti‐TNF agents in a Thai population. Methods: We retrospectively reviewed all infectious incidences from 100 consecutive medical records of patients who were treated with either etanercept or infliximab for any rheumatologic and non‐rheumatologic conditions. Results: Indications for anti TNF‐α agents were mainly rheumatoid arthritis (46%) and spondyloarthropathy (SpA) (41%). Seventy‐seven patients were treated with etanercept and 23 with infliximab. For those whose initial treatment was etanercept, there were two events of suspected active pulmonary tuberculosis (TB) and suspected hepatitis‐B virus (HBV) reactivation. Two out of 23 patients (8.7%) who were firstly treated with infliximab had herpes zoster skin infection. Incidence of overall infection before anti‐TNF treatment were significantly higher in patient who started with etanercept (0.065 vs. 0.019 cases per person‐years in etanercept and infliximab respectively, P < 0.0001). Incidence of overall infection post‐anti‐TNF treatment were 0.122 and 0.201 cases per person‐years in patients who started with etanercept and infliximab with no significant difference (P > 0.05). The overall infection rates were significantly increased after infliximab treatment (P < 0.0001). Conclusion: Even thought there were two new events of TB and HBV reactivation after etanercept treatment, incidence of overall infection seemed to be increased after infliximab treatment. The infectious screening and monitoring with high index of suspicion as well as the pre‐emptive treatment are still important whenever either etanercept or infliximab is started.     

Extended indications for anti‐tumor necrosis factor‐α therapy

Tumour necrosis factor‐α is a pleiotropic cytokine which has a broad range of actions in inflammation, infection and immunity. TNF‐α is supposed to play a crucial role in the pathogenesis of various autoimmune diseases. TNF‐α blocking agents have been demonstrated to be highly effective in the treatment of rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and juvenile rheumatoid arthritis. TNF‐α inhibitors also have been tried with other rheumatic diseases and have emerged as promising treatments. We here review the current evidences of effectiveness of the anti‐TNF‐α therapy in various autoimmune diseases.     

αIIbG236E causes Glanzmann thrombasthenia by impairing association with β3

Glanzmann thrombasthenia (GT) is a recessively inherited bleeding disorder caused by the quantitative or qualitative deficiency of the platelet fibrinogen receptor, integrin αIIbβ3. The N-terminal domain of the αIIb subunit is folded in a β-propeller that plays the role of binding fibrinogen and associating with the ligand-binding region of β3. Analysing the mutations of Italian GT patients we found that a patient had a αIIb G236E missense substitution that substitutes a glycine from the highly conserved ΦΦGΦ motif of blade 4 of the β-propeller. To verify experimentally the effect of the substitution of glycine 236 human embryonic kidney (HEK) cells were transfected with normal or mutated αIIb in conjunction with normal β3. Using flow cytometry analysis we found the percentage of HEK cells transfected with αIIbG236Eβ3 that reacted with anti αIIbβ3 was very low. In HEK cells transfected with either αIIbβ3 or αIIbG236Eβ3 and lysed, when immunoblotting was done in non-reducing conditions a band reacting with an antibody against αIIb was present in both lysates, although less intense in cells transfected with αIIbG236Eβ3. In reducing condition αIIb from cells transfected with αIIbβ3 was nearly all mature, while in cells transfected with αIIbG236Eβ3 the ratio pro-αIIb: αIIb was 1 : 1, with signs of degradation of the mutated protein. Cell lysates were then immunoprecipitated with antibodies against αIIb and immunoblotted with an antibody reacting with β3. While in immunoblots from cells transfected with αIIbβ3 a band corresponding to β3 was strongly detectable, in immunoblots originating from cells transfected with αIIbG236Eβ3 no band at the same level of normal β3 was detected. Immunofluorescence studies showed accumulation of αIIbG236Eβ3 in the endoplasmic reticulum and minimal transport to the Golgi. In conclusion we demonstrated that the αIIbG236E mutation causes GT by impairing the association with β3 during biogenesis of the receptor.     

Platelet Alloimmunization after Transfusion

Background and objectives: The frequency of platelet-specific antibodies after one series of blood transfusions has not been reported, and in multiply transfused patients is controversial. Materials and methods: We studied the frequency of alloimmunization against platelet antigens in 117 patients who received a single series of blood transfusions. They received mostly saline-adenine-glucose + mannitol red blood cell components (poor in leukocytes and platelets) in connection with cardiac surgery. Platelet-specific antibodies were detected with the platelet ELISA and the monoclonal-antibody-specific immobilization of platelet antigen assay. HLA antibodies were detected by the standard lymphocyte cytotoxicity techniques. Results: We found platelet-specific anti-HPA-5b (anti-Br^a) in 2 cases (1.7%). One antibody was the result of de novo immunization. We detected lymphocytotoxic HLA antibodies in 21 patients (17.9%), of whom 18 (15.4%) had had no detectable antibodies before transfusion. There was a positive correlation between the transfused load of immunogenic materials and the frequency of alloimmunization against HLA antigens. In one third of the immunized patients, there was no history of previous immunization. Conclusion: There was a low incidence of platelet-specific antibodies after one series of blood transfusions in this group of patients. This is similar to the results of some previous studies in multiply transfused patients, but not with those of others who found a higher incidence.     

