Identification and functional characterization of a novel 27-bp deletion in the macroglycopeptide-coding region of the GPIBA gene resulting in platelet-type von Willebrand disease

Abstract

Interaction between the platelet glycoprotein Ib␣ (GPIb␣) receptor and its adhesive ligand von Willebrand factor (VWF) has a critical role in the process of hemostasis. Platelet-type von Willebrand disease (PT-VWD) is a rare bleeding disorder that results from gain-of-function mutations in the GPIBA gene. We studied this gene from 5 members of a previously unreported family with a PT-VWD phenotype. We identified a novel in-frame deletion of 27 base pair (bp) in the macroglycopeptide region. This deletion was not found in the unaffected family members or in 50 healthy controls. The patients' platelets expressed normal quantities of GPIb/IX/V complex on their surface and the mutant (Mut) GPIb␣ was expressed at levels indistinguishable from the wild-type (WT) receptor on the surface of transfected Chinese hamster ovary ( IntroductionPlatelet-type von Willebrand disease (PT-VWD) is a rare autosomal dominant bleeding disorder first described in 1982. 1 It results from an abnormally high affinity interaction between the platelet membrane glycoprotein (GP) Ib/IX/V complex and von Willebrand factor (VWF), leading to characteristic platelet hyperaggregability. 1,2 Patients with PT-VWD have frequent and severe nosebleeds and excessive bleeding following tooth extraction, tonsillectomy, and other surgical operations. 3 The condition shares most of the clinical and laboratory features of type IIB VWD and the final discrimination between them requires either platelet-mixing studies or a molecular genetic approach. Rapid clearance of highmolecular-weight (HMW) VWF multimers from plasma together with thrombocytopenia caused by an increased removal of the aggregated platelets contribute to the bleeding diathesis in PT-VWD. 3,4 The GPIb/IX/V platelet receptor comprises 4 transmembrane polypeptides, each of which belongs to the leucine-rich repeat (LRR) family of proteins. They are GPIb␣ disulfide linked to GPIb␤, GPIX, and GPV, each of which is noncovalently associated with the complex with a stoichiometry of 2:2:2:1. 5,6 The 2 proteins, GPIb␤ and GPIX, are required for efficient cell-surface expression of GPIb␣. 7 GPIb␣ is the largest component of the receptor (610 amino acids) and carries the VWF-binding site. The structure of GPIb␣ reveals 4 functional domains: the 45-kDa amino-terminal domain; a heavily glycosylated mucinlike stalk known as the macroglycopeptide region, a single transmembrane domain, and an intracytoplasmic domain. [8][9][10] Four regions within the amino-terminal domain contribute to VWF binding: N-terminal flank (His1-lle35), LRRs (Leu36-Ala200 residues), C-terminal disulfide loop (Phe201-Gly268), and an anionic tyrosine sulfated region (Asp269-Glu282). [11][12][13] The interaction between VWF and its platelet receptor GPIb␣ is essential for platelet adhesion at sites of high-shear stress. Structural changes affecting GPIb␣ are responsible for the PT-VWD phenotype. 14,15 To date, 5 families and one isolated case of PT-VWD have been reported and in these cases the responsible mutations reside within the ...

Diagnosis of platelet-type von Willebrand disease by flow cytometry

“…[1][2][3][4][5] As a consequence, platelets from patients with PT-VWD bind spontaneously high molecular weight (HMW) multimers of VWF and are then cleared from the circulation, resulting in thrombocytopenia and the loss of HMW VWF multimers.…”

“…8 Differential diagnosis is important because the two diseases must be treated differently: patients with type 2B VWD with normal exogenous VWF while patients with PT-VWD with platelet transfusions. 5 We have recently described a flow cytometric assay to quantify VWF-binding induced by ristocetin to fresh autologous or to formalin-fixed donor platelets. 9 The assay is useful in the diagnosis of VWD and for the monitoring of DDAVP treatment and in particular is able to detect enhanced affinity of VWF for GPIb, similarly to the RIPA, and therefore allows the diagnosis of type 2B VWD.…”

Visualizing the von Willebrand factor/glycoprotein Ib-IX axis with a platelet-type von Willebrand disease mutation

“…To this end, platelet-type von Willebrand disease (PT-VWD) is a hereditary bleeding disorder caused by point mutations within the extracellular ␣-subunit domain of the GP Ib-IX receptor. [3][4][5][6] A documented result of PT-VWD mutations is an increased affinity between mutant GP Ib-IX and soluble VWF. 7 A mechanistically similar situation exists with type 2B VWD where single mutations in VWF have an increased affinity for a normal platelet GP Ib-IX receptor.…”

Platelet Dysfunction and a High Bone Mass Phenotype in a Murine Model of Platelet-Type von Willebrand Disease

“…6,11,12 One report describes a 27-bp deletion within the GP Ib␣ macroglycopeptide domain that results in a Pt-vWD phenotype. 13 Both the 233 and 239 residues reside within a ␤-hairpin loop in the crystal structure of GP Ib␣. 14 These mutations have been proposed to stabilize the loop conformation leading to an increased affinity for vWF ( Figure 1).…”