Flow-based measurements of von Willebrand factor (VWF) function: Binding to collagen and platelet adhesion under physiological shear rate

Fuchs, Birte; Budde, Ulrich; Schulz, Andrea; Kessler, Craig M.; Fisseau, Claudine; Kannicht, Christoph

doi:10.1016/j.thromres.2009.08.020

« Previous Next »Thrombosis Research
Volume 125, Issue 3 , Pages 239-245, March 2010

Flow-based measurements of von Willebrand factor (VWF) function: Binding to collagen and platelet adhesion under physiological shear rate

Birte Fuchs
- Octapharma Research & Development, Molecular Biochemistry Department, Berlin, Germany
Ulrich Budde
- AescuLabor-Hamburg, Institut der Labormedizin, Hamburg, Germany
Andrea Schulz
- Institute of Chemistry and Biochemistry, Organic Chemistry, Freie Universität Berlin, Germany
Craig M. Kessler
- Lombardi Cancer Center, Georgetown University Medical Center, Washington, USA
Claudine Fisseau
- Octapharma Research & Development, Molecular Biochemistry Department, Berlin, Germany
Christoph Kannicht
- Octapharma Research & Development, Molecular Biochemistry Department, Berlin, Germany
- Corresponding author. Octapharma R&D, Molecular Biochemistry Department, Arnimallee 22, D-14195 Berlin, Germany. Tel.: +49 30 83 22 73 60; fax: +49 30 84 10 98 62.

Received 19 December 2008; received in revised form 19 August 2009; accepted 31 August 2009.

Abstract

Introduction

VWF circulates in plasma as a series of heterogeneous multimers, mediating platelet tethering, translocation and finally adhesion to areas of injured endothelium under physiological high arterial blood flow. VWF-platelet binding requires conformational changes in VWF, which are induced by immobilization and shear. Because of unavailability of a simple flow-based measurement system, VWF activity assays are generally performed under static conditions. We describe an easily reproducible in vitro flow-chamber model using commercially available flow devices to examine VWF-collagen binding and VWF-mediated platelet adhesion under physiological flow conditions.

Methods

The collagen surface of the flow-chamber was analyzed by atomic force microscopy. Collagen-bound VWF was characterized by multimer analysis and multi labelling immunofluorescence detection of exposed GPIb binding domains. Platelet adhesion was captured by time-lapse microscopy.

Results

The described flow-chamber system facilitates multimer analysis of collagen-bound VWF, whereas all VWF multimers bound to collagen under physiological low to high shear rates. Multi labelling immunofluorescence detection exhibited exposed GPIb binding domains co-localized with VWF molecules. VWF-dependent platelet adhesion using time-lapse microscopy showed values comparable to experiments done with whole blood, and platelet adhesion was dependent on the VWF concentration.

Conclusions

The established flow-chamber model represents an easy-to-set-up and customized tool for the characterization of VWF-binding to collagen as well as the determination of VWF-dependent platelet adhesion under defined flow conditions in real-time.

Abbreviations: AFM, atomic force microscopy, ECM, extracellular matrix, FVIII, coagulation factor VIII, GPIb, glycoprotein Ib, RBCs, red blood cells, VWD, von Willebrand disease, VWF, von Willebrand factor, VWF:Ag, von Willebrand factor antigen, VWF:CB, von Willebrand factor collagen binding activity, VWF:RCo, von Willebrand factor ristocetin cofactor activity