A new polymorphism, '"leu/phe, within the leucine-rich repeat sequence of platelet glycoprotein ibo

Yumiko Matsubara, Mitsuru Murata, Takanori Moriki, Kenji Yokoyama, Naohide Watanabe, Hideaki Nakajima, Makoto Handa, Yasuo Ikeda

Research output: Contribution to journalArticle

Abstract

The a chain of platelet glycoprotein Ib/IX/V complex (GPIba), a subunit of von Willebrand factor (vWF) receptor, has leucine-rich repeat (LRR) sequence within the Nterminal 45kDa domain which contains the vWF-binding site. LRR has a consensus sequence with leucines in conserved positions and is believed to be essential to maintain the proper conformation and function of the receptor. Here we report a new polymorphism within the LRR sequence of GPIba. By a direct DNA sequencing analysis, we found a Cto-T transition at nucleotide #792, which would result in a substitution of Phe at residue 70, that is conserved for Leu. The genotype frequency among 100 healthy Japanese subjects was 91% ("Leu/Leu). 9% ((tm)Leu/Phe) and 0% ((tm)Phe/Phe). The (tm)Leu/Phe polymorphism was not apparently linked to the two known polymorphisms of GPIba, "5T/C or'45Thr/Met. Because the 70Leu/Phe polymorphism would disrupt the consensus sequence in the second repeat of the seven LRRs, we examined ristocetin-induced and shear-induced platelet aggregation (RIPA and SIPA), which are dependent on GPIbo/ vWF interaction, using platelets from 2 available subjects with the 70Leu/Phe genotype. The maximum aggregation for RIPA (1.2mg/ml) and SIPA (108dyn/cm2) were within the normal ranges. Next, we constructed two plasmids containing cDNA encoding a partial GPIba sequence (codon 1-302) which includes the 45kDa domain but lacks the transmembrane domain, wild-type (WT) and mutant (L70F). These plasmids were separately transfected into Chinese hamster ovary cells to establish stable cell lines. WTand L70F-transfected cells secreted similar amount of GPIba fragments into culture media (CM). After adjustment for antigen concentration, serum-free CM from WT- and L70F-transfected cells were analyzed by dot-blotting for the immunological reactivity toward a panel of anti-GPIba monoclonal antibodies, LJ-P3 (a generous gift of Dr ZM Ruggeri), GUR83-35, and GUR20-5, all of which recognize conformation-specific epitopes within the 45kDa domain, and Hipl that recognizes an epitope within the second repeat of the LRR. The reactivity of L70F as compared to WT was similar for LJ-P3 and GUR83-35, but was lower for GUR20-5 and Hip 1, both of which inhibit RIPA. In conclusion, we found a (tm)Leu/Phe polymorphism within the LRR sequence of GPIba. Platelets from subjects with the (tm)Leu/Phe genotype maintained an ability to mediate RIPA and SIPA. Experiments using recombinant GPIba fragments, however, suggested that the (tm)Leu/Phe polymorphism might affect conformation of the N-terminal domain of GPIba.

Original languageEnglish
JournalBlood
Volume96
Issue number11 PART II
Publication statusPublished - 2000

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Platelet Membrane Glycoproteins
Polymorphism
Leucine
Platelets
Platelet Glycoprotein GPIb-IX Complex
Conformations
Genotype
Consensus Sequence
von Willebrand Factor
Epitopes
Plasmids
Blood Platelets
Agglomeration
Cells
Ristocetin
Gift Giving
Aptitude
Serum-Free Culture Media
Cricetulus
DNA Sequence Analysis

ASJC Scopus subject areas

  • Hematology

Cite this

Matsubara, Y., Murata, M., Moriki, T., Yokoyama, K., Watanabe, N., Nakajima, H., ... Ikeda, Y. (2000). A new polymorphism, '"leu/phe, within the leucine-rich repeat sequence of platelet glycoprotein ibo. Blood, 96(11 PART II).

A new polymorphism, '"leu/phe, within the leucine-rich repeat sequence of platelet glycoprotein ibo. / Matsubara, Yumiko; Murata, Mitsuru; Moriki, Takanori; Yokoyama, Kenji; Watanabe, Naohide; Nakajima, Hideaki; Handa, Makoto; Ikeda, Yasuo.

In: Blood, Vol. 96, No. 11 PART II, 2000.

