BACKGROUND: The leukocyte serine proteases (LSPs) elastase, proteinase 3 and cathepsin G cleave von Willebrand factor (VWF) near or at the same cleavage site (Tyr1605-Met1606) as ADAMTS-13, the metalloprotease that specifically controls the proteolytic processing of VWF. Recent studies have shown that oxidation of VWF at Met1606 with formation of methionine sulfoxide (MetSO) severely impairs its proteolysis by ADAMTS-13. METHODS: This study was aimed at assessing whether or not oxidation of VWF by reactive oxygen species (ROS) can also affect its cleavage by elastase, proteinase 3, and cathepsin G. In this study, the catalytic specificity of hydrolysis by LSPs of the VWF peptide substrate VWF74 and full-length VWF, both unaltered and in the oxidized form, was measured by RP-HPLC, electrophoretic and mass spectrometry methods. RESULTS: LSPs cleaved both VWF multimers and VWF74 near or at the same peptide bond as is cleaved by ADAMTS-13, with k(cat)/K(m) values similar to those of the metalloprotease. However, unlike ADAMTS-13, cathepsin G cleaved VWF74 containing a MetSO residue at position 1606 with a k(cat)/K(m) value higher than that for VWF74, whereas the catalytic efficiencies of both elastase and proteinase 3 were unaffected by the replacement of Met1606 with MetSO. Likewise, oxidation of VWF multimers by hypochlorous acid and ROS, produced by activated leukocytes, improved their hydrolysis by LSPs. CONCLUSIONS: Oxidation by leukocyte ROS has a net positive effect on the cleavage of VWF multimers by LSPs, under conditions where high concentrations of oxidant species would severely reduce the proteolytic efficiency of ADAMTS-13.
BACKGROUND: The leukocyte serine proteases (LSPs) elastase, proteinase 3 and cathepsin G cleave von Willebrand factor (VWF) near or at the same cleavage site (Tyr1605-Met1606) as ADAMTS-13, the metalloprotease that specifically controls the proteolytic processing of VWF. Recent studies have shown that oxidation of VWF at Met1606 with formation of methionine sulfoxide (MetSO) severely impairs its proteolysis by ADAMTS-13. METHODS: This study was aimed at assessing whether or not oxidation of VWF by reactive oxygen species (ROS) can also affect its cleavage by elastase, proteinase 3, and cathepsin G. In this study, the catalytic specificity of hydrolysis by LSPs of the VWF peptide substrate VWF74 and full-length VWF, both unaltered and in the oxidized form, was measured by RP-HPLC, electrophoretic and mass spectrometry methods. RESULTS: LSPs cleaved both VWF multimers and VWF74 near or at the same peptide bond as is cleaved by ADAMTS-13, with k(cat)/K(m) values similar to those of the metalloprotease. However, unlike ADAMTS-13, cathepsin G cleaved VWF74 containing a MetSO residue at position 1606 with a k(cat)/K(m) value higher than that for VWF74, whereas the catalytic efficiencies of both elastase and proteinase 3 were unaffected by the replacement of Met1606 with MetSO. Likewise, oxidation of VWF multimers by hypochlorous acid and ROS, produced by activated leukocytes, improved their hydrolysis by LSPs. CONCLUSIONS: Oxidation by leukocyte ROS has a net positive effect on the cleavage of VWF multimers by LSPs, under conditions where high concentrations of oxidant species would severely reduce the proteolytic efficiency of ADAMTS-13.
Authors: Emma Birnie; Gavin C K W Koh; Ester C Löwenberg; Joost C M Meijers; Rapeephan R Maude; Nicholas P J Day; Sharon J Peacock; Tom van der Poll; W Joost Wiersinga Journal: PLoS Negl Trop Dis Date: 2017-03-15
Authors: Michael Schwameis; Christian Schörgenhofer; Alice Assinger; Margarete M Steiner; Bernd Jilma Journal: Thromb Haemost Date: 2014-12-11 Impact factor: 5.249