Autodegradation of murine activated protein C due to cleavage at Lys43

1. Autodegradation of murine activated protein C due to cleavage at Lys43 Laurent Burnier 1,2, José A. Fernández 2 and John H. Griffin 2. 1. The Swiss National Science Foundation; 2. The Scripps Research Institute, La Jolla, CA, USA. MES TBS Background: Multiple protective effects of pharmacologic activated protein C (APC) are reported in several pathologies, mostly using mouse models. Murine APC mutants are valuable for mechanistic studies and proof-of-concept in vivo studies. Murine APC production requires multiple challenging steps due to autodegradation of APC. This is especially important when comparing in vitro and in vivo potencies of different APC variants. A B Zn 0.25 mM Zn 0.25 mM EDTA Mg 2+ Mn 2+ Ca 2+ Zymogen Zn 1 mM Zn 1 mM EDTA EDTA HC Aim: To develop a new methodology that will decrease degradation of murine APC during activation and that will enable active site titration of APC. LC Figure 4: After thrombin inhibition with hirudin, APC was purified by FPLC to remove thrombin from preparation, and then quantified. We compared the amidolytic activity of APC activated in EDTA or zinc buffer. Assay has been performed in a buffer containing 5 mM EDTA or 1 mM Zn2+, since zinc ions inhibit amidolytic activity of human APC. Direct comparison showed that APC generated in presence of Zn2+ had a higher activity than APC made in the presence of EDTA. Figure 3: A. Other metal ions than Zinc (Ca2+, Mg2+, Mn2+) also dampen cleavage of the light chain (arrow). B. The PC activation rate by thrombin is equivalent in a buffer containing EDTA or Zinc ions, but slower with different metal ions, as revealed by amidolytic activity assay at different time point during the activation phase. Material and methods: Recombinant murine APC was activated with human recombinant thrombin in different buffers. After purification to remove thrombin for the preparation, APC was concentrated and loaded onto SDS-PAGE (TGX gels, Bio-Rad) and stained with Fastblue Coomassie Staining. Stained proteins were revealed using Odyssey near-infrared scanner (LiCor). Amydolytic activity was performed in TBS-BSA buffer (pH 8.0) with Pefachrome PCa substrate (Pentapharm). Figure 2: Activation of PC in presence of zinc (ZnCl2, 0.25 mM to 1 mM) at pH 6.0 (MES) showed a significant reduction of APC autodegradation in comparaison with EDTA in TBS (pH 7.4). We did not observed differences of activation rates in the presence of EDTA or zinc (Fig. 3B). A B HC HC LC C Heavy chain (HC) 15 kDa LC Light chain (LC) Figure 5: A. Activated protein C SDS-PAGE profil showing the degrated bands (red square) at about 15 kDa. B.Protein sequence of murine protein C, a red arrow highlights the cleavage site revealed by N-terminal sequence analysis (C.). Degraded product MES TBS Citrate Zymogen Conclusion:Murine APC autodegradation involves cleavage at Lys43 that can be avoided by simple buffer modifications. Mutation of Lys43 in murine PC should simplify the production of quality APC for mechanistic and in vivo studies. Figure 1: Citrate or MES buffers (pH 6.0), used during the activation step, showed a reduced APC autodegradation in comparison to Tris buffer (TBS, pH 7.4).