Novel gain-of-function mutations of platelet glycoprotein Ib alpha by valine mutagenesis in the Cys(209)-Cys(24)8 disulfide loop

Platelet-type von Willebrand disease is a bleeding disorder resulting from gain-of-function mutations of glycoprotein (GP) Ib alpha that increase its affinity for von Willebrand factor (vWf). The two known naturally occurring mutations, G233V and M239V, both enrich the valine content of an already v aline-rich region within the Cys(209)-Cys(248) disulfide loop. We tested th e effect of converting other non-valine residues in this region to valine. Of 10 mutants expressed in CHO cells as components of GP nb-M complexes, fo ur displayed a gain-of-function phenotype (G233V, D235V, K237V, and M239V) based on I-125-vWf binding and adhesion to immobilized vWf. The remainder d isplayed loss-of-function phenotypes. The gain-of-function mutants bound vW f spontaneously and had a heightened response to low concentrations of rist ocetin or botrocetin, whereas the loss-of-function mutants bound vWf more p oorly than wild-type GP Ib alpha. No distinct gain- or loss-of-function con formations were identified with conformation-sensitive antibodies. Compared with cells expressing wild-type GP Ib alpha, cells expressing the gain of- function mutants rolled significantly more slowly over immobilized vWf unde r flow than wild-type cells and were able to adhere to vWf coated at lower densities. In aggregate, these data indicate that the region of GP Ib alpha bounded by Asn(226) and Ala(244) regulates the affinity for vWf.