Identification and Functional Characterization of a Novel 27bp Deletion in the Macroglycopeptide-Coding Region of the GPIbα Gene Resulting in Platelet-Type Von Willebrand Disease.
Authors: Othman Maha , Elbatarny Hisham S. , Notley Colleen , Lavender Louise F. , White Helen , Byrne Christopher D. , O’Shaughnessy Denise F. , Lillicrap David P. 
Published: April 2020

Abstract
Platelet-type Von Willebrand disease (PT-VWD) is a rare autosomal dominant bleeding disorder. It results from an abnormally high affinity interaction between the platelet membrane glycoprotein Ib/IX/V complex and Von Willebrand factor (VWF), leading to characteristic platelet hyperaggregability. The condition shares most of the clinical and laboratory features of type 2B VWD and the final discrimination between these two diseases requires either platelet mixing studies or a molecular genetic approach. Five members of a British family with PT-VWD were studied; three affected and two unaffected. The proposita, had a long history of bleeding with recurrent epistaxis, postoperative and dental bleeding and menorrhagia. An initial series of hematological investigations lead to a diagnosis of type 2B VWD but a significant fall in her platelet count following therapy with a VWF/FVIII concentrate resulted in the consideration of PT-VWD as the correct diagnosis. This diagnosis was subsequently confirmed with appropriate FVIII and VWF assays. Affected individuals had VWF:Ag values in the range 0.34–0.47 U/mL, FVIII 0.45–0.64 U/mL and VWF:RCo ranging from 0.18–0.42 U/mL. These subjects showed loss of the HMW VWF multimers from plasma and enhanced ristocetin-induced platelet agglutination (RIPA). Also, washed normal platelets did not show enhanced RIPA when mixed with the patient’s plasma. On molecular genetic analysis of the GPIbα locus, the three affected family members were heterozygous for a 27 bp in-frame deletion (nucleotides 4374–4400). This corresponds to residues 421–429 in the macroglycopeptide region of GPIbα. This deletion was not identified in any of 50 normal controls. Flow cytometric quantitative analysis of the platelet GPIb/IX/V complex showed that the number of molecules for each of GPIb, GPIX and GPV, in each of the affected and non-affected family members fell within the normal range. Wild type (WT) GPIbα and the 27bp deletion mutant GPIba were then expressed in CHO b/IX cells. To examine the effect of the 27bp deletion on the conformation of the receptor, the cells were incubated with monoclonal antibodies that bind to different sites on GPIbα. We demonstrated that each of the three monoclonal antibodies bound WT and Mut GPIba to a similar extent and that the mutant protein was expressed at a normal level on the CHO cell surface. Finally, the effect of the 27bp deletion on VWF binding was analysed in CHO b/IX cells expressing the mutant and WT GPIbα receptor with 125I-VWF in the presence of varying ristocetin concentrations. We showed that the mutant receptor binds significantly more VWF than the WT GPIbα in the absence of ristocetin and also displays heightened sensitivity to low ristocetin concentrations compared to the WT cells. This study reports the first mutation resulting in PT-VWD that does not directly involve the VWF binding region of GPIbα. Our functional studies with the mutant receptor confirm that the deletion of 9 amino acids from the macroglycopeptide region of the protein enhances its interaction with VWF.