Cloning and expression of canine glycoprotein Ib alpha

The interaction of the glycoprotein (GP) Ib-IX-V complex with von Willebran d factor (vWF) is critical in initiation of haemostasis and thrombosis thro ugh platelet adhesion to damaged endothelium. The binding site for vWF resi des within the GPIb alpha subunit of the complex. To further define the phy siological function of platelet GPIb alpha we cloned and expressed the cani ne GPIb alpha cDNA. A canine platelet cDNA library was constructed and scre ened with a randomly primed P-32-labeled 1041-base-pair restriction fragmen t of the human GPIb alpha cDNA. Analysis of 23 clones demonstrated that the canine GPIb alpha cDNA is 2530 nucleotides in length and includes a short 5' untranslated segment of 42 nucleotides followed by a signal peptide of 1 6 amino acids, a mature peptide of 645 amino acids and a 3' noncoding regio n of 455 nucleotides. A single intron of 142 nucleotides, 6 nucleotides ups tream from the ATG translation initiation codon was identified in the canin e gene in a similar location to that present in the human gene. Chinese hamster ovary cells that stably express human GPIb beta and GPIX we re transfected with the canine GPIb alpha cDNA. Canine GPIb alpha was expre ssed on the surface of these cells and bound vWF in the presence of botroce tin. The binding of vWF was inhibited by an anti-vWF human monoclonal antib ody known to inhibit vWF binding to GPIb alpha. The results of this investi gation will allow the development of reagents to study the physiological fu nction of GPIb alpha in an animal model.