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Thromb Haemost 2019; 119(07): 1102-1111
DOI: 10.1055/s-0039-1687878
Cellular Haemostasis and Platelets
Georg Thieme Verlag KG Stuttgart · New York

Alteration in GPIIb/IIIa Binding of VWD-Associated von Willebrand Factor Variants with C-Terminal Missense Mutations

Gesa König
1   Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf (UKE), University of Hamburg, Hamburg, Germany
,
Tobias Obser
1   Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf (UKE), University of Hamburg, Hamburg, Germany
,
Olivier Marggraf
2   Center for Anesthesiology and Intensive Care Medicine, Department of Intensive Care Medicine, University Medical Center Hamburg-Eppendorf (UKE), University of Hamburg, Hamburg, Germany
,
Sonja Schneppenheim
3   Medilys Central Laboratory Coagulation, Asklepios Clinic Altona, Hamburg, Germany
,
Ulrich Budde
3   Medilys Central Laboratory Coagulation, Asklepios Clinic Altona, Hamburg, Germany
,
Reinhard Schneppenheim
1   Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf (UKE), University of Hamburg, Hamburg, Germany
,
Maria A. Brehm
1   Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf (UKE), University of Hamburg, Hamburg, Germany
› Institutsangaben
Funding This study was supported by research funding from the German Research Foundation (DFG) to the Research Group FOR1543: “Shear flow regulation of hemostasis - bridging the gap between nanomechanics and clinical presentation.”
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Publikationsverlauf

08. November 2018

08. März 2019

Publikationsdatum:
29. April 2019 (online)

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Abstract

The platelet receptor glycoprotein (GP) IIb/IIIa, formed by integrins αIIb and β3, plays an important role in platelet adhesion and aggregation. Its major binding site is the arginine-glycine-aspartic acid (RGD) sequence present in several adhesive proteins. Upon platelet activation, inside-out signaling activates the complex permitting binding to RGD motif containing proteins, such as von Willebrand factor (VWF). VWF is a large multidomain plasma GP essential to primary hemostasis, which can directly interact with platelets because it exhibits binding sites for GPIbα and GPIIb/IIIa in its A1 and C4 domain, respectively. A vast variety of VWF variants have been identified in which domain-specific mutations affect distinct functions of VWF but reduced GPIIb/IIIa binding has barely been studied so far. Here, we strived to investigate the influence of C domain mutations, which have been identified in patients diagnosed with von Willebrand disease (VWD), on VWF–GPIIb/IIIa interaction. To determine binding to membrane-incorporated GPIIb/IIIa in the absence of GPIbα, we developed and validated a cell-based binding assay which uses HEK293 cells stably expressing a constitutively active form of the GPIIb/IIIa receptor complex on their plasma membrane. By employing this assay, we measured GPIIb/IIIa binding of 14 VWF C domain mutants identified in VWD patients. Mutants p.Cys2257Arg, p.Gly2441Cys, p.Cys2477Tyr, and p.Pro2722Ala exhibited significantly reduced binding. Summarizing, we have developed a useful research tool to specifically investigate GPIIb/IIIa interaction with its protein binding partners and identified four VWF variants that exhibit impaired GPIIb/IIIa binding. At least in the homozygous state, this defect could contribute to the VWD phenotype.

Keywords

GPIIb/IIIa - αIIbβ3 - von Willebrand factor - von Willebrand disease

Supplementary Material