Presentation Details
| Novel Mechanism of Thrombosis and Hemostasis Involving Platelet Alpha-Dystroglycan Viktor Prifti1, 2, Aron A.Shoara2, 3, Reid C.Gallant4, Sladjana Slavkovic2, 3, Kanwal Singh3, Margaret Rand1, Walter Kahr5, Yiming Wang2, Heyu Ni1, 2, 3. 1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.2Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute, St.Michael’s Hospital, Toronto, ON, Canada.3Canadian Blood Services Centre for Innovation, Toronto, ON, Canada.4Department of Medicine, University of Toronto, Toronto, ON, Canada.5Department of Biochemistry, University of Toronto, Toronto, ON, Canada |
Abstract
Background: Alpha-dystroglycan (αDG) is a cell-surface adhesion glycoprotein. Patients with Duchenne muscular dystrophy (DMD) who have reduced αDG experience increased bleeding during major surgeries. DMD patients have also shown reduced platelet aggregation and adhesion responses. Furthermore, αDG is known to possess matrix metalloprotease cleavage sites and is shed during platelet activation. However, the precise mechanism of platelet expressed αDG in the processes of hemostasis and pathological thrombosis remain unclear. Objectives: We investigated the functional role of platelet αDG in hemostasis and thrombosis, by blocking αDG with inhibitory antibodies and observing the effect on platelet aggregation and thrombus size and stability. We then studied candidate binding proteins of αDG which were hypothesized to be major platelet activation/adhesion receptors. Methods: ADP-, thrombin-, and collagen-induced aggregation assays were done with human platelet rich plasma (PRP) and gel-filtered platelets utilizing polyclonal (H-300) and monoclonal (VIA4-1) anti-αDG antibodies. Ristocetin- and VWF A1-induced agglutination assays were performed with VIA4-1 in human PRP and gel-filtered platelets. Cremaster arteriole thrombosis in vivo mouse model was utilized to investigate the inhibitory effect of anti-αDG antibody on thrombus formation. Bio-layer interferometry (BLI) was used to detect αDG binding to human αIIbβ3, fibronectin (Fn), αIIbβ3-Fn complex and also to GPIbα. Isothermal titration calorimetry (ITC) was utilized to quantify αDG-GPIbα binding thermodynamic parameters. Results: ADP-, thrombin-, and collagen-induced human platelet aggregation was inhibited with H-300 and VIA4-1. Ristocetin- and VWF A1-induced human platelet agglutination was inhibited with VIA4-1. In vivo mouse thrombus formation was inhibited by H-300. BLI detected a weak interaction between αDG and conformationally active αIIbβ3 enhanced by the presence of fibronectin. Importantly, a high affinity interaction was detected between αDG and GPIbα, which was subsequently confirmed with ITC. Conclusion: Platelet αDG has a critical role in thrombosis and hemostasis. αDG was found to be a potential cognate receptor for GPIbα. αDG was found to have a mild binding affinity to αIIbβ3 and Fn in isolation. However, αDG demonstrated enhanced affinity to the Fn-αIIbβ3 complex. GPIbα may propagate outside-in signaling following interaction with αDG to support platelet activation. Our results suggest that anti-thrombotic pharmaceutical agents could be developed which may also enhance the care of muscular dystrophy patients.
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