Expression of the amino-terminal domain of platelet glycoprotein Ib alpha:Exploitation of a calmodulin tag for determination of its functional activity

Platelet glycoprotein (GP) Ib alpha is a component of the GPIb-IX receptor complex, which is involved in multiple physiological and pathological proce sses, including platelet adhesion at sites of vascular injury, thrombin bin ding, Bernard-Soulier syndrome, platelet-type von Willebrand disease, and i mmune-mediated thrombocytopenias. The amino-terminal domain of similar to 3 00 residues of GPIb alpha mediates both normal biological function (by prov iding the sites for direct ligand interaction) and aberrant function (throu gh amino acid substitutions). To investigate the molecular interactions med iated by this region of GPIb alpha we have developed a recombinant baculovi rus to facilitate its expression as a calmodulin fusion protein from insect cells. By employing the calmodulin tag, the fusion protein could be obtain ed at > 90% purity after a single isolation step at yields of 8 mg/L of ins ect cell medium (purified fusion protein). The recombinant GPIb alpha fragm ent was shown to be posttranslationally sulfated and glycosylated, although its glycosylation differed from that of the equivalent GPIb alpha fragment isolated from human platelets. The differential glycosylation, however, di d not affect the function of the recombinant GPIb alpha fragment in either von Willebrand factor (vWf) or thrombin binding as these were both found to be identical to those of the same-length GPIb alpha fragment derived from human platelets, The calmodulin tag was also exploited in the development o f assays to measure directly vWf and thrombin binding, since it did not int erfere with either demonstrating the feasibility for the use of this solubl e receptor fusion protein in detailed biophysical assays to investigate the molecular mode of binding of platelet glycoprotein Ib alpha to these ligan ds. (C) 2001 Academic Press.