Heterozygous ITGA2B R995W mutation inducing constitutive activation of the αIIbβ3 receptor affects proplatelet formation and causes congenital macrothrombocytopenia

Congenital macrothrombocytopenia is a genetically heterogeneous group of rare disorders.^1-4  The most frequent forms include MYH9 disorders and Bernard-Soulier syndrome. In approximately half of cases of congenital macrothrombocytopenia, the pathogenesis remains unknown; thus, a definite diagnosis is unavailable. Glanzmann thrombasthenia is the most common congenital platelet disorder caused by qualitative or quantitative abnormality of the integrin αIIbβ3, in which the platelet counts and morphology are normal.⁵  However, ITGA2B R995Q mutation has been reported in a patient with Glanzmann thrombasthenia-like phenotype and macrothrombocytopenia.^6,7  Recently, heterozygous ITGB3 mutations were found in patients with congenital macrothrombocytopenia.^8-10  We report here a novel, conserved heterozygous ITGA2B R995W mutation in 4 unrelated families.

Twenty-seven patients with congenital macrothrombocytopenia, in whom MYH9 disorders, heterozygous and homozygous Bernard-Soulier syndrome, type 2B von Willebrand disease, and TUBB1 mutations were excluded, underwent mutational analysis of ITGA2B and ITGB3. Fifty-five consecutive patients were prospectively analyzed for the surface expression of platelet αIIbβ3. Written informed consent was obtained from all patients or their parents in accordance with the Declaration of Helsinki. Institutional review boards of Nagoya Medical Center and each of the participating institutions/hospitals approved this study.

The entire coding sequence of exons and exon-intron boundaries of ITGA2B (supplemental Table 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article) and ITGB3 was amplified by polymerase chain reaction and sequenced. The disease-associated ITGA2B haplotype was determined by cloning and sequencing the polymerase chain reaction products.

Flow cytometry and immunoblotting were performed as described previously.^11,12  The activation state of αIIbβ3 was evaluated by the binding of the ligand-mimetic antibody PAC-1 (BD Biosciences) and FITC-labeled fibrinogen.¹³ 

ITGA2B and ITGB3 sequences were amplified from the patient's platelet cDNA and cloned into pcDNA3.1 (Invitrogen). T562N¹³  and D723H⁸  were introduced into ITGB3 cDNA using site-directed mutagenesis. ITGA2B and ITGB3 expression plasmids were cotransfected into 293T and CHO cells. Transfected cells were subjected to flow cytometry, FAK phosphorylation, and spreading assay.^13,14 

ITGA2B and ITGB3 cDNAs were inserted upstream of internal ribosome entry site (IRES)-enhanced green fluorescent protein (EGFP) and IRES-Kusabira-Orange in the retroviral vector pGCDNsamIRES/EGFP and pGCDNsamIRES/huKO, respectively.^15,16  Each plasmid was transfected into 293gp packaging cells with a vesicular stomatitis virus G expression plasmid. Supernatants were used for the transduction of 293gpg producer cells harboring a tetracycline-inducible vesicular stomatitis virus G expression cassette,¹⁷  and virus-bearing supernatant was harvested under tetracycline-deficient conditions.

Mouse fetal liver cells were harvested from embryonic day 13.5 embryos and cultured in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum and 50 ng/mL human thrombopoietin. The next day, cells were infected with retroviruses expressing ITGA2B and ITGB3 on recombinant human fibronectin fragment CH-296 (RetroNectin, Takara)-coated plates. After transduction, proplatelet formation was monitored for the next 4 days on EGFP and Kusabira-Orange double-positive megakaryocytes in suspension by inverted fluorescence microscopy. The Experimental Animal Committee of Nagoya Medical Center approved the animal studies.

We searched for ITGA2B and ITGB3 mutations in 27 patients with macrothrombocytopenia and identified a novel, conserved heterozygous ITGA2B R995W mutation in one patient (patient 1; Figure 1A). The decreased surface expression of platelet αIIbβ3 prompted us to prospectively screen its expression by flow cytometry. We detected decreased αIIbβ3 expression level (50%-70% of control) in 3 of 55 consecutive patients with macrothrombocytopenia of unknown etiology (patients 2-4 in Table 1). Immunoblotting showed a normal electrophoretic mobility of αIIb, but the total expression level relative to β1-tubulin was decreased to 0.7 (Figure 1B; Table 1). Sequence analysis identified the same heterozygous ITGA2B R995W mutation. In total, we identified 11 patients in 4 unrelated Japanese families. In each family, the disease-associated ITGA2B haplotype was unique, indicating independent occurrence (supplemental Table 2). Patients had larger platelets, approximately 30% increase of control, and moderate thrombocytopenia (Figure 1C; Table 1). These results indicate that macrothrombocytopenia shows a dominant inheritance.

Bleeding tendency was absent or mild (eg, patient 1 had undergone total colectomy without platelet transfusion). Platelet aggregation induced by adenosine diphosphate and collagen was reduced, although the bleeding time was within the normal limit (Table 1). Platelet spreading on immobilized fibrinogen was partially impaired: the number of fully spread platelets was decreased (supplemental Figure 1). These findings indicate that patients are asymptomatic or exhibit a marginal bleeding tendency and that the clinical and laboratory phenotype is distinct from Glanzmann thrombasthenia.

