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Glycoprotein Ib-IX-V Complex Transmits Cytoskeletal Forces That Enhance Platelet Adhesion.

ABSTRACT: Platelets bind to exposed vascular matrix at a wound site through a highly specialized surface receptor, glycoprotein (GP) Ib-IX-V complex, which recognizes von Willebrand factor (VWF) in the matrix. GPIb-IX-V is a catch bond for it becomes more stable as force is applied to it. After attaching to the wound site, platelets generate cytoskeletal forces to compact and reinforce the hemostatic plug. Here, we evaluated the role of the GPIb-IX-V complex in the transmission of cytoskeletal forces. We used arrays of flexible, silicone nanoposts to measure the contractility of individual platelets on VWF. We found that a significant proportion of cytoskeletal forces were transmitted to VWF through GPIb-IX-V, an unexpected finding given the widely held notion that platelet forces are transmitted exclusively through its integrins. In particular, we found that the interaction between GPIbα and the A1 domain of VWF mediates this force transmission. We also demonstrate that the binding interaction between GPIbα and filamin A is involved in force transmission. Furthermore, our studies suggest that cytoskeletal forces acting through GPIbα are involved in maintaining platelet adhesion when external forces are absent. Thus, the GPIb-IX-V/VWF bond is able to transmit force, and uses this force to strengthen the bond through a catch-bond mechanism. This finding expands our understanding of how platelets attach to sites of vascular injury, describing a new, to the best of our knowledge, mechanism in which the catch bonds of GPIb-IX-V/VWF can be supported by internal forces produced by cytoskeletal tension.

SUBMITTER: Feghhi S

PROVIDER: S-EPMC4982925 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Reconstitution of the platelet glycoprotein Ib-IX complex in phospholipid bilayer Nanodiscs.

Project description:The glycoprotein Ib-IX (GPIb-IX) complex expressed on platelet plasma membrane is involved in thrombosis and hemostasis via the initiation of adhesion of platelets to von Willebrand factor (VWF) exposed at the injured vessel wall. While most of the knowledge of the GPIb-IX complex was obtained from studies on platelets and transfected mammalian cells expressing the GPIb-IX complex, there is not an in vitro membrane system that allows systematic analysis of this receptor. The phospholipid bilayer Nanodisc composed of a patch of phospholipid surrounded by membrane scaffold protein is an attractive tool for membrane protein study. We show here that the GPIb-IX complex purified from human platelets has been reconstituted into the Nanodisc. The Nanodisc-reconstituted GPIb-IX complex was able to bind various conformation-sensitive monoclonal antibodies. Furthermore, it bound to VWF in the presence of botrocetin with an apparent K(d) of 0.73 ± 0.07 nM. The binding to VWF was inhibited by anti-GPIb? antibodies with epitopes overlapping with the VWF-binding site, but not by anti-GPIb? monoclonal antibody RAM.1. Finally, the Nanodisc-reconstituted GPIb-IX complex exhibited ligand binding activity similar to that of the isolated extracellular domain of GPIb?. In conclusion, the GPIb-IX complex in Nanodiscs adopts a native-like conformation and possesses the ability to bind its natural ligands, thus making a Nanodisc a suitable in vitro platform for further investigation of this hemostatically important receptor complex.

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Transmembrane domains are critical to the interaction between platelet glycoprotein V and glycoprotein Ib-IX complex.

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Specific heteromeric association of four transmembrane peptides derived from platelet glycoprotein Ib-IX complex.

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