Susan R. Russell

Point mutation in a Leucine-rich repeat of platelet glycoprotein Ibα resulting in the Bernard-Soulier syndrome

Leucine-rich repeats are a conserved structural motif, of yet undefined significance, found in a group of proteins from different species. Among these are the four components of the human platelet glycoprotein Ib-IX-V complex, a membrane receptor that performs an essential role in the thrombogenic function of platelets by interacting with the adhesive protein, von Willebrand factor. We have found that a single amino acid substitution (Ala156-->Val) within one of the six leucine-rich repeats in the alpha-subunit of glycoprotein Ib results in a variant form of the congenital bleeding disorder, Bernard-Soulier syndrome, characterized by giant dysfunctional platelets. Genetic studies of the propositus and his family members were complemented by immunological and functional analysis of expressed recombinant GP Ib alpha fragments to demonstrate that the observed mutation is the cause of defective von Willebrand factor binding. These studies define the molecular basis of the Bernard-Soulier syndrome within this family and demonstrate that structural integrity of a leucine-rich repeat is necessary for normal function of the glycoprotein Ib-IX-V receptor complex and, possibly, for normal platelet morphology.

Expression of the phenotypic abnormality of platelet-type von Willebrand disease in a recombinant glycoprotein Ib alpha fragment.

The platelet GP Ib-IX receptor supports platelet adhesion and activation by binding to vWf in the exposed subendothelial matrix. An abnormal GP Ib-IX complex exists in platelet-type or pseudo-von Willebrand disease and has a characteristic increased affinity for soluble vWf resulting in impaired hemostatic function due to the removal of larger vWf multimers from the circulation. Genetic studies within an afflicted family have demonstrated that the disease is linked to a Gly233-->Val amino acid substitution within the alpha-subunit of the oligomeric GP Ib-IX complex (Miller, J.L., D. Cunningham, V.A. Lyle, and C. L. Finch. 1991. Proc. Natl. Acad. Sci. USA. 88:4761-4765). To evaluate the functional consequences of this mutation, we constructed a recombinant analogue of the alpha-subunit of GP Ib containing Val233. Experiments comparing molecules with either Gly233 or Val233 revealed that the Val substitution generates a molecule with increased affinity for vWf. The recombinant fragment reproduces the functional abnormality of the GP Ib-IX complex in platelet-type von Willebrand disease, thus establishing the molecular basis of the bleeding disorder within this family. Moreover, it becomes apparent that structural elements responsible for the regulation of hemostasis through modulation of vWf affinity for platelets reside within the alpha-subunit of the GP Ib-IX complex.