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 plate