We previously identified AHR as a novel regulator of megakaryocytic (Mk) polyploidization and differentiation (Lindsey et al. Brit J of Haem, 2011). Best known as a mediator of toxicological signals, we propose that AHR influences multiple aspects of normal hematopoietic differentiation (Lindsey et al. Stem Cell Rev, 2012), including unpublished data suggesting a role for AHR in mediating platelet function. AHR-null mice had 9% fewer platelets and 10.4% fewer reticulated, young RNA-containing platelets than WT mice. Abnormal Mk maturation played a role in this phenotype, as AhR-null mice had ca. 25% fewer high ploidy (= 32n) Mks residing within the murine bone marrow niche compared to WT mice. While investigating if AHR influenced platelet function, we found that AHR-null mice bleed 5.3 times longer (8 minutes for AHR-null mice compared to 1.5 minutes for WT mice) and lose 3 times as much blood as WT mice during bleeding time assays. Although significant, we felt that the decreased Mk polyploidization and resulting reduced platelet counts were not enough to explain the drastic bleeding phenotype in AHR-null mice. In agreement with our hypothesis that AHR impacts platelet function, others have suggested AHR is critical for blood clotting during Oryzias latipes embryogenesis (Kawamura et al. Zoolog Sci, 2002). We previously showed that treatment of bone marrow-derived progenitor cells with AHR ligands such as TCDD (dioxin, a prototypic AHR ligand and activator) during ex vivo expansion could produce polyploid CD41-expressing cells in the absence of any cytokines. Here, we show that ex vivo expansion of murine progenitor cells with 10 mM of the AHR inhibitor 6',2',4'-trimethoxyflavone (TMF) resulted in 37% fewer highly polyploid (≥32n) Mks by day 7 (n=3, p=0.017), effectively blocking the effects of TPO on Mk differentiation and suggesting that AHR activation is downstream of TPO signaling.
To examine the dramatic bleeding phenotype present in AHR-null mice, we next turned our attention toward platelet function, mediated by both outside-in and inside-out signaling. Defects in either or both of these signaling cascades could result in the bleeding defect present in AHR-null mice. In our initial experiments, we found that platelets from AHR-null mice bind fibrinogen equivalently to WT platelets (n=3), suggesting that AHR is not involved in inside-out platelet signaling. As we investigated other measures of platelet activation, we found that although platelets from AHR-null mice efficiently aggregated in response to ADP and the PAR-4 agonist AYPGKF (n=3 p=0.897 and 0.914, respectively), only 20 percent of AHR-null murine platelets (compared to 60 percent for WT platelets) aggregated in response to collagen (n=3, p=0.013). Spreading assays further demonstrated defective collagen-dependent outside-in signaling in AHR-null mice. We found that 5 times as many AHR-null platelets remained round (lacking filipodia or lamellipodia) as WT platelets after resting on collagen-coated slides (100 ug/mL) for 5 minutes. Over 60 percent of WT platelets were fully spread after a 20 minute incubation on collagen, compared to only 35 percent of AHR-null platelets. Additionally, roughly 20 percent of AHR-null platelets failed to respond and maintained a round morphology, representing 8 times as many unresponsive platelets as WT mice. The extent of spreading also appeared altered in AHR-null platelets, as the surface area of AHR-null platelets spreading on collagen was reduced by 42% after 5 minutes and 39% after 20 minutes compared to WT (n=3; p<0.001 for both experiments). Similar responses were seen when AHR-null platelets were allowed to spread on fibrinogen (100 ug/mL) after activation by 1 ug/mL collagen, with a 39% and 28% reduction in the surface area of AHR-null platelets after 5 and 20 minutes, respectively (n=3; p<0.001 for both experiments). Based on these findings, we are now investigating the molecular mechanisms of the collagen signaling defects present in AHR-null platelets, beginning with known interactions between AHR and vav genes, critical mediators of collagen-dependant platelet outside-in signaling. Our work is significant in that it builds upon our previously reported data and provides evidence that AHR is a critical component of the physiologic response platelets undergo in response to collagen. This information may provide novel treatment options for patients with bleeding disorders.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.