On The Localization Of The Cleavage Site In Human Alpha-2-Antiplasmin, Involved In The Generation Of The Non-Plasminogen Binding Form

Background Alpha-2-antiplasmin (alpha 2AP) is the main natural inhibitor of plasmin. The C-terminus of alpha 2AP is crucial for the initial interaction with plasmin(ogen) and the rapid inhibitory mechanism. Approximately 35% of circulating alpha 2AP has lost its C-terminus (non-plasminogen binding alpha 2AP/NPB-alpha 2AP) and thereby its rapid inhibitory capacity. The C-terminal cleavage site of alpha 2AP is still unknown. A commercially available monoclonal antibody against alpha 2AP (TC 3AP) detects intact but not NPB-alpha 2AP, suggesting that the cleavage site is located N-terminally from the epitope of TC 3AP.Objectives To determine the epitope of TC 3AP and then to localize the C-terminal cleavage site of alpha 2AP.Methods For epitope mapping of TC 3AP, commercially available plasma purified alpha 2AP was enzymatically digested with Asp-N, Glu-C, or Lys-N. The resulting peptides were immunoprecipitated using TC 3AP-loaded Dynabeads (R) Protein G. Bound peptides were eluted and analyzed by liquid chromatography-tandem mass spectometry (LC-MS/MS). To localize the C-terminal cleavage site precisely, alpha 2AP (intact and NPB) was purified from plasma and analyzed by LC-MS/MS after enzymatic digestion with Arg-C.Results We localized the epitope of TC 3AP between amino acid residues Asp428 and Gly439. LC-MS/MS data from plasma purified alpha 2AP showed that NPB-alpha 2AP results from cleavage at Gln421-Asp422 as preferred site, but also after Leu417, Glu419, Gln420, or Asp422.Conclusions The C-terminal cleavage site of human alpha 2AP is located N-terminally from the TC 3AP epitope. Because C-terminal cleavage of alpha 2AP can occur after multiple residues, different proteases may be responsible for the generation of NPB-alpha 2AP.