Three Novel Integrin b3 Subunit Missense Mutations (H280P, C560F, and G579S) in Thrombasthenia, Including One (H280P) Prevalent in Japanese Patients Hironobu Ambo,* ,1 Tetsuji Kamata,§ ,1 Makoto Handa,² ,2 Masashi Taki, ¶ Minoru Kuwajima,\ Yohko Kawai,‡ Atsushi Oda,* Mitsuru Murata,* Yoshikazu Takada,§ Kiyoaki Watanabe,‡ and Yasuo Ikeda* *Department of Internal Medicine, ²Blood Center, ‡Laboratory Medicine, School of Medicine, Keio University, Tokyo 160-8582, Japan; §The Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037; ¶ The Department of Pediatrics, School of Medicine, St. Marianna University, Kanagawa, 216-8511, Japan; and \The Department of Clinical Pathology, Kagawa Prefectural Central Hospital, Kagawa, 760-8557, Japan Received September 17, 1998 We analyzed three unrelated Japanese patients with type II Glanzmann thrombasthenia (GT) for associ- ated mutations. Polymerase chain reaction and subse- quent direct sequencing of platelet RNA and genomic DNA revealed three single nucleotide substitutions of the integrin b3 subunit gene (His (CAT)-280 to Pro (CCT), Cys (TGT)-560 to Phe (TTT), and Gly(GGC)-579 to Ser(AGC)). Interestingly, the three unrelated pa- tients all had the H280P mutation; one was homozy- gous and the other two heterozygous for this muta- tion. Ectopic expression of wild type and mutant complexes in Chinese hamster ovary cells revealed decreased surface expression of the mutated aIIbb3 complexes, thus demonstrating that these mutations may result in the mild GT phenotypes. The identifica- tion of three unrelated patients having the same mu- tation (H280P) suggests that this mutation might be prevalent in the Japanese thrombasthenic population. © 1998 Academic Press The platelet membrane glycoprotein (GP) IIb-IIIa complex, known as aIIbb3, is a member of the integrin family and consists of two distinct membrane glyco- proteins, GPIIb (aIIb) and GPIIIa (b3), which are non- covalently bound in a divalent-cation dependent man- ner. After cellular activation, the complex becomes an adhesion receptor for RGD-bearing adhesive proteins such as fibrinogen or von Willebrand factor and medi- ates platelet aggregation and subsequent platelet plug formation at sites of injured vasculature. Glanzmann thrombasthenia (GT) is an autosomal recessive hemorrhagic disorder that is the result of quantitative or qualitative abnormalities in aIIbb3. This bleeding diathesis is caused by the failure of platelet thrombus formation in physiologic primary he- mostasis. GT is subclassified into type I (severely quan- titative; surface expression of aIIbb3 , 5% of normal), type II (mildly quantitative; aIIbb3 5-20%) and variant (qualitative), although a patient with both quantitative and qualitative abnormalities has been recently re- ported (1). With the recent elucidation of the structural organi- zation of the aIIb and b3 genes (2-5), a number of genetic defects associated with GT have been identified in both aIIb and b3 (6-7). A defect in either subunit blocks expression of the aIIbb3 complex on the cell surface, thus producing quantitative GT phenotypes. It has been found that the avb3 complex, another b3 integrin expressed on platelets, is defective in quanti- tative GT patients with b3 mutations but not in pa- tients with aIIb mutations (1). Herein, we have identified three novel b3 missense mutations (H280P, C560F and G579S) in three unre- lated Japanese patients with GT type II. Of particular interest was the observation that these three patients had the same mutation (H280P), suggesting that this mutation may be significant in Japanese thrombas- thenic patients. MATERIALS AND METHODS Patients. We studied three Japanese patients from different fam- ilies diagnosed with type II GT. Patient TK was a 5 year-old boy, patient NT a sixty-year-old woman and patient HJ a fifty two-year- 1 H.A. and T.K. contributed equally to this study and should be regarded as cofirst authors. 2 To whom correspondence should be addressed. Makoto Handa, M.D., Blood Center, School of Medicine, Keio University, 35 Shinano- machi, Shinjuku-ku, Tokyo 160-8582, Japan. Fax: 81-3-3353-9706. E-mail: mhanda@mc.med.keio.ac.jp. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 251, 763–768 (1998) ARTICLE NO. RC989526 763 0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.