A Chemical APC Mimetic Protects Endothelium from Thromboinflammatory Injury

Stimulation of protease-activated receptor 1 (PAR1) on endothelium by activated protein C (APC) is protective in animal models of inflammation and APC has been used clinically in sepsis and wound healing. Clinical use of APC in sepsis, however, was terminated as it was compromised by APC's anticoagulant activity, which is associated with bleeding and limits its dosing in patients. We used a small molecule approach to circumvent this problem. With support from the Molecular Libraries Program, we screened 302,457 compounds to identify small molecules that modulated PAR1-mediated platelet activation. One class of PAR1-targeted compounds, which we termed parmodulins, was found to act at the cytosolic face of PAR1, at the G-protein binding sites. When evaluated in endothelial cell cultures,parmodulin 1 (PM1) and parmodulin 2 (PM2) inhibited apoptosis induced by thrombin, TNF-α, or staurosporine, in a manner similar to APC. PAR1 knockdown using siRNA abolished these protective effects demonstrating that parmodulins elicit a cytoprotective pathway by acting through PAR1. To assess the mechanism of action of parmodulin cytoprotection, we first evaluated proximal signaling mechanisms. Parmodulins stimulated phosphorylation of PI3-kinase and Akt in endothelium. Inhibition of Gβγ blocked parmodulin-induced phosphorylation of Akt, indicating that parmodulins act at the cytosolic face of PAR1 by releasing Gβγ. Transcriptional profiling of over 30,000 genes and specific evaluation of NF-kB transcriptional activation showed that exposure to PM2 blocked TNF-α-induced transcriptional activation. In addition to interfering with inflammatory signaling, parmodulins also stimulated the upregulation of cytoprotective proteins such as stanniocalcin-1. Since our premise was that parmodulins could achieve cytoprotective effects without anticoagulant effects, we compared dose curves of APC versus PM2 in both apoptosis assays and standard clotting assays. APC prolonged the aPTT at concentrations lower than those required to achieve cytoprotection of the endothelium. The low APC concentration used in our study was similar to plasma concentrations measured in clinical studies. Hence, these data were consistent with the fact that clinical bleeding was observed at doses of APC used for sepsis. In contrast, despite inhibiting apoptosis as effectively as APC, PM2 had no effect on plasma aPTT at any concentration. Nonetheless, PM2 was able to inhibit LPS- and TNF-α-induced thrombin generation and FXa activation on endothelium owing to its cytoprotective effect. PM2 also prevented TNF-α-induced accumulation of platelets on endothelium in bioengineered microvessels. These data demonstrate that PM2 can reduce inflammation-induced endothelial pro-thrombotic phenotype even without directly inhibiting coagulation factors. To assess the endothelial protective effects of PM2 in vivo we evaluated leukocyte rolling in cremaster venules of mice. Infusion of PM2 significantly reduced surgery-induced leukocyte rolling flux compared to vehicle-treated mice. As selectins are critically involved in leukocyte rolling we monitored soluble E-selectin levels in LPS-induced inflammation. Treatment of mice with PM2 significantly reduced the LPS-induced release of soluble E-selectin. Previously, we demonstrated that PM2 blocks platelet accumulation in a mouse laser injury model of thrombus formation. We here show that infusion of PM2 also significantly reduces fibrin accumulation to 25% of control (p<0.001) at the site of laser injury. Together our data show that PM2 exerts endothelial-mediated anti-inflammatory, anti-coagulant and anti-thrombotic effects in vitro and in vivo. These results demonstrate that modulating PAR1 at the cytosolic face could represent an alternative approach to APC in the treatment of thromboinflammatory disorders like sepsis.