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

Abstract

Background: 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. Objectives:To determine the epitope of TC 3AP and then to localize the C-terminal cleavage site of α2AP.Methods: 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. Results: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. Conclusions: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. K E Y W O R D Salpha-2-antiplasmin, epitope mapping, mass spectrometry, proteolysis, western blot | 1163 ABDUL et AL.

Method development and characterization of the low molecular weight peptidome of human wound fluids

“…Interestingly, C-terminal regions of antiplasmin were only observed in normally healing wounds, and it is notable that this region of plasmin contains bioactive epitopes that can modulate urokinase activity 53 and interact with endothelial cells 54 . Recently, C-terminal fragments generated in vivo were indeed identified 55 . Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) is a 120 kDa serum glycoprotein secreted primarily by the liver.…”

Method development and characterisation of the low-molecular-weight peptidome of human wound fluids

“…Interestingly, C-terminal regions of antiplasmin were only observed in normally healing wounds, and it is notable that this region of plasmin contains bioactive epitopes that can modulate urokinase activity ( Lee et al, 2002 ) and interact with endothelial cells ( Thomas et al, 2007 ). Recently, C-terminal fragments generated in vivo were indeed identified ( Abdul et al, 2020 ). Inter-alpha-trypsin inhibitor heavy chain H4 is a 120 kDa serum glycoprotein secreted primarily by the liver.…”

Fibrinolytic drugs induced hemorrhage: mechanisms and solutions

“…Serine proteinase plasmin, a group of plasmin with a serine in its core catalytic domain by catalyzing the fibrin degradation process of fibrin clots [83]. Compared with the metalloprotease plasmin, serine proteinase plasmin is ineffective when administered by the intravenous route because it is rapidly neutralized by plasma antiplasmin [84]. Thus, few serine proteinase plasmin was used clinically up to now.…”