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Lawrence L K Leung - One of the best experts on this subject based on the ideXlab platform.
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both plasma basic Carboxypeptidases Carboxypeptidase B2 and Carboxypeptidase n regulate vascular leakage activity in mice
Journal of Thrombosis and Haemostasis, 2021Co-Authors: Zhifei Shao, Lawrence L K Leung, Qin Zhou, Lei Zhao, Paul Declerck, John MorserAbstract:BACKGROUND Kallikrein is generated when the contact system is activated, subsequently cleaving high molecular weight kininogen to bradykinin (BK). BK binds to bradykinin receptor 2 (B2R) causing vascular leakage. BK is inactivated by proteolysis by the plasma Carboxypeptidase B2 and N (CPB2 and CPN). CPN is constitutively active but CPB2 is generated from its zymogen, proCPB2. OBJECTIVES Determine the role of CPB2 and CPN in the regulation of vascular leakage. METHODS Mice deficