Glanzmann thrombasthenia (GT) is a bleeding disorder characterized by impaired platelet aggregation in response to most physiological agonists and caused by either a complete lack or dysfunction of the platelet integrin αIIbβ3 (glycoprotein IIb/IIIa). In this study we identified a novel homozygous non-synonymous variant (c.995A>T, p.Asp332Val) in exon 11 of ITGA2B leading to replacement of Aspartic Acid by Valine. According to the ACMG (American College of Medical Genetics and Genomics) variant classification guidelines, this variant is classified as likely pathogenic. This amino acid change affects the second Calcium-binding domain of αIIb integrin. The Index patient’s platelet aggregation induced by all agonist except Ristocetin is absent, and the expression of the GPIIb/IIIa-complex is severely decreased.

Keywords: Glanzmann thrombasthenia, ITGA2B, Calcium-binding domain, Novel likely pathogenic variant

INTRODUCTION

Glanzmann thrombasthenia (GT) is the most common inherited platelet disorder characterized by a deficiency or functional defect of platelet integrin αIIbβ3. Most often GT is inherited autosomal recessively, a few patients with an autosomal dominant inheritance of GT have been identified. GT was first described by Glanzmann in 1918 as a platelet abnormality presenting with haemorrhage and thrombasthenia (weak) platelets. Characteristical for this disease is severely impaired or absent platelet aggregation in response to multiple physiological agonists. Platelets lacking integrin αIIbβ3 may adhere to the subendothelium after injury, however, platelet spreading on the exposed surface is defective as well as thrombus formation [1]. Patients suffer from a lifelong moderate to severe haemorrhagic syndrome which can manifest rapidly after birth. Bleeding symptoms comprise haematomas, petechiae, gastrointestinal and mucocutaneous bleeding (i.e., epistaxis). GT cannot be clinically distinguished from other platelet disorders; therefore, comprehensive diagnostic investigation is important to elucidate the underlying cause of the haemorrhagic diathesis [2-4]. The integrins are a family of type I transmembrane heterodimeric glycoprotein receptors. Integrin αIIbβ3 (also called glycoprotein (GP) IIb/IIIa complex) is an important, highly expressed platelet receptor which binds fibrinogen as well as Von Willebrand factor, vitronectin and fibronectin [5]. Both subunits of αIIbβ3, αIIb and β3, are encoded by separate genes less than 200 kb apart on chromosome 17q21. The gene coding for integrin αIIb subunit (ITGA2B) spans 17 kb and comprises 30 exons, whereas the gene that encodes the integrin β3 subunit (ITGB3) spans 59 kb and contains 15 exons. The polypeptide precursors of αIIb and β3 associate as a non-covalent heterodimer in the endoplasmic reticulum. Subsequently, single-chain pro-αIIb is cleaved into mature αIIb in the Golgi apparatus, comprising a heavy and light chain linked through a disulfide bridge [6]. Crystal structure analyses revealed that the head of the integrin contains a seven-bladed β-propeller structure from the α-subunit (comprising seven ~60-amino-acid N-terminal repeats) and a von Willebrand factor A-domain from the β-subunit (termed the β A-domain). The β A-domain is anchored to the upper face of the β-propeller, with an arginine residue in a 310 helical segment of the β A-domain (between β D and α5) linked to a hydrophobic ‘cage’ in the central shaft of the β-propeller. The remainder of the head composes an immunoglobulin module into which the β A-domain is inserted [7]. These two subunits associate in the membrane and in the cytoplasm with a low affinity [8]. In the inside-out signaling pathway, the cytoskeletal protein talin9 has been reported to be able to bind specifically to the cytoplasmic domain of β3 and in turn activate integrin αIIbβ3 [9]. The αIIb heavy chain includes four tandem domains, each containing a 12 amino acids stretch homologous to the calcium binding loops (EF-hand motif) of proteins such as calmodulin and parvalbumin [10]. The Calcium binding domain is part of the β-propeller domain at the N-terminus of the αIIb subunit and is of critical importance for αIIbβ3 biogenesis; at this location many mutations giving rise to type I and type II GT [11].

