Cloning and characterization of the gene encoding the human platelet glycoprotein V. A member of the leucine-rich glycoprotein family cleaved during thrombin-induced platelet activation.
Glycoprotein V (GPV) is a major platelet membrane 82-kDa glycoprotein, missing in the Bernard-Soulier syndrome, that is cleaved when platelets are treated with thrombin. We report the cloning and sequencing of the GPV cDNA and gene obtained by a combination of polymerase chain reaction amplification of platelet mRNA and genomic library screening. The single-copy gene for GPV is contained within 6.5 kilobase pairs (kb) of genomic sequence and has a simple structure with a single intron of 958 base pairs in the 5'-untranslated sequence; the coding sequence is contained within a single exon. The promoter region contains a canonical TATA box, and putative GATA, Ets-1, and Sp1 cis-acting elements. Reverse transcription-polymerase chain reaction analysis on RNAs from cells of different hematopoietic origins revealed that GPV was specifically transcribed from platelets and from cells of the megakaryocytic lineage (megakaryocytes, HEL cells). A single transcript of 4.5 kb for GPV was detected in human platelets by Northern blot analysis. The entire amino acid sequence of GPV was deduced from the cDNA and genomic sequences. Mature GPV was composed of 544 amino acids which contained a single transmembrane domain, a short cytoplasmic domain (16 residues), and a large extracellular domain with 8 potential N-glycosylation sites. Analysis of the extracellular domain revealed the presence of 15 tandem Leu-rich repeats of 24 amino acids with homology to GPIb alpha and identified a cleavage site for thrombin near the COOH terminus with similarity to the A alpha chain of fibrinogen, but no hirudin-like sequence was found.
Lanza
F
F
Centre Régional de Transfusion Sanguine, Institut National de la Santé et de la Recherche Médicale Unité 311, Strasbourg, France.
Morales
M
M
de La Salle
C
C
Cazenave
J P
JP
Clemetson
K J
KJ
Shimomura
T
T
Phillips
D R
DR
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D007930:Leucine / Q000032:analysis*; D008969:Molecular Sequence Data; D015539:Platelet Activation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013917:Thrombin / Q000494:pharmacology*
D018076:DNA, Complementary; D010980:Platelet Membrane Glycoproteins; D013917:Thrombin; D007930:Leucine