Reelin supports Glycoprotein VI and integrin outside-in signaling of platelets

Objective Reelin is known as an extracellular matrix protein mediating cell migration upon brain development. Recent studies provide evidence for reelin to be expressed in platelets, to co-localize to F-actin and to bind to amyloid precursor protein (APP) and apolipoprotein E receptor 2 (ApoER2) at the platelet surface. Furthermore, reelin is important for glycoprotein Ib (GPIb) mediated platelet adhesion and thrombus formation under high shear. Consequently, defective thrombus formation protects reelin-deficient mice (reeler) against arterial thrombosis.

We aim to define the role and signaling mechanisms of reelin in platelet cytoskeletal reorganization and glycoprotein VI (GPVI) mediated platelet activation and aggregation.

Material and Methods In vitro and in vivo analysis of reeler mice.

Results Reelin activated the small GTPases RhoA and Rac1 in platelets and was important for PAK1/2 phosphorylation thereby supporting lamellipodia formation upon spreading on fibrinogen. Experiments revealed strongly reduced clot retraction using platelet rich plasma (PRP) from reeler mice, which could be rescued by the addition of extracellular reelin, emphasizing the role of reelin for integrin outside-in signaling. Furthermore, adhesion and cytoskeletal reorganization of reeler platelets were reduced on immobilized collagen-related peptide (CRP) and collagen. Likewise significantly reduced phosphorylation of PLCγ2 and Syk as well as platelet aggregation and ATP release were detected using reeler platelets that have been stimulated with GPVI agonists. A significant reduction of platelet adhesion to immobilized reelin was observed when GPVI has been inhibited on platelets. Additionally, immunoprecipitation of reelin using recombinant GPVI clearly showed a direct interaction of GPVI and reelin. A direct interaction was confirmed with bio layer interferometry showing an interaction between reelin and GPVI with subnanomolar affinity. Consequently, in vivo thrombus formation was completely abrogated in reeler mice treated with a GPVI depleting antibody.

Conclusion Taken together these data provide first evidence for reelin to support GPVI signaling and integrin outside-in signaling of platelets. Thus, reelin affects platelet activation via different signaling pathways involving GPIb, GPVI and the classical reelin receptors APP and ApoER2 pointing to a promising role of reelin as therapeutic target for antithrombotic therapy.

Publication History

Article published online:
18 June 2021