Immunization against αIIbβ3 and αvβ3 in Glanzmann thrombasthenia patients carrying the French Gypsy mutation

Abstract

The c.1544+1G>A mutation was identified in Gypsy Glanzmann thrombasthenia (GT) patients. Gypsy GT patients express normal αvβ3 carrying HPA‐1b epitopes. To demonstrate HPA‐1a alloimmunization by modified antigen capture assays. Gypsy GT patients could develop anti‐HPA‐1a alloantibodies against β3 and αvβ3. Abstract BackgroundGlanzmann thrombasthenia (GT) is a rare bleeding disorder caused by the absence or the dysfunction of the platelet αIIbβ3 integrin. A founder mutation in the ITGA2B gene was previously identified in French Gypsy patients. Interestingly, this mutation was strongly linked to the human platelet antigen‐1b (HPA‐1b). The HPA‐1bb Gypsy patients are at risk of isoimmunization against αIIbβ3, as this complex is not expressed at their platelet surface. Tentatively, they would, however, not have an increased risk of developing anti‐HPA‐1a alloantibodies by exposure of αIIbβ3 on platelets from random platelet transfusions. However, the β3 chain can also associate with the αv subunit expressed at the platelet surface. Because Gypsy GT patients express normal αvβ3 carrying HPA‐1b epitopes, these patients might develop anti‐HPA‐1a alloantibodies reacting with αvβ3 and/or β3. Objectives/Patients/MethodsTo demonstrate this hypothesis, sera from HPA‐1bb (n = 5) and HPA‐1ab (n = 1) Gypsy GT patients were investigated by modified antigen capture assay using platelets or stable transfected cells. Furthermore, stable transfected cells expressing either αIIbβ3 or αvβ3 together with soluble monomeric chimeric β3 (as absorbent) were used to differentiate anti‐β3 and anti‐αvβ3 reactivity. ResultsOnly HPA‐1bb patients developed alloantibodies reacting with HPA‐1a cells. Further analysis showed that HPA‐1bb patients developed anti‐HPA‐1a alloantibodies reacting with β3 and/or αvβ3. ConclusionIn this study, we found that HPA‐1bb patients who failed to express αIIbβ3 on the platelet surface can develop alloantibodies against HPA‐1a reacting with β3 as well as αvβ3. This is of particular importance as anti‐HPA‐1a alloantibodies might cause fetal neonatal alloimmune thrombocytopenia and/or platelet transfusion refractoriness.

Maternal Anti-HPA-1a Antibodies Increase Endothelial Cell Apoptosis and Permeability

“…Our study demonstrates that maternal anti-HPA-1a antibodies derived from individual mothers vary in their effect on EC function, most probably reflecting variability in the repertoire of anti-β3 epitopes. Extensive studies exploring the possible subtypes of anti-HPA-1a antibodies revealed that these antibodies may be directed against epitopes located on the β3 chain, aIIbβ3, or αvβ3 integrins [12,13]. As suggested by the results of the antigen capture assay, anti-HPA-1a antibodies derived from mothers 1 and 2 bind to both EC αvβ3 and platelet aIIbβ3 integrins, whereas anti-HPA-1a antibodies derived from mothers 3-10 bind to the platelet aIIbβ3 integrin only (Fig.…”

“…Antigen capture assay was performed as described previously [11‒13]. In brief, 100 μL aliquots of 5 × 10 5 hCMEC/D3 cells or 1 × 10 8 washed HPA-1a platelets were incubated with 10 μL anti-HPA-1a serum and mouse anti-human β3 mAb (20 μg/mL; RayBiotech, GA, USA) for 1 h at 37°C, washed, and solubilized in 0.5% NP-40 lysis buffer.…”

“…Following the incubation, the inserts were removed from the wells, and 100 μL samples of the media from each well (lower compartment) were transferred to a 96-well plate for fluorescence measurement at 485 and 535 nm excitation and emission, respectively, using a fluorescence microplate reader (Infinite 200 PRO; Tecan, Switzerland). Antigen capture assay was performed as described previously [11][12][13]. In brief, 100 μL aliquots of 5 × 10 5 hCMEC/D3 cells or 1 × 10 8 washed HPA-1a platelets were incubated with 10 μL anti-HPA-1a serum and mouse anti-human β3 mAb (20 μg/mL; Ray-Biotech, GA, USA) for 1 h at 37°C, washed, and solubilized in 0.5% NP-40 lysis buffer.…”

Profiling the Genetic and Molecular Characteristics of Glanzmann Thrombasthenia: Can It Guide Current and Future Therapies?

“…Santoso et al 105 found that intracranial hemorrhage in mothers with fetal/neonatal alloimmune thrombocytopenia (FNAIT) caused by anti-HPA-1a antibodies, was predominately due to their reactivity with αvβ3 in endothelial cells. Evidence for anti-HPA-1 alloantibodies reactive with αvβ3 in GT was provided by Fiore et al 106 who detected them in polytransfused patients with the French Manouche gypsy mutation (on ITGA2B ) with αvβ3 in platelets and other cells expressing β3 homozygous for the rare HPA-1b allele. Thus HPA typing is recommended in GT and especially for affected women.…”

Активність Катепсинів В, L, Н У Плазмі Крові Пацієнтів Із Хронічними Дифузними Захворюваннями Печінки

“…Причому катепсин L більшою мірою, ніж катепсин В, проявляє еластолітичну, колагенолітичну і протеогліка нолітичну дію. Відомо, що ці тіолпротеази впли вають на активацію та проліферацію зірчастих клітин легень, їх диференціацію в міофібробласти і контролюють процеси фіброгенезу [5]. Вплив ISSN 2410-681X.…”