Alpha-Dystroglycan Supports Platelet Aggregation and Thrombus Formation

Introduction:

Fibrinogen (Fg) and von Willebrand factor (VWF) have been considered essential for platelet adhesion and aggregation. However, platelet aggregation still occurs in mice lacking Fg and/or VWF or plasma fibronectin but not β3 integrin (JCI, 2000; JTH, 2006; Blood, 2009; JCI, 2014). This suggests that other non-classical αIIbβ3 integrin ligand(s) mediate platelet aggregation. α-dystroglycan (α-DG) is a component of the dystrophin-glycoprotein complex that binds extracellular matrix proteins containing laminin-G like domains via unique heteropolysaccharide [-GlcA-β1,3-Xyl-α1,3-]_n called matriglycan, which can be targeted specifically with monoclonal antibody IIH6C4. Although α-DG was identified in a recent proteomic study of platelet releasate, its membrane expression and function in platelets have never been investigated.

Methods and Results:

Using the anti-α-DG monoclonal antibody IIH6C4, we found expression of α-DG in mouse and human resting platelets in Western blots. α-DG expression was also identified on the non-permeabilized mouse and human resting platelets by flow cytometry, indicating that α-DG is constitutively expressed on the platelet surface.

We next examined whether disruption of the integrity of the dystrophin-glycoprotein complex affects the platelet aggregation. In a dystrophin-deficient mouse model of Duchenne muscular dystrophy with reduced α-DG expression (mdx mice), we found that ADP induced platelet aggregation in platelet-rich plasma (PRP) decreased 50%, suggesting that the integrity of the dystrophin-glycoprotein complex is required for normal platelet aggregation.

To test whether inhibition of platelet aggregation can be achieved by targeting α-DG, we applied the well-established polyclonal (H300) and monoclonal (IIH6C4 and VIA4) anti-α-DG antibodies. Mouse gel-filtered platelet aggregation induced by thrombin was significantly inhibited by all three antibodies. Mouse platelet aggregation in PRP was also inhibited by H300. For platelets from healthy human donors, the inhibitive effect was more profound. Using a lower concentration of H300 (1 µg/mL in human vs. 2 µg/mL in mouse), ADP induced human platelet aggregation in PRP was inhibited to less than 50% of control and quickly de-aggregated within 5 minutes, while no de-aggregation was observed in the controls. Human gel-filtered platelet aggregation was also inhibited in a dose-dependent manner by these antibodies. Our results thus revealed a vital role of α-DG in platelet aggregation.

In an ex vivo perfusion chamber model, human platelet adhesion and thrombus formation on collagen were markedly decreased at an arterial shear rate of 1800/s by anti-α-DG antibodies. Interestingly, although α-DG was found to be a ligand of laminin, platelet adhesion on laminin was not significantly altered by these antibodies, suggesting that contribution of α-DG to thrombus formation is not through its classical ligand laminin but other previously unidentified mechanisms. Next, we tested the role of α-DG in thrombus formation in vivo. Using a mouse cremaster artery laser-injury intravital microscopy model, we found that the anti-α-DG antibodies significantly delayed and decreased thrombus formation.

To investigate the underlying mechanism of the surprisingly profound impact of α-DG on platelet aggregation and thrombus formation, we performed the co-immunoprecipitation assay and found that α-DG interacts with both β3 integrin and fibronectin, even in the absence of Fg and VWF, suggesting that α-DG may directly or form an α-DG-fibronectin complex to bind αIIbβ3 integrin, contributing to platelet aggregation and thrombus formation.

Conclusion:

Our data demonstrated that α-DG and likely other components of the dystrophin-glycoprotein complex are expressed on the platelet surface, and play a vital role in platelet aggregation and thrombus formation. α-DG may contribute to platelet aggregation independent of VWF and Fg through direct or indirect interaction with αIIbβ3 integrin. It is likely that patients with muscular dystrophies, such as those with Duchenne muscular dystrophy, are protected from thrombosis. More importantly, our data established α-DG, and potentially other components of the dystrophin-glycoprotein complex, as novel targets for the treatment of thrombotic disorders.