This study describes the clinical and biochemical phenotype of a patient with GT caused by a novel homozygous likely pathogenic variant which results in an amino acid change located in the second Calcium-binding domain of αIIb integrin.

PATIENT, MATERIALS AND METHODS

The index patient is a 6 year old boy who came for counseling before a planned dental restoration.  He and his family came originally from Iran. The parents are first cousins. He suffers from increased bleeding symptoms since early childhood, including petechiae on the entire skin and easy bruising (Figure 1A). He reported prolonged epistaxis: 2-3 nose bleeding episodes during the year lasted as minor bleeding even for a couple of days. After minor injuries or blood drawing the bleeding symptoms persist for 2-3 h. His parents reported major bleeding and prolonged wound healing after circumcision as an infant. In Iran he successfully received repeated platelet concentrates after sustained bleeding episodes. After Glanzmann Thrombasthenia was diagnosed here, the index patient underwent tooth extraction (5 teeth) under treatment with activated recombinant factor VII (rFVIIa) without any major bleeding. He did not need any platelet concentrate.

The mother reported that she had minor bleeding symptoms as a child like epistaxis and easy bruising. She had no increased bleeding symptoms giving birth to her children. The father and the 4 year old brother are unaffected. One brother underwent an adenoidectomy without bleeding complications.

Platelet aggregometry analyses

Platelet-rich plasma (PRP) was prepared from citrated blood. Platelet aggregometry (APACT4) was performed using 2.0 and 10.0 μg/ml collagen (Takeda, Linz, Austria), 4.0 and 10.0 μmo/l/l adenosine diphosphate (ADP) (Sigma-Aldrich, St. Louis, MO, USA), 8.0 μmol/l epinephrine (Sanofi-Aventis, Frankfurt, Germany), 0.3 and 0.5 mg/ml arachidonic acid (MöLab GmBH, Langenfeld, Germany) and 1.2 mg/ml ristocetin (American Biochemical and Pharmaceutical LTD, Frankfurt, Germany).

Flow cytometry analyses

Flow cytometry analyses were performed according to Lahav et al. [12] using FACSCalibur (Becton Dickinson, Heidelberg, Germany). Aliquots of diluted PRP (5 × 10⁷ to 5 × 10⁷ platelets/ml) were fixed and stained with FITC-labelled monoclonal surface antibody against CD41 (fibrinogen receptor GPIIb/IIIa-complex), CD42a (von Willebrand factor (VWF) receptor GPIb/IX) and CD42b (VWF receptor GPIb) (Coulter, Immunotech, Marseille, France), respectively.

For VWF-binding analyses, diluted PRP (5 × 10⁷ to 5 × 10⁷ platelets/ml) was stimulated with different concentrations of ristocetin (0-1 mg/ml) and ADP (0-2 µmol/l) for 3 min at RT, respectively. Platelets were stained with FITC-labeled anti-VWF (Bio-Rad AbD Serotec, Puchheim, Germany) and Alexa Fluor 488-labelled anti-fibrinogen (Invitrogen, Waltham, MA, USA).

For secretion analyses, diluted PRP (5 × 10⁷ platelets/ml) was stimulated with different concentrations of thrombin (0, 0.05, 0.1, 0.2, 0.5 and 1 U/ml; Siemens Healthineers, Marburg, Germany) in the presence of 1.25 mM fibrinolysis inhibiting factor Gly-Pro-Arg-Pro (Bachem, Bubendorf, Switzerland). Platelets were stained with monoclonal FITC-labelled anti-CD62 antibody (α-granule secretion) and anti-CD63 antibody (δ-granule secretion) (both Immunotech, Marseille, France).

Molecular genetic analysis

Sanger sequencing was performed for the coding region and splice sites of ITGA2B (NM_000419.4) and ITGB3 (NM_000212.2) as described previously [13] and aligned using CodonCode^® software. Variants were evaluated using mutation analyzing software Alamut^®, in silico pathogenicity prediction and occurrence in Glanzmann database (Sinai central), NCBI ClinVar and HGMD^® [Access 11/2018].

