Sriram Ambadapadi, Ganesh Munuswamy-Ramanujam, Donghang Zheng, Colin Sullivan, Erbin Dai, Sufi Morshed, Baron McFadden, Emily Feldman, Melissa Pinard, Robert McKenna, Scott Tibbetts, Alexandra Lucas

Serpins regulate coagulation and inflammation, binding serine proteases in suicide inhibitory complexes. Target proteases cleave the serpin reactive center loop (RCL) scissile P1-P1' bond resulting in serpin-protease suicide inhibitory complexes. This inhibition requires a near full-length serpin sequence. Myxomavirus Serp-1 inhibits thrombolytic and thrombotic proteases while mammalian neuroserpin (NSP) inhibits only thrombolytic proteases. Both serpins markedly reduce arterial inflammation and plaque in rodent models after single dose infusion. In contrast Serp-1 but not NSP improves survival in a lethal murine gammaherpesvirus68 (MHV68) infection in interferon gamma receptor deficient mice (IFNγR-/-). Serp-1 has also been successfully tested in a Phase 2a clinical trial. We postulated that proteolytic cleavage of the RCL produces active peptide derivatives with expanded function. Eight peptides encompassing predicted protease cleavage sites for Serp-1 and NSP were synthesized and tested for inhibitory function in vitro and in vivo. In engrafted aorta, selected peptides containing R or RN, not RM, with 0 or +1 charge, significantly reduced plaque. Conversely, S-6 a hydrophobic peptide of NSP, lacking R or RN with -4 charge, induced early thrombosis and mortality. S-1 and S-6 also significantly reduced CD11b+ monocyte counts in mouse splenocytes. S-1 peptide had increased efficacy in PAI-1 serpin deficient transplants. Plaque reduction correlated with mononuclear cell activation..In a separate study Serp-1 peptide, S-7 improved survival in the MHV68 vasculitis model whereas an inverse S-7 peptide was inactive. Reactive center peptides derived from Serp-1 and NSP with suitable charge and hydrophobicity have the potential to extend immunomodulatory functions of serpins.