Platelet glycoprotein VI has evolutionary conserved immunoglobulin domains, but a highly polymorphic stem and cytoplasmic domain

Abstract number: P1299

Watkins^* N. A., Rankin† A., O'Connor† M., Jennings† N., Wilson† E., Joutsi-Korhonen† L., Mcbride† S., Smethurst† P. A., Harmer† I. J., Farndale† R. W., Ouwehand† W. H.

†UK ^*University of Cambridge, UK;

Glycoprotein (GP) VI plays a pivotal role in platelet activation by collagen in the subendothelial matrix. GPVI, whose expression is restricted to platelets and megakryocytes, is 314 amino acids in length and contains two amino-terminal Ig-like domains (residues 1–189) that have a 38% sequence homology with the KIR. Recently, we showed that CRP, a triple helical collagen-derived synthetic peptide, binds the apical surface of the first Ig-like domain of GPVI. The GPVI gene is located in the leucocyte receptor cluster at Chr 19q13.4 and has two alleles in Caucasoids, ‘a’ and ‘b’ with frequencies of 0.8 and 0.16, respectively. Studies with platelets from individuals homozygous for these two alleles demonstrated that the low frequency allele is associated with a reduced platelet aggregation response to CRP. This observation prompted us to generate a high-resolution single nucleotide polymorphism (SNP) map of the 8 GPVI exons encoding the mature protein by sequencing genomic DNA from 94 Caucasoids. A total of 8 silent and 6 replacement SNPs distributed evenly throughout the coding sequence were observed. Five of the replacement SNPs have been reported previously, 3 in the stem and 2 in the cytoplasmic domain. The 6th nonsynonymous SNP, which has a low frequency (0.01), encodes a novel Leu83Val substitution in the first Ig-like domain of GPVI. We also sequenced the GPVI gene in a nonhuman primate and observed 99% conservation at the nucleotide level in the two exons encoding the Ig-like domains. Taqman based typing for 8 of the 14 SNPs (6 replacement and 2 silent) in 1000 Caucasoid DNA samples confirmed the presence of two dominant alleles and revealed additional ‘hybrid’ alleles in 6% of individuals that have most likely arisen by crossing-over. Our study shows that the two Ig-like domains of GPVI, which are involved in ligand binding, are highly conserved, demonstrating a strong evolutionary pressure to maintain the amino acid sequence. We have identified a panel of individuals homozygous for the rare hybrid alleles that will be used delineate the effect of the different replacement mutations on the aggregation response to CRP. Comparisons of allele frequencies between different ethnic populations may elucidate the evolution of the GPVI gene diversification.