Abstract 659: Inhibition of vWF Secretion with Novel G alpha12 N-terminal alpha-SNAP Binding Domain Peptide Increases Survival in Septic Rodents

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.

Expression cDNA cloning of a transforming gene encoding the wild-type G alpha 12 gene product

Using an expression cDNA cloning approach, we examined human tumor cell lines for novel oncogenes that might evade detection by conventional techniques. We isolated a transforming sequence that was highly efficient in transforming NIH 3T3 mouse fibroblasts. DNA sequence analysis identified the gene as the human homolog of a recently cloned alpha subunit of mouse GTP-binding protein G alpha 12. NIH 3T3 cells transfected with G alpha 12 cDNA grew in soft agar and were tumorigenic in nude mice. There were no apparent mutations in the cloned cDNA in comparison with a G alpha 12 cDNA clone isolated from a normal human epithelial cell library, implying that overexpression alone was sufficient to cause NIH 3T3 cell transformation. The observed altered growth properties mediated by G alpha 12 showed a certain degree of dependency on serum factors, and its mitogenic potential was also potently inhibited by suramin treatment.

Expression cDNA cloning of a transforming gene encoding the wild-type G alpha 12 gene product.

Using an expression cDNA cloning approach, we examined human tumor cell lines for novel oncogenes that might evade detection by conventional techniques. We isolated a transforming sequence that was highly efficient in transforming NIH 3T3 mouse fibroblasts. DNA sequence analysis identified the gene as the human homolog of a recently cloned alpha subunit of mouse GTP-binding protein G alpha 12. NIH 3T3 cells transfected with G alpha 12 cDNA grew in soft agar and were tumorigenic in nude mice. There were no apparent mutations in the cloned cDNA in comparison with a G alpha 12 cDNA clone isolated from a normal human epithelial cell library, implying that overexpression alone was sufficient to cause NIH 3T3 cell transformation. The observed altered growth properties mediated by G alpha 12 showed a certain degree of dependency on serum factors, and its mitogenic potential was also potently inhibited by suramin treatment.