The Role of Ligand Density in the Binding of Von Willebrand Factor by the Glycoprotein Ib-IX-V Complex in Platelets

Abstract

The initial arrest of platelets at a wound site requires the binding of glycoprotein Ib-IX-V complex (GPIb-IX-V) to the extracellular matrix protein von Willebrand factor (VWF). Increasing forces on these bonds increases the bond lifetime, known as a catch bond. Recently, we have shown that platelets are able to transmit cytoskeletal forces through the GPIbα subunit of GPIb-IX-V, which binds to the A1 domain of VWF. This provides an internal force on the bond to maintain adhesion in the absence of external forces. Integrin engagement and force transmission are known to require receptor clustering. We therefore investigate whether the clustering of GPIb-IX-V receptor is needed for the transmission of cytoskeletal forces to VWF. We first examined whether GPIb-IX-V clusters formed in spreading platelets. GPIbα-positive punctate structures were visualized by confocal microscopy and correlated with dark regions imaged using interference reflection microscopy (IRM), indicating that GPIb-IX-V forms adhesive contacts that are similar in size to integrin-related focal adhesions in platelets. To investigate the effect of ligand density on platelet spreading, we prepared molecularly-defined adhesive ligand spots, which were separated 28 or 108 nm apart by nonadhesive regions using self-assembling gold nanoparticles. These nanoparticles were coated with A1 domain of VWF. When seeded on nanoparticles with 28 nm spacing, platelets appeared well-spread and formed punctate adhesions. However, when seeded on 108 nm spacing, platelet adhesion was significantly reduced and those platelets that did adhere spread poorly. These findings demonstrate that an upper limit for ligand density exists where platelet adhesion and spreading are impeded. Investigating the effects of ligand spacing will facilitate an understanding of GPIbα clustering in platelet adhesion and spreading, thus providing insight into thrombotic diseases and congenital bleeding disorders.