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Gene cloning of rat and mouse platelet glycoprotein V: identification of
megakaryocyte-specific promoters and demonstration of functional thrombin
cleavage
C Ravanat, M Morales, DO Azorsa, S Moog, S Schuhler, P Grunert, D Loew, A Van Dorsselaer, JP Cazenave and F Lanza
INSERM Unite U.311, Etablissement de Transfusion Sanguine de Strasbourg,
France.
Platelet glycoprotein (GP) V is a major surface protein cleaved during
thrombin-induced platelet activation. GPV associates noncovalently with the
GPIb-IX complex to form GPIb-V-IX, a receptor for von Willebrand factor and
thrombin. We describe the cloning of the genes coding for rat and mouse GPV
and compare them with the human gene. The two rodent genes have a similar
structure and resemble the human GPV gene with a coding sequence
(approximately 1,700 nucleotides) entirely contained in one exon and a
single intron (approximately 900 nucleotides) in the 5' untranslated
region. Both genes have megakaryocyte-type promoters with conserved tandem
Ets and GATA recognition motifs and lack a TATA box. The mature rat and
mouse proteins comprise 551 amino acids, have 70% sequence identity, and
contain an additional 8-amino acid intracellular segment as compared with
the human protein. As in human GPV, there is an NH2-terminal leucine-rich
region of 15 repeats and a thrombin cleavage recognition sequence. Whereas
the rat and human thrombin cleavage sites are similar, the mouse cleavage
site resembles that of the human thrombin receptor. Functionality of these
sites was demonstrated by thrombin cleavage of synthetic peptides and
analysis by high-performance liquid chromatography (HPLC) or mass
spectrometry. Cleavage of native rat GPV was confirmed by means of a
polyclonal antibody directed against the new NH2-terminal peptide exposed
after thrombin cleavage. This antibody specifically recognized thrombin-
activated rat platelets by fluorescence-activated cell sorting (FACS)
analysis. In addition, we raised monoclonal antibodies specific for rat GPV
(88 kD), which recognized the NH2-terminal soluble fragment (70 kD)
liberated after thrombin cleavage. Knowledge of these rodent GPV genes and
availability of species-specific peptides and antibodies will be essential
to further studies aiming to define the exact in vivo function of platelet
GPV using animal models of thrombosis and gene inactivation experiments.
Volume 89,
Issue 9,
pp. 3253-3262,
05/01/1997
Copyright © 1997 by The American Society of Hematology
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