Assessing the Role of GPIb-IX-V Complex in Transmitting Platelet Cytoskeletal Forces

Thesis (Ph.D.)--University of Washington, 2015Platelets rapidly seal wounds in blood vessels, preventing blood loss. They bind to ex-posed vascular matrix at the wound site through a highly specialized surface receptor, the glycoprotein (GP) Ib-IX-V complex, which recognizes von Willebrand factor (VWF) in the matrix. This bond is unique in that it becomes more stable as force is applied to it, i.e. a “catch” bond. After attaching to the wound site, platelets generate cytoskeletal forces to compact and reinforce the hemostatic clot. Here, we evaluated the role of the GPIb-IX-V complex in the transmission of these forc-es, using arrays of flexible 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 integrins. In particular, the GPIbα sub-unit of GPIb-IX-V mediates this force transmission through its interaction with the A1 domain of VWF on the platelet exterior and by binding the cytoskeletal actin-binding protein filamin A in the interior. We then used Chinese hamster ovary (CHO) cells that express GPIb-IX complex to investigate the mechanism by which GPIb-IX-V transmits cytoskeletal forces. We found that, these forces are myosin-dependent and are required to maintain cell attachment through the GPIb-IX-V complex. Using CHO cells that lacked the filamin A binding site, a cytoskeletal protein that links the complex to the actin cyto-skeleton of the platelets, we found that, this complex plays a major role in mediating the cytoskeletal force transmission through the GPIb-IX-V complex. Thus, the GPIb-IX-V/VWF bond is able to transmit cytoskeletal force, and uses this force to strengthen the bond through a catch-bond mechanism. This finding expands our understanding of how cells attach to matrices, describing a new mechanism in which catch bonds can be sup-ported by internal forces in the absence of external forces to reinforce attachment. We further studied the role of GPIb-IX-V complex in supporting contractile forces of hemostatic clots that form under flow condition, using a microfluidic device. We embed-ded a multitude of block and micropost pairs at the bottom of the microfluidic channel to measure the contractile forces of hemostatic clots as they formed between the block and micropost at pathological shear rates. We found that, in addition to supporting the clot at the wound site, GPIb-IX-V complex plays a significant role in supporting the contractile forces of hemostatic clots. Together these results identify the first example of a non-integrin receptor that is capable of transmitting forces in platelets