Associations of HLA‐DRB1 alleles with anti‐citrullinated protein antibody‐positive and anti‐citrullinated protein antibody‐negative rheumatoid arthritis in northern east part of Turkey

Aim: The aim of this study was to investigate the associations between human leukocyte antigen (HLA)‐DRB1 alleles with genetic susceptibility to rheumatoid arthritis (RA) and production of antibodies against cyclic citrullinated peptide (anti‐CCP antibody) and rheumatoid factor (RF) in Turkish RA patients. Methods: We studied 291 RA patients and 253 controls. Genotyping was performed by polymerase chain reaction with sequence‐specific oligonucleotide probes hybridization method. Serum levels of anti‐CCP antibody, IgM‐RF and high sensitive C‐reactive protein titers were measured by commercial kits using immunological methods. Results: We found that HLA‐DRB1*04 and *09 alleles were associated in anti‐CCP+ and anti‐CCP+ RA patients (P < 0.0001 and P < 0.001, respectively), while DRB1*01 and *04 were determined to be higher in RF+ RA patients (P < 0.001 and P < 0.0001, respectively). Moreover, DRB1*11 and DRB1*13 alleles were determined to be lower in RF and anti‐CCP/RF+ RA patients (P < 0.001 for both). HLA‐DRB1*04 was identified as a common responsible allele for susceptibility to the disease in anti‐CCP, RF and anti‐CCP/RF? RA patients (P = 0.0018, P = 0.0004 and P = 0.0023, respectively). HLA‐DRB1*13 allele alone was found to be protective against to anti‐CCP+ and RF? RA (P = 0.0003 and P = 0.006, respectively). On the contrary, there was no protective allele in anti‐CCP/RF? RA as well as anti‐CCP? RA patients. Conclusion: This study indicates that associate and protective HLA‐DRB1 allele distributions are different in autoantibody (anti‐CCP or RF or anti‐CCP/RF)+ RA and autoantibody? RA patients, with exceptions of DRB1*04 and DRB1*13.     

‘It's magic stuff’: The experiences of patients with ankylosing spondylitis taking anti‐TNF‐α medication

Introduction: Several studies have identified the efficacy of anti‐tumour necrosis factor‐alpha (anti‐TNF‐α) treatment in ankylosing spondylitis (AS). However, few studies have explored the perceptions of patients taking this new medication. The aim of this study was to explore the impact of anti‐TNF‐α on the quality of life of people with AS. Methods: A qualitative approach was adopted to provide a holistic understanding of participants' views and experiences in the context of their overall lives. Semi‐structured interviews were undertaken and transcribed verbatim. Data were analysed using thematic analysis. Ethical approval and informed consent were obtained. Results: Eight people participated and described a significant improvement in their physical and psychological status, leading to a more positive outlook on their life. Specific areas highlighted were employment, activities of daily living, hobbies and relationships with partners and family, some of which are not captured by current AS‐specific outcome measures. Negative aspects of anti‐TNF‐α use were described as the inconvenience of monitoring and issues relating to travelling abroad. All participants expressed concern about the possibility of being withdrawn from treatment and the perceived impact this would have on their lives. Conclusions: Anti‐TNF‐α treatment has a positive impact on the lives of people with AS, such that a major concern is being withdrawn from treatment, highlighting the need to provide tailored support to people being withdrawn from treatment. To capture the full impact of anti‐TNF‐α treatment, further consideration needs to be given to the choice of appropriate outcome measures. Copyright © 2008 John Wiley & Sons, Ltd.     

Regulation of pulmonary inflammation and fibrosis through expression of integrins αVβ3 and αVβ5 on pulmonary T lymphocytes

Objective

Pulmonary diseases associated with fibrosis, including scleroderma lung disease, are characterized by the accumulation of T cells in the lungs. These cells are thought to facilitate lung fibrosis, but the exact mechanisms of their profibrotic action are not clear. Several αV‐containing integrins, including αVβ3 and αVβ5, have been shown to directly activate transforming growth factor β (TGFβ) and promote collagen accumulation. The aim of this study was to investigate whether pulmonary T cells express profibrotic integrins and regulate collagen accumulation.

