Platelet Cytoskeletal Force Transmission Through The GPIb-IX-V Complex

Platelets are the primary cellular components of the hemostatic plug that forms during primary hemostasis. The first step in this process is platelet adhesion from the flowing blood to a surface, carried out by the platelet glycoprotein (GP) Ib-IX-V binding to immobilized von Willebrand factor (VWF). Adhesion is followed by activation of integrin αIIbβ3, which mediates the attachment of platelets to each other by binding multivalent ligands such as VWF or fibrinogen. To stabilize the hemostatic plug and strengthen its attachment to the wound site, platelets must transmit contractile forces from actin and myosin proteins in their cytoskeleton to extracellular matrix proteins within the vessel wall or to the adhesive proteins between adjacent platelets. Integrin αIIbβ3 is one of the membrane proteins capable of transmitting these forces, having a direct link to the platelet cytoskeleton through talin and other focal adhesion related proteins. In the current study, we investigated whether the GPIb-IX-V complex is also capable of force transmission after binding ligand. The GPIb-IX-V complex contains 4 polypeptides, GPIbα, GPIbβ, GPIX and GPV. Only GPIbα binds VWF, which it does through VWF's A1 domain. GPIbα also attaches the complex to the actin and membrane skeletons through its cytoplasmic domain, with the large skeletal protein filamin functioning as the intermediary. There is strong evidence that the GPIbα-A1 bond is force sensitive, becoming stronger as force is applied to it, a property that defines it as a “catch bond”. For this reason, we investigated the role of GPIbα in transmitting platelet forces using a new tool that we have developed to measure contractile forces generated by platelets. This tool, composed of arrays of nanoposts separated by 2 μm (Figure 1), was fabricated using e-beam lithography. VWF was adsorbed to the tips of the nanoposts and platelets were allowed to adhere, spread, and contract. To assess the contribution of αIIbβ3 and GPIbα to force generation, we blocked these receptors with the antibodies 7E3 and AK2, respectively. Treatment with 7E3 significantly lowered the force generated, but did not eliminate it completely (57% reduction). AK2 had a smaller effect (20% reduction), and the combination of the two usually abolished force generation. We observed a similar force reduction (30%) as AK2 treatment when we blocked the VWF A1 domain with recombinant GPIbα N-terminus. Because VWF contains binding sites for more than one platelet receptor, and although purified, could have trace amounts of other plasma proteins, we also evaluated force generation on nanoposts coated with recombinant VWF A1 domain, which should only bind GPIbα. In this case, the platelets generated forces similar to those observed when αIIbβ3 was blocked by 7E3, providing further evidence that GPIbα can transmit forces by binding the A1 domain. Figure 1.Platelet bending nanoposts.Figure 1. Platelet bending nanoposts. As a final test of the ability of GPIbα to support force generation, we examined whether Chinese hamster ovary (CHO) cells expressing the GPIb-IX complex (CHOαβIX, fully functional but lacking GPV) could generate force on VWF or A1 domain (Figure 2). CHOαβIX cells adhered, spread and generated forces of similar magnitude on microposts (larger because of the larger cell size) coated with either substrate. CHOβIX cells, lacking GPIbα, did not adhere to either substrate.Figure 2.CHO cell bending microposts.Figure 2. CHO cell bending microposts. To investigate the requirement for cytoskeletal attachment of the complex in force generation, we studied a CHOαβIX line containing GPIbα truncated after residue 518 and therefore lacking almost the entire cytoplasmic domain. These cells adhered and spread on VWF-coated microposts, but generated minimal contractile force. Together, these results indicate that the GPIb-IX-V complex is able to transmit cytoskeletal contractile forces to its ligand, VWF, in a process requiring the cytoplasmic domain of GPIbα. This is the first example of a non-integrin transmitting force to an external substrate. Disclosures: Sniadecki: Stasys Medical Corporation: Equity Ownership, Founder Other.