There was spontaneous PAC-1 binding to resting patients' platelets as well as to αIIb-W995/β3-transfected 293T cells. Although fibrinogen did not bind to platelets in whole blood, increased fibrinogen binding to the washed platelets was observed (Figure 1D; supplemental Figure 2). The activation state, quantified as an activation index in 293T cells, was higher for αIIb-W995 than for β3-H723 but was weaker than that for a strong activating mutant, β3-N562¹³ (Figure 1E). CD62p expression was absent on the resting platelets (supplemental Figure 2). Spontaneously phosphorylated FAK, a downstream effector of integrin signaling, was not evident in resting platelets in suspension, probably because of low expression level of abnormal αIIbβ3 receptor. However, FAK phosphorylation occurred in αIIb-W995/β3-transfected 293T cells in suspension, indicating constitutively activated αIIbβ3 (Figure 1F). These results indicate that R995W mutation changes αIIbβ3 to a constitutively, albeit partially, activated conformation, but does not induce platelet activation.

αIIb-R995 forms a salt bridge with β3-D723 in the membrane-proximal region and maintains the inactive conformation of the αIIbβ3.^20,21  Disruption of the interaction because of partially activated αIIb/β3-H723 or αIIb/β3-A723 mutants but not fully activated mutants, such as αIIb/β3-N562, was reported to cause microtubule-dependent abnormal proplatelet-like cytoplasmic extensions in megakaryocytes and CHO cells.^8,22  We found that αIIb-W995/β3-transfected CHO cells exhibited membrane ruffling and abnormal cytoplasmic protrusions with the bulbous tips on fibrinogen-coated surfaces (Figure 1G), indicating that the salt bridge-disrupting mutations exert the same influence on the integrin activation and cytoskeletal events. Abnormal clustering of αIIbβ3, which was reported in ITGB3 L718P mutation,¹⁰  was not observed in these cells or in platelets spread on immobilized fibrinogen (supplemental Figure 1). It is worth noting that macrothrombocytopenia-associated ITGB3 mutations in the ectodomain and the cytoplasmic membrane-proximal region have different properties in terms of outside-in signaling and bleeding tendency.^9,10 

Finally, to determine the functional consequences of R995W mutation on platelet production, we coexpressed αIIb and β3 in mouse fetal liver cells by retroviral transfer and differentiated them into megakaryocytes (Figure 1H). There was an early increase and decrease in the percentage of proplatelet formation-positive megakaryocytes in αIIb-W995/β3-transfected megakaryocytes. The number of proplatelet tips was decreased, and the size of the tips increased. These results are consistent with thrombocytopenia and the increased platelet size in patients, indicating that the stimulation of mutant αIIbβ3 leads to abnormal proplatelet formation. However, not all ITGA2B- and ITGB3-activating mutations are associated with macrothrombocytopenia. Patients with homozygous ITGB3 C549R or C560R mutation inducing constitutively active αIIbβ3 have a normal platelet count and size,^23,24  suggesting different molecular mechanisms for the induction of abnormal proplatelet formation.

αIIbβ3 has not been implicated in an abnormal platelet count or morphology.⁵  Our data support and extend the recent reports that heterozygous, activating mutations in ITGA2B and ITGB3, in the juxtamembrane region, cause macrothrombocytopenia.^6-10  We thus propose that such mutations represent the etiology of a subset of congenital macrothrombocytopenias. It is also probable that homozygosity causes Glanzmann thrombasthenia, as demonstrated in the original report of macrothrombocytopenia-associated ITGA2B R995Q mutation.^6,7  The creation of a knock-in mouse model and/or use of an in vivo megakaryocyte infusion model²⁵  should clarify the mechanism underlying the production and processing of giant platelets.

The authors thank Dr R. C. Mulligan (Children's Hospital Boston, Harvard Medical School, Boston, MA) for 293gp and 293gpg cells, Dr M. Handa (Department of Transfusion Medicine & Cell Therapy, Keio University School of Medicine, Tokyo, Japan) for PT25-2 antibodies, Dr A. Saito (Department of Clinical Research Promotion, Clinical Research Center, National Hospital Organization Nagoya Medical Center) for statistical analysis, and Yoshimi Ito-Yamamura for her skillful technical assistance.

This work was supported by the Japan Society for the Promotion of Science (Grant-in-Aid for Scientific Research), the Ministry of Health, Labor and Welfare, Academic Frontier Project in Japan, Mitsubishi Pharma Research Foundation, the 24th General Assembly of the Japanese Association of Medical Sciences Promotion Fund, the Mother and Child Health Foundation, and the National Hospital Organization Research Fund.

Contribution: S.K. designed and performed research, analyzed data, and wrote the paper; H.K. and Y. Tomiyama performed platelet experiments and interpreted the results; M. Onodera constructed retrovirus vectors; M. Otsu, N.T., K.E., and M. Onodera designed the retroviral transfection experiments; Y.M., Y. Takamatsu, J.S., and K.M. contributed patient samples; and H.S. supervised the research.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Shinji Kunishima, Department of Advanced Diagnosis, Clinical Research Center, National Hospital Organization Nagoya Medical Center, 4-1-1 Sannomaru, Naka-ku, Nagoya 4600001, Japan; e-mail: kunishis@nnh.hosp.go.jp.