Severe sepsis is associated with disseminated intravascular coagulation (DIC) as a result of interdependent mechanisms of systemic intravascular inflammation, microvascular thrombosis, and thrombocytopenia. Currently, no drug is available to concomitantly treat these events in sepsis. A poorly understood mechanism and yet critical determinant of sepsis-induced microvascular thrombosis is von Willebrand Factor (vWF) secretion by activated endothelial cells. We recently discovered that heterotrimeric G protein alpha subunit G alpha 12 plays a critical role in basal and evoked vWF secretion by endothelial cells by promoting Weibel-Palade body (WPB) exocytosis. Based on the observed interaction of G alpha 12 with alpaSNAP, a critical member of the exocyst complex required for plasma membrane fusion and exocytosis of WPB contents, we generated a myristoylated Galpha12 N-terminal alphaSNAP Binding Domain (Myr-SBD) blocking peptide and tested the hypothesis that this would selectively and potently inhibit Galpha12 interaction with alphaSNAP and thereby block vWF secretion, limit platelet adhesion and prevent microvascular thrombosis associated with cecal ligation and puncture (CLP) induced sepsis. CLP-induced fulminant sepsis in rats and mice was associated with a 2-3-fold increase in plasma vWF within first 24 hrs. Importantly, we observed reduced plasma vWF levels 24 hrs after CLP surgery in mice given a one-time i.v. bolus (2 μmol/kg) of micellar Myr-SBD at the time of surgery as compared to Myr-scrambled peptide or vehicle only group. Strikingly, this was associated with increased survival without adversely inducing hemorrhage and vascular leakage. Furthermore, and consistent with the hypothesis that Gα12-dependent increase in vWF secretion during sepsis leads to poor outcome, control WT mice succumbed to sepsis in less than 96 hrs whereas 80% of Gα12-/- mice shown previously to have significantly reduced plasma vWF levels survived. Inhibition of Galpha12/alphaSNAP dependent vWF secretion may therefore be an effective strategy for blocking microvascular thrombosis, disseminated intravascular coagulation, and death due to sepsis.
Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4 dimerisation and transcriptional activity
