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.
|Issue number||11 PART II|
|Publication status||Published - 2000 Dec 1|
ASJC Scopus subject areas
- Cell Biology