Research output: Contribution to journalArticle

Matsubara, Y, Murata, M, Moriki, T, Yokoyama, K, Watanabe, N, Nakajima, H, Handa, M & Ikeda, Y 2000, 'A new polymorphism, '"leu/phe, within the leucine-rich repeat sequence of platelet glycoprotein ibo', Blood, vol. 96, no. 11 PART II.
Matsubara Y, Murata M, Moriki T, Yokoyama K, Watanabe N, Nakajima H et al. A new polymorphism, '"leu/phe, within the leucine-rich repeat sequence of platelet glycoprotein ibo. Blood. 2000;96(11 PART II).
Matsubara, Yumiko ; Murata, Mitsuru ; Moriki, Takanori ; Yokoyama, Kenji ; Watanabe, Naohide ; Nakajima, Hideaki ; Handa, Makoto ; Ikeda, Yasuo. / A new polymorphism, '"leu/phe, within the leucine-rich repeat sequence of platelet glycoprotein ibo. In: Blood. 2000 ; Vol. 96, No. 11 PART II.
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abstract = "The a chain of platelet glycoprotein Ib/IX/V complex (GPIba), a subunit of von Willebrand factor (vWF) receptor, has leucine-rich repeat (LRR) sequence within the Nterminal 45kDa domain which contains the vWF-binding site. LRR has a consensus sequence with leucines in conserved positions and is believed to be essential to maintain the proper conformation and function of the receptor. Here we report a new polymorphism within the LRR sequence of GPIba. By a direct DNA sequencing analysis, we found a Cto-T transition at nucleotide #792, which would result in a substitution of Phe at residue 70, that is conserved for Leu. The genotype frequency among 100 healthy Japanese subjects was 91{\%} ({"}Leu/Leu). 9{\%} ((tm)Leu/Phe) and 0{\%} ((tm)Phe/Phe). The (tm)Leu/Phe polymorphism was not apparently linked to the two known polymorphisms of GPIba, {"}5T/C or'45Thr/Met. Because the 70Leu/Phe polymorphism would disrupt the consensus sequence in the second repeat of the seven LRRs, we examined ristocetin-induced and shear-induced platelet aggregation (RIPA and SIPA), which are dependent on GPIbo/ vWF interaction, using platelets from 2 available subjects with the 70Leu/Phe genotype. The maximum aggregation for RIPA (1.2mg/ml) and SIPA (108dyn/cm2) were within the normal ranges. Next, we constructed two plasmids containing cDNA encoding a partial GPIba sequence (codon 1-302) which includes the 45kDa domain but lacks the transmembrane domain, wild-type (WT) and mutant (L70F). These plasmids were separately transfected into Chinese hamster ovary cells to establish stable cell lines. WTand L70F-transfected cells secreted similar amount of GPIba fragments into culture media (CM). After adjustment for antigen concentration, serum-free CM from WT- and L70F-transfected cells were analyzed by dot-blotting for the immunological reactivity toward a panel of anti-GPIba monoclonal antibodies, LJ-P3 (a generous gift of Dr ZM Ruggeri), GUR83-35, and GUR20-5, all of which recognize conformation-specific epitopes within the 45kDa domain, and Hipl that recognizes an epitope within the second repeat of the LRR. The reactivity of L70F as compared to WT was similar for LJ-P3 and GUR83-35, but was lower for GUR20-5 and Hip 1, both of which inhibit RIPA. In conclusion, we found a (tm)Leu/Phe polymorphism within the LRR sequence of GPIba. Platelets from subjects with the (tm)Leu/Phe genotype maintained an ability to mediate RIPA and SIPA. Experiments using recombinant GPIba fragments, however, suggested that the (tm)Leu/Phe polymorphism might affect conformation of the N-terminal domain of GPIba.",
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T1 - A new polymorphism, '"leu/phe, within the leucine-rich repeat sequence of platelet glycoprotein ibo

