Page 156 - 2019_06-Haematologica-web

R. Vollenberg et al.
vided some evidence that autoantibodies may also trigger more complex processes, such as platelet activation, platelet desialylation, or platelet apoptosis, all of which could lead to Fc-independent platelet clearance.8-11 More recently, there has also been evidence that the glycopro- tein specificity of the autoantibodies could be important; for example, in a study by Li et al.,10 desialylation occurred in the presence of anti-GPIbα, but not in the presence of anti-GP IIb/IIIa antibodies.
In general, antibody identification (or the use of mono- clonal antibodies in animal models) in these studies was restricted to two types of autoantibody specificities: anti- GP IIb/IIIa and anti-GP Ib/IX. This was because these two glycoproteins are currently considered to be the most important autoimmune targets in ITP.12,13 However, glyco- protein V (GP V) is a major protein on the platelet mem- brane, with approximately 10,000 copies per platelet.14 More than 30 years ago, GP V was first thought to be the immune target of quinidine-related platelet antibodies by Stricker and Shulman,15 and Garner et al.16 described GP V as the antigen in a gold-triggered autoimmune response in patients with rheumatoid arthritis. GP V was also described as the target protein in pediatric varicella-associ- ated thrombocytopenia.17 Some evidence for a potential role of GP V in ITP came from preliminary studies in patients with different types of thrombocytopenia.18,19 A valuable systematic study on GP V in patients with ITP was recently published,20 but whether or not anti-GP V autoantibodies contribute to thrombocytopenia in ITP remains unknown.
Here, we investigated the potential of anti-GP V autoan- tibodies in patients with ITP. Our work shows that autoantibodies to GP V are found in a majority of patients with ITP and can potentially cause platelet clearance mechanisms. This new information helps fill in some of the missing pathophysiological events in ITP.
Methods
Adult patients with a suspected diagnosis of ITP were identi- fied, as previously described.7 In brief, standardized questionnaires covering relevant criteria to refute or confirm a diagnosis of ITP according to the British guidelines were used.21 Patients with rele- vant other diagnoses that could explain thrombocytopenia, such as aplastic anemia, leukemia, lymphoma, myelodysplastic syn- drome, solid tumors, liver cirrhosis, recent cardiac surgery, BM/blood stem cell transplantation, sepsis, and drug-induced thrombocytopenia, were not included. Platelet-bound and free anti-platelet autoantibodies of the IgG type were detected by the monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay, as described by Kiefel et al.22 Assay sensitivity was controlled by the use of the anti-HPA-1a World Health Organization (WHO) standard (NIBSC, Potters Bar, UK).
Leftover material was used for the additional experiments per- formed. All anti-GP V sera used in these experiments were nega- tive for the presence of anti-GP IIb/IIIa, anti-GP Ib/IX, anti-GP Ia/IIa, and anti-GP IV by MAIPA. Immunoglobulin G (IgG) frac- tions were isolated using a commercial purification kit (MelonTM-Gel IgG Spin Purification Kit, Thermo Fisher Scientific, Waltham, MA, US).
Surface plasmon resonance (SPR) analysis allows label-free, real- time investigation of antigen-antibody interactions. This was per- formed on a protein interaction array system (ProteOn XPR36, Bio-Rad, Munich, Germany). Recombinant His-tagged GP V as
the target protein and GP IV as an irrelevant control (R&D Systems; Life Technologies, Carlsbad, CA, USA) were immobi- lized onto flow cells of an HTE sensor chip. Phosphate buffered saline-tween (PBS-T) was used as running buffer for all steps. The SPR signal originates from changes in the refractive index at the chip’s surface. For antigen-antibody interactions, changes in the refractive index are linear to the number of antibodies bound. Data were acquired with the computer software (ProteOn Manager Software, BioRad). Interaction curves were referenced by interspot, second flow cell with immobilized GP IV and mon- oclonal anti-GP V (MAB42, R&D Systems, 6 μg/mL) as standard. The R700/R350 ratio was used to differentiate high-avidity (>0.5) and low-avidity (<0.5) antibody binding.23
A phagocytosis assay was performed using CD14+ positively- selected macrophages (autoMACS Pro Separator; Miltenyi Biotec, Germany) from cryogenically stored human spleen specimens obtained from ITP patients. Healthy donor platelets were fluores- cently labeled with CellTracker Green 5-chloromethylfluorescein diacetate (Thermo Fisher Scientific, MA, USA), washed, then opsonized with the ITP serum samples and added to the splenic macrophages for phagocytosis. Macrophages were observed by spinning-disc confocal microscopy under 63x objective oil immer- sion with differential interference contrast (DIC) and laser fluores- cence (488, 647 excitation) on a Quorum multi-modal imaging system (Quorum Technologies, ON, Canada) equipped with a 50 micrometer pinhole spinning disc and an ORCA-Flash 4.0 V2 PLUS sCMOS camera. Four images were taken at the center of each well with Z-stacking every 0.33 μm with >30 stacks. Images were reconstructed in 3D for analysis using Imaris 8.0.2 (Bitplate, UK) and phagocytic index was calculated as (total engulfed platelets / splenic macrophages counted) x 100.
A NOD/SCID mouse model was used to investigate the elimi- nation of human platelets by anti-GPV autoantibodies in ITP patients.24 In brief, NOD/SCID mice (NOD.CB17-Prkdcscid/J; Stock No. complexes, 001303) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) via Charles River, Research Models and Services (Sulzfeld, Germany). Sex- and age-matched (8-16-week old) animals were used in this study. Human platelets (200 μL, 2x109/mL) were injected into the lateral mouse tail vein. After 30 minutes (min) a blood sample was collected by tail vein puncture to determine the baseline of circulating human platelets (100%). Subsequently, IgG fractions isolated from human sera containing anti-GPV antibodies or control sera from healthy donors were injected into the other lateral tail vein (2 mg/g body weight). The survival of human platelets in the mouse circulation was analyzed over time using flow cytometry (Cytomics FC 500; Beckman Coulter) after staining platelets with anti-human CD41- PE-Cy5 (Beckman Coulter) and anti-mouse CD41-FITC (BD Biosciences, San Diego, CA, USA). Animal experiments were per- formed with the approval of the local authorities in Tuebingen, Germany. The study was conducted in accordance with the Declaration of Helsinki, and the use of human material was approved by the local ethics committees in Giessen, Germany and Toronto, ON, Canada.
Results
Prevalence of platelet-bound autoantibodies against GP V
A total of 1645 patients with no alternative reason for a low platelet count were included. The amount of autolo- gous platelets was sufficient for a complete direct test (including all 3 glycoprotein specificities) in 1140 patients (69.3% of n=1645 patients with a clinical suspicion of
1238
haematologica | 2019; 104(6)