Novel Endothelial Cell Targeted Peptide Nanoformulation for Inhibiting von Willebrand Factor Secretion to Reduce Thrombotic Complications in Sepsis

Abstract

RationaleMicrovascular thrombosis and disseminated intravascular coagulation (DIC) are primary determinants of ischemia, organ failure and mortality/morbidity in sepsis. Interestingly, elevated plasma level of von Willebrand factor (vWF), the glue to which platelets adhere to form a clot, is associated with reduced survival and poor outcome in septic patients. Previously, we demonstrated that endothelial cell (EC) Gα12 is required for soluble N‐ethylmaleimide‐sensitive‐fusion factor attachment protein α (αSNAP)‐dependent vWF secretion from Weibel Palade bodies. Here, we report that sterically stabilized simple micelles (SSM) containing cell‐permeable myristoylated αSNAP binding domain (Myr‐SBD) peptide based on the unique N‐terminal Gα12 sequence that binds to αSNAP, inhibits vWF secretion.MethodsStructural complex of αSNAP/SBD was determined by blind‐ and data‐driven docking using AutoDock and HADDOCK, respectively. After incorporating Myr‐SBD into SSM (Myr‐SBD‐SSM) by film hydration method, micelle size and shape were confirmed by dynamic light scattering and transmission electron microscopy (TEM), respectively. Intracellular delivery of Myr‐SBD‐SSM was evaluated by fluorescence microscopy and fluorometry in cultured ECs. Cecal ligation and puncture (CLP) in C57BL/6 mice (14–16 wks, 25 g) was used to induce severe inflammation, microvascular thrombosis and DIC, and blood was collected 6 h and 24 h post CLP. The effect of Myr‐SBD‐SSM on plasma vWF, platelet count and inflammatory cytokines was assessed following addition of 0, 2 or 4 μmol Myr‐SBD/kg given i.v. at the time of surgery. Bleeding was assessed by tail‐snip assay. Glomerular fibrin deposition was scored by IHC analysis at 24 h post CLP.ResultsThe αSNAP/SBD structure showed the potential interaction between Arg of SBD peptide and Glu139 of αSNAP by H‐bond. Spherical shape of Myr‐SBD‐SSM (14.1 ± 2.7 nm in diameter) was confirmed by negative staining TEM. Addition of Myr‐SBD‐SSM to ECs resulted in the intracellular accumulation of Myr‐SBD which blocked thrombin‐induced vWF secretion. Intravenous delivery of Myr‐SBD‐SSM to mice at the time of CLP blunted the increase in plasma vWF and associated microvascular thrombosis in the kidney glomerulus. Importantly, Myr‐SBD‐SSM treatment resulted in increased platelet and WBC counts 6 h after surgery and improved survival compared to untreated or scrambled peptide treated mice with severe vascular inflammation suggesting Myr‐SBD‐SSM reduced clotting, platelet consumption and systemic inflammation in intraperitoneal sepsis. Moreover, Myr‐SBD‐SSM treatment did not increase tail‐snip bleeding time suggesting it did not prevent hemostasis.ConclusionOur studies demonstrate that Myr‐SBD‐SSM nanoformulation reduces vWF secretion and attenuates the thrombotic complications of severe inflammation.Support or Funding InformationThis work was supported by MPI R01 HL125356 (RDM, XD), Michael Reese Research and Education Foundation (RDM) and American Heart Association (MB, LR).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.