Methods

Expression of integrins was assessed by immunohistochemical analysis of lung tissue, by flow cytometry using bronchoalveolar lavage fluid from patients with systemic sclerosis (SSc), and in a CCL18 overexpression animal model of pulmonary T cell infiltration. Experiments in cell cultures were performed to determine whether integrin‐expressing T cells are profibrotic in cocultures with pulmonary fibroblasts and, if so, through what possible mechanism.

Results

Lymphocytes and integrin‐positive cells were present in the lungs, and pulmonary T cells expressed integrins αVβ3 and αVβ5 in patients with SSc and in the animal model. Systemic administration of neutralizing anti–integrin αV antibody or a genetic deficiency of integrin β3 in the CCL18 overexpression model significantly attenuated CCL18‐driven pulmonary lymphocytic infiltration and collagen accumulation. Jurkat T cells overexpressing integrin αVβ3 or integrin αVβ5 in cocultures with primary pulmonary fibroblasts stimulated collagen accumulation and Smad2 nuclear translocation. Neutralizing anti‐TGFβ antibody attenuated the profibrotic effect of integrin‐expressing T cells.

Conclusion

Pulmonary infiltrating T lymphocytes may express integrins αVβ3 and αVβ5 that are necessary for lymphocytic infiltration and T cell–associated TGFβ activation and collagen accumulation.

     

Mutations of the integrin αIIb/β3 intracytoplasmic salt bridge cause macrothrombocytopenia and enlarged platelet α‐granules

Rare gain‐of‐function mutations within the ITGA2B or ITGB3 genes have been recognized to cause macrothrombocytopenia (MTP). Here we report three new families with autosomal dominant (AD) MTP, two harboring the same mutation of ITGA2B, αIIbR995W, and a third family with an ITGB3 mutation, β3D723H. In silico analysis shows how the two mutated amino acids directly modify the salt bridge linking the intra‐cytoplasmic part of αIIb to β3 of the integrin αIIbβ3. For all affected patients, the bleeding syndrome and MTP was mild to moderate. Platelet aggregation tended to be reduced but not absent. Electron microscopy associated with a morphometric analysis revealed large round platelets; a feature being the presence of abnormal large α‐granules with some giant forms showing signs of fusion. Analysis of the maturation and development of megakaryocytes reveal no defect in their early maturation but abnormal proplatelet formation was observed with increased size of the tips. Interestingly, this study revealed that in addition to the classical phenotype of patients with αIIbβ3 intracytoplasmic mutations there is an abnormal maturation of α‐granules. It is now necessary to determine if this feature is a characteristic of all mutations disturbing the αIIb R995/β3 D723 salt bridge.     

Glanzmann thrombasthenia-like syndromes associated with Macrothrombocytopenias and mutations in the genes encoding the αIIbβ3 integrin

Glanzmann thrombasthenia (GT) is the most widely studied inherited disorder of platelets; it is caused by the absence of platelet aggregation due to quantitative and/or qualitative deficiencies of the αIIbβ3 integrin coded by the ITGA2B and ITGB3 genes located at 17q21-23. Although platelet count and platelet volume (and morphology) are normal in classic GT, some reports have inferred a role for αIIbβ3 in megakaryocytopoiesis and some novel but rare point mutations in either of the ITGA2B and ITGB3 genes have been associated with an altered platelet production and selective deficiencies in platelet function. This was brought to light by the discovery of mutations at Arg995 in αIIb and Asp723 in β3 that lead to platelet anisotropy (increased size variation) and thrombocytopenia. Significantly, Arg995 and Asp723 form a salt linkage binding the cytoplasmic tails of αIIbβ3 together keeping the integrin in a bent resting state. Mutations weakening this link (if not abolishing it) increase the activation state of αIIbβ3 and interfere with megakaryocytopoiesis. Other mutations affecting platelet production involve extracellular but membrane proximal domains of β3. Our purpose is to review the mutations in the ITGA2B and ITGB3 genes that lead to anisotropy and to discuss mechanisms by which this can be brought about.     

Rapid characterization of hybridomas producing monoclonal antibodies against platelet β3 integrin using ELIspot

Generally, B-cell responses against human platelet antigens are assessed by the serological detection of specific platelet antibodies, mostly against β3 integrin. However, this approach seems to be of low sensitivity, since platelet autoantibodies against αIIbβ3 are detected in only 50% of all patients with immune thrombocytopenia (ITP). In this study, a novel B-cell ELIspot method was established to characterize the specificity of mouse monoclonal antibodies (moabs) against human β3 integrin. Moabs produced by hybridomas were immobilized on membrane and bound antibodies were visualized as spots using biotinylated recombinant proteins αIIbβ3 or αvβ3 and the enzyme labeled streptavidin-substrate system. Three hybridomas, Gi5, Gi16 and AP3, designated previously as anti-αIIbβ3, anti-αIIb and anti-β3, respectively, were investigated. Hybridoma producing moab against CD177 was used as the negative control. Whereas AP3 reacted with αIIbβ3 and αvβ3, Gi5 only formed spots with αIIbβ3. Titration analysis showed that the number of spots correlated significantly with the number of seeded cells. Approximately 15 antibody producing hybridoma cells could be identified among 10³ nonproducing B-cells. Furthermore, superior correlation with the total number of IgG producing cells was obtained. Analysis of the third hybridoma, Gi16 (anti-αIIb), showed only few spots with αIIbβ3, indicating that this hybridoma contained different clones (producer and non-producer). Significant increased number of spots could be identified after re-cloning of these clones by limiting dilution method. Our results demonstrate that this B-cell ELIspot assay can be used for the identification of a small number of hybridoma cells producing moabs against β3 integrin, verification of their monoclonality, productivity and for determining their specificity in the early state of workup steps. In the future, this approach may be useful to define B-cell clones in patients who developed platelet antibodies against different β3-integrins and to differentiate their diversities.     

Use of allogeneic stem cell transplantation for moderate–severe Glanzmann thrombasthenia

Glanzmann thrombasthenia (GT) is a rare, autosomal recessive coagulopathy characterized by either qualitative or quantitative abnormalities of the membrane glycoprotein αIIbβ3 complex leading to bleeding tendencies, ranging from purpura to life-threatening hemorrhage. Although patients can be managed with supportive measures including platelet transfusions, complications such as alloimmunization are possible. Allogeneic stem cell transplantation (ASCT) can be indicated in severe cases of GT. We report the case of an eight-month-old girl diagnosed with moderate–severe GT, who was successfully treated with a reduced-intensity, human leukocyte antigen (HLA)-identical ASCT.     

A novel Pro126His beta propeller mutation in integrin alphaIIb causes Glanzmann thrombasthenia by impairing progression of pro-alphaIIbbeta3 from endoplasmic reticulum to Golgi

BackgroundGlanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation in response to most physiological agonists and caused by either a lack or dysfunction of the platelet integrin αIIbβ3 (glycoprotein IIb/IIIa).PatientsMucocutaneous bleeding manifestations and platelet dysfunction consistent with GT were observed in a 20-year-old proband of a Chinese family.ObjectivesTo determine the molecular basis of GT and characterize the mutation by in vitro expression studies.ResultsAnalysis of the patient's platelets by fluorescence-activated cell sorting demonstrated the presence of trace amounts of β3, exposed on her platelet surface, but a complete absence of αIIbβ3. Sequence analysis revealed a novel C470A transversion in exon 4 of the αIIb gene predicting a Pro126His alteration in the blade 2 of the αIIb β propeller domain. The proband was homozygous for the mutation, the mother and the father were heterozygous, whereas 100 healthy subjects lacked this transversion. Chinese hamster ovary cells cotransfected with cDNAs of mutated αIIb and wild-type β3 failed to express αIIbβ3 on the cell surface as shown by FACS. Western blot analysis of the cell lysates showed no detectable mature αIIb. Immunoprecipitation with antibody against β3 demonstrated pro-αIIb in the cells expressing the mutant αIIbβ3, indicating pro-αIIbβ3 complex formation. Intracellular immunofluorescence studies demonstrated the pro-αIIbβ3 complex that co-localized with an ER marker, but showed minimal co-localization with a Golgi marker.ConclusionsA novel Pro126His mutation in αIIb compromised transport of the pro-αIIbβ3 complex from the endoplasmic reticulum to the Golgi, leading to intracellular retention. The impaired αIIbβ3 transport is responsible for the thrombasthenia in this patient.     

The effect of rituximab on anti‐platelet autoantibody levels in patients with immune thrombocytopenia

Rituximab is an effective therapy resulting in a platelet count improvement in 60% of patients with immune thrombocytopenia (ITP). Rituximab depletes B cells; thus, a reduction in platelet autoantibody levels would be anticipated in patients who achieve a clinical response to this treatment. The objectives of this study were to determine whether rituximab was associated with a reduction in platelet autoantibody levels, and to correlate the loss of autoantibodies with the achievement of a treatment response. We performed a case‐control study nested within a previous randomized controlled trial of standard therapy plus adjuvant rituximab or placebo. We measured platelet‐bound anti‐glycoprotein (GP) IIbIIIa and anti‐GPIbIX using the antigen capture test. Of 55 evaluable patients, 25 (45%) had a detectable platelet autoantibody at baseline. Rituximab was associated with a significant reduction in anti‐GPIIbIIIa levels (P = 0·02) but not anti‐GPIbIX levels (P = 0·51) compared with placebo. Neither the presence of an autoantibody at baseline nor the loss of the autoantibody after treatment was associated with a response to rituximab. The subset of patients with persistent autoantibodies after treatment failed to achieve a platelet count response, suggesting that persistence of platelet autoantibodies can be a marker of disease severity.