RESULTS

1.       Nurden AT, Pillois X, Wilcox DA (2013) Glanzmann thrombasthenia: State of the art and future directions. Semin Thromb Hemostasis 39: 642-655.

2.       Boeckelmann D, Hengartner H, Greinacher A, Nowak-Gottl U, Sachs UJ, et al. (2017) Patients with Bernard-Soulier syndrome and different severity of the bleeding phenotype. Blood Cells Mol Dis.

3.       Lecchi A, Femia EA, Paoletta S, Dupuis A, Ohlmann P, et al. (2016) Inherited dysfunctional platelet P2Y12 receptor mutations associated with bleeding disorders. Hamostaseologie 36: 279-283.

4.       Sandrock-Lang K, Wentzell R, Santoso S, Zieger B (2016) Inherited platelet disorders. Hamostaseologie 36: 178-186.

5.       Bray PF, Barsh G, Rosa JP, Luo XY, Magenis E, et al. (1988) Physical linkage of the genes for platelet membrane glycoproteins IIb and IIIa. Proc Natl Acad Sci U S A 85: 8683-8687.

6.       Rosenberg N, Yatuv R, Sobolev V, Peretz H, Zivelin A, et al. (2003) Major mutations in calf-1 and calf-2 domains of glycoprotein IIb in patients with Glanzmann thrombasthenia enable GPIIb/IIIa complex formation, but impair its transport from the endoplasmic reticulum to the Golgi apparatus. Blood 101: 4808-4815.

7.       Humphries MJ (2002) Insights into integrin-ligand binding and activation from the first crystal structure. Arthritis Res 4: S69-78.

8.       Huang H, Vogel HJ (2012) Structural basis for the activation of platelet integrin alphaIIbbeta3 by calcium- and integrin-binding protein 1. J Am Chem Soc 134: 3864-3872.

9.       Wegener KL, Partridge AW, Han J, Pickford AR, Liddington RC, et al. (2007) Structural basis of integrin activation by talin. Cell 128: 171-182.

10.    Bennett JS (1996) Structural biology of glycoprotein IIb-IIIa. Trends Cardiovasc Med 6: 31-36.

11.    Nurden AT, Fiore M, Nurden P, Pillois X (2011) Glanzmann thrombasthenia: A review of ITGA2B and ITGB3 defects with emphasis on variants, phenotypic variability and mouse models. Blood 118: 5996-6005.

12.    Lahav J, Jurk K, Hess O, Barnes MJ, Farndale RW, et al. (2002) Sustained integrin ligation involves extracellular free sulfhydryl and enzymatically catalyzed disulphide exchange. Blood 100: 2472-2478.

13.    Sandrock-Lang K, Oldenburg J, Wiegering V, Halimeh S, Santoso S, et al. (2015) Characterisation of patients with Glanzmann thrombasthenia and identification of 17 novel mutations. Thromb Hemostasis 113: 782-791.

14.    Gidwitz S, Temple B, White GC 2nd (2004) Mutations in and near the second calcium-binding domain of integrin alpha IIb affect the structure and function of integrin alphaIIbbeta3. Biochem J 379: 449-459.

15.    Mitchell WB, Li JH, Singh F, Michelson AD, Bussel J, et al. (2003) Two novel mutations in the alpha IIb calcium-binding domains identify hydrophobic regions essential for alpha IIb beta 3 biogenesis. Blood 101: 2268-2276.

16.    Nelson EJ, Li J, Mitchell WB, Chandy M, Srivastava A, et al. (2005). Three novel beta-propeller mutations causing Glanzmann thrombasthenia result in production of normally stable pro-alpha IIb, but variably impaired progression of pro-alphaIIbbeta3 from endoplasmic reticulum to Golgi. J Thromb Hemostasis 3: 2773-2783.

17.    Shen WZ, Ding QL, Jin PP, Wang XF, Jiang YZ, et al. (2009) A novel Pro126His beta propeller mutation in integrin alpha IIb causes Glanzmann thrombasthenia by impairing progression of pro-alphaIIbbeta3 from endoplasmic reticulum to Golgi. Blood Cells Mol Dis 42: 44-50.