Platelets from mice lacking the aryl hydrocarbon receptor exhibit defective collagen-dependent signaling

Abstract

Background: We previously identified aryl hydrocarbon receptor (AHR) as a novel regulator of megakaryocytic differentiation and polyploidization and reported that AHR-null mice have approximately 15% fewer platelets than do wild-type mice, yet they exhibit a dramatic, unexplained bleeding phenotype. Objectives: The current work tests our hypothesis that AHR-null platelets are functionally deficient, contributing to the previously reported (yet unexplained) bleeding phenotype present in AHR-null mice. Methods: AHR-null bone marrow was ex vivo differentiated with thrombopoietin with or without AHR ligands or AHR inhibitors and analyzed for degree of megakaryopoiesis and polyploidization. Platelet function of AHR-null mice was assessed with aggregation and spreading assays. Platelet signaling was examined using Western analysis and Rac activity assays. Results: AHR ligands differentiate murine bone marrow-derived progenitors into polyploid megakaryocytes in the absence of thrombopoietin, and AHR inhibitors block thrombopoietin-induced megakaryocytic differentiation. Despite their responsiveness toward thrombin, AHR-null platelets demonstrate decreased aggregation and spreading in response to collagen compared with wild-type platelets. AHR-null platelets bind fibrinogen after stimulation with thrombin or AYPGKF and aggregate in response to AYPGKF and adenosine diphosphate. Mechanistically, AHR absence led to down-regulation of Vav1 and Vav3, altered phospholipase Cγ2 phosphorylation, decreased Rac1 activation, and reduced platelet activation in response to collagen. Conclusions: These results are consistent with a role for AHR in platelet function, especially as it relates to platelet aggregation and spreading in response to collagen. Our work suggests AHR is a critical component of the physiologic response that platelets undergo in response to collagen and may provide novel treatment options for patients with bleeding disorders. © 2013 International Society on Thrombosis and Haemostasis.