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The PAI-1-vitronectin-vimentin ternary complex : mechanism of extracellular assembly and role in transplant vasculopathy

University of British Columbia

The active state of plasminogen activator inhibitor type-1 (PAI-1) is prolonged when it forms a complex with vitronectin (VN), a major serum protein. Active PAI-1 in the PAI-1:VN complex serves many functions related to fibrinolysis and cell migration but key to these effects is its extracellular distribution. PAI-1:VN complexes can bind to exposed vimentin (VIM) on activated platelet and platelet microparticles, resulting in the assembly of PAI-1:VN:VIM ternary complexes. However, the manner in which the vimentin cytoskeleton is presented extracellularlyi s not well understood. I hypothesized that PAI-1:VN:VIM ternary complex assembly occurs on cell surfaces when microparticle release leads to exposure of vimentin cytoskeleton which can lead to either assembly of the ternary complex or become involved in an autoimmune response specific for vimentin. To follow the intracellular and extracellular fate of PAI-1, I constructed an expression vector encoding PAI-1-dsRed, a fluorescent form of PAI-1, which would permit live cell tracking of PAI-1 in megakaryocytes and endothelial cells. Secondly, to study how vimentin is expressed on platelets and platelet microparticles, flow cytometry was used to isolate vimentin positive platelets or PMP's and atomic force microscopy was performed to image platelets or PMP's at nanoscale resolution. From these studies, I propose a model of vimentin expression in which the junction of microparticle release results in the exposure of cytoskeletal vimentin on both the cell and the microparticle. This exposed vimentin could potentially induce VN multimerization on the same cell surface leading to incorporation of multiple PAI-1:VN complexes. Finally, I investigated how anti-vimentin antibodies can induce platelet:leukocyte conjugate formation. To achieve this, in vitro tests were performed to determine the binding site of anti-vimentin antibodies (AVA's) and how they induce blood cell activation. Overall, my results suggest that vimentin exposure in our model of microparticle release can lead to ternary complex assembly if suitable quantities of PAI-1 are released during platelet activation. In the setting of transplant vasculopathy with high titres of AVA's, vimentin-positive granulocytes can bind these autoantibodies, which then leads to platelet activation and the formation of platelet:leukocyte conjugates.

Thesis/Dissertation

application/pdf

2008-10-09

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/2509

Pathology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/