AU - Matsubara, Yumiko

AU - Murata, Mitsuru

AU - Moriki, Takanori

AU - Yokoyama, Kenji

AU - Watanabe, Naohide

AU - Nakajima, Hideaki

AU - Handa, Makoto

AU - Ikeda, Yasuo

PY - 2000

Y1 - 2000

N2 - The a chain of platelet glycoprotein Ib/IX/V complex (GPIba), a subunit of von Willebrand factor (vWF) receptor, has leucine-rich repeat (LRR) sequence within the Nterminal 45kDa domain which contains the vWF-binding site. LRR has a consensus sequence with leucines in conserved positions and is believed to be essential to maintain the proper conformation and function of the receptor. Here we report a new polymorphism within the LRR sequence of GPIba. By a direct DNA sequencing analysis, we found a Cto-T transition at nucleotide #792, which would result in a substitution of Phe at residue 70, that is conserved for Leu. The genotype frequency among 100 healthy Japanese subjects was 91% ("Leu/Leu). 9% ((tm)Leu/Phe) and 0% ((tm)Phe/Phe). The (tm)Leu/Phe polymorphism was not apparently linked to the two known polymorphisms of GPIba, "5T/C or'45Thr/Met. Because the 70Leu/Phe polymorphism would disrupt the consensus sequence in the second repeat of the seven LRRs, we examined ristocetin-induced and shear-induced platelet aggregation (RIPA and SIPA), which are dependent on GPIbo/ vWF interaction, using platelets from 2 available subjects with the 70Leu/Phe genotype. The maximum aggregation for RIPA (1.2mg/ml) and SIPA (108dyn/cm2) were within the normal ranges. Next, we constructed two plasmids containing cDNA encoding a partial GPIba sequence (codon 1-302) which includes the 45kDa domain but lacks the transmembrane domain, wild-type (WT) and mutant (L70F). These plasmids were separately transfected into Chinese hamster ovary cells to establish stable cell lines. WTand L70F-transfected cells secreted similar amount of GPIba fragments into culture media (CM). After adjustment for antigen concentration, serum-free CM from WT- and L70F-transfected cells were analyzed by dot-blotting for the immunological reactivity toward a panel of anti-GPIba monoclonal antibodies, LJ-P3 (a generous gift of Dr ZM Ruggeri), GUR83-35, and GUR20-5, all of which recognize conformation-specific epitopes within the 45kDa domain, and Hipl that recognizes an epitope within the second repeat of the LRR. The reactivity of L70F as compared to WT was similar for LJ-P3 and GUR83-35, but was lower for GUR20-5 and Hip 1, both of which inhibit RIPA. In conclusion, we found a (tm)Leu/Phe polymorphism within the LRR sequence of GPIba. Platelets from subjects with the (tm)Leu/Phe genotype maintained an ability to mediate RIPA and SIPA. Experiments using recombinant GPIba fragments, however, suggested that the (tm)Leu/Phe polymorphism might affect conformation of the N-terminal domain of GPIba.

AB - The a chain of platelet glycoprotein Ib/IX/V complex (GPIba), a subunit of von Willebrand factor (vWF) receptor, has leucine-rich repeat (LRR) sequence within the Nterminal 45kDa domain which contains the vWF-binding site. LRR has a consensus sequence with leucines in conserved positions and is believed to be essential to maintain the proper conformation and function of the receptor. Here we report a new polymorphism within the LRR sequence of GPIba. By a direct DNA sequencing analysis, we found a Cto-T transition at nucleotide #792, which would result in a substitution of Phe at residue 70, that is conserved for Leu. The genotype frequency among 100 healthy Japanese subjects was 91% ("Leu/Leu). 9% ((tm)Leu/Phe) and 0% ((tm)Phe/Phe). The (tm)Leu/Phe polymorphism was not apparently linked to the two known polymorphisms of GPIba, "5T/C or'45Thr/Met. Because the 70Leu/Phe polymorphism would disrupt the consensus sequence in the second repeat of the seven LRRs, we examined ristocetin-induced and shear-induced platelet aggregation (RIPA and SIPA), which are dependent on GPIbo/ vWF interaction, using platelets from 2 available subjects with the 70Leu/Phe genotype. The maximum aggregation for RIPA (1.2mg/ml) and SIPA (108dyn/cm2) were within the normal ranges. Next, we constructed two plasmids containing cDNA encoding a partial GPIba sequence (codon 1-302) which includes the 45kDa domain but lacks the transmembrane domain, wild-type (WT) and mutant (L70F). These plasmids were separately transfected into Chinese hamster ovary cells to establish stable cell lines. WTand L70F-transfected cells secreted similar amount of GPIba fragments into culture media (CM). After adjustment for antigen concentration, serum-free CM from WT- and L70F-transfected cells were analyzed by dot-blotting for the immunological reactivity toward a panel of anti-GPIba monoclonal antibodies, LJ-P3 (a generous gift of Dr ZM Ruggeri), GUR83-35, and GUR20-5, all of which recognize conformation-specific epitopes within the 45kDa domain, and Hipl that recognizes an epitope within the second repeat of the LRR. The reactivity of L70F as compared to WT was similar for LJ-P3 and GUR83-35, but was lower for GUR20-5 and Hip 1, both of which inhibit RIPA. In conclusion, we found a (tm)Leu/Phe polymorphism within the LRR sequence of GPIba. Platelets from subjects with the (tm)Leu/Phe genotype maintained an ability to mediate RIPA and SIPA. Experiments using recombinant GPIba fragments, however, suggested that the (tm)Leu/Phe polymorphism might affect conformation of the N-terminal domain of GPIba.

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