GENE CLONING OF RAT AND MOUSE PLATELET GLYCOPROTEIN-V - IDENTIFICATION OF MEGAKARYOCYTE-SPECIFIC PROMOTERS AND DEMONSTRATION OF FUNCTIONAL THROMBIN CLEAVAGE

Platelet glycoprotein (GP) V is a major surface protein cleaved during thrombin-induced platelet activation. GPV associates noncovalently wi th the GPIb-IX complex to form GPIb-V-IX, a receptor for von Willebran d 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 roden t genes have a similar structure and resemble the human GPV gene with a coding sequence (approximate to 1,700 nucleotides) entirely containe d in one exon and a single intron (approximate to 900 nucleotides) in the 5' untranslated region. Both genes have megakaryocyte-type promote rs with conserved tandem Ets and GATA recognition motifs and lack a TA TA box. The mature rat and mouse proteins comprise 551 amino acids, ha ve 70% sequence identity, and contain an additional 8-amino acid intra cellular segment as compared with the human protein. As in human GPV, there is an NH2-terminal leucine-rich region of 15 repeats and a throm bin 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 demonstr ated by thrombin cleavage of synthetic peptides and analysis by high-p erformance liquid chromatography (HPLC) or mass spectrometry. Cleavage of native rat GPV was confirmed by means of a polyclonal antibody dir ected against the new NH2-terminal peptide exposed after thrombin clea vage. This antibody specifically recognized thrombin-activated rat pla telets by fluorescence-activated cell sorting (FAGS) analysis. In addi tion, we raised monoclonal antibodies specific for rat GPV (88 kD), wh ich recognized the NH2-terminal soluble fragment (70 kD) liberated aft er thrombin cleavage. Knowledge of these rodent GPV genes and availabi lity of species-specific peptides and antibodies will be essential to further studies aiming to define the exact in vivo function of platele t GPV using animal models of thrombosis and gene inactivation experime nts. (C) 1997 by The American Society of Hematology.