On the localization of the cleavage site in human alpha-2-antiplasmin, involved in the generation of the non-plasminogen binding form.

Shiraazkhan Abdul, Dick H W Dekkers, Robert A S Ariëns, Frank W G Leebeek, Dingeman C Rijken, Shirley Uitte de Willige

Journal of thrombosis and haemostasis : JTH 2020 May

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Alpha-2-antiplasmin (α2AP) is the main natural inhibitor of plasmin. The C-terminus of α2AP is crucial for the initial interaction with plasmin(ogen) and the rapid inhibitory mechanism. Approximately 35% of circulating α2AP has lost its C-terminus (non-plasminogen binding α2AP/NPB-α2AP) and thereby its rapid inhibitory capacity. The C-terminal cleavage site of α2AP is still unknown. A commercially available monoclonal antibody against α2AP (TC 3AP) detects intact but not NPB-α2AP, suggesting that the cleavage site is located N-terminally from the epitope of TC 3AP. To determine the epitope of TC 3AP and then to localize the C-terminal cleavage site of α2AP. For epitope mapping of TC 3AP, commercially available plasma purified α2AP was enzymatically digested with Asp-N, Glu-C, or Lys-N. The resulting peptides were immunoprecipitated using TC 3AP-loaded Dynabeads® 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, α2AP (intact and NPB) was purified from plasma and analyzed by LC-MS/MS after enzymatic digestion with Arg-C. We localized the epitope of TC 3AP between amino acid residues Asp428 and Gly439. LC-MS/MS data from plasma purified α2AP showed that NPB-α2AP results from cleavage at Gln421-Asp422 as preferred site, but also after Leu417, Glu419, Gln420, or Asp422. The C-terminal cleavage site of human α2AP is located N-terminally from the TC 3AP epitope. Because C-terminal cleavage of α2AP can occur after multiple residues, different proteases may be responsible for the generation of NPB-α2AP. © 2020 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

Citation

Shiraazkhan Abdul, Dick H W Dekkers, Robert A S Ariëns, Frank W G Leebeek, Dingeman C Rijken, Shirley Uitte de Willige. On the localization of the cleavage site in human alpha-2-antiplasmin, involved in the generation of the non-plasminogen binding form. Journal of thrombosis and haemostasis : JTH. 2020 May;18(5):1162-1170