Platelet mTOR is a regulator of sterile immunothrombosis

Abstract

Abstract Background: Immunothrombosis entails a tight interplay between thrombotic and inflammatory pathways and plays a pathological role in ischemic stroke and venous thrombosis. The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase involved in platelet signaling and thrombus stabilization in vitro. Platelet mTOR is upregulated in aging and inflammatory disorders, however, its role in vivo is poorly understood. Methods: A platelet-specific mTOR-deficient mouse model (mTORplt-/-) and wild-type littermate controls (mTORplt+/+) were used to assess platelet activation and platelet leukocyte interactions in response to a wide range of platelet agonists. Additionally, we examined the role of platelet mTOR in models of hemostasis, thrombosis, inflammatory bleeding and immunothrombosis including ischemic stroke and venous thrombosis. Results: Platelets lacking mTOR had a small platelet activation defect upon low dose agonist stimulation as measured by aIIbb3 activation. This was overcome at higher concentrations of agonists. Additionally, mTORplt-/- platelets became less procoagulant upon dual agonist stimulation (p<0.001). In the absence of mTOR, platelets also interacted significant less with monocytes (p<0.05) and neutrophils (p<0.01) upon stimulation in vitro. Similar to platelet activation, this was most apparent with lower dose agonists and could be overcome at high dose agonists. Interestingly, we found that platelet mTOR drives the activation of neutrophils based on CD11b expression (p<0.05) in platelet-neutrophil complexes. In vivo, the absence of platelet mTOR did not impact hemostasis or arterial thrombosis as measured through the tail-transection bleeding model and the FeCl3-induced carotid artery thrombosis model, respectively. Also, inflammatory bleeding was not affected by platelet mTOR-deficiency as determined in the lung and skin using an LPS-induced acute lung injury model and the reverse passive Arthus reaction model, respectively. Moreover, in the acute lung injury model, the number of neutrophils recruited to the lung was similar in both genotypes. In contrast, mTORplt-/- mice were protected from ischemic stroke brain injury as evidenced by reduced infarct volumes (p<0.01) and improved motor function (p<0.05). These beneficial functional outcomes were associated with significantly enhanced cerebral reperfusion (p<0.05) and significantly less neutrophil infiltration into ischemic brain tissue (p<0.05). As reperfusion injury in ischemic stroke brain injury and clot burden in venous thrombosis share common mechanistic pathways, we next evaluated the impact of platelet mTOR on venous thrombosis outcomes. mTORplt-/- mice were also protected from stasis-induced deep vein thrombosis, as evidenced by fewer thrombi and reduced thrombus weights (p<0.05). Together, these results support an immunothrombotic role for platelet mTOR, implicating it as a common mediator of both arterial and venous immunothrombosis. Conclusions: Platelet mTOR is a critical mediator of immunothrombosis, while it is dispensable for hemostasis in mice. The immunothrombotic specific effects of mTOR make it an attractive therapeutic target with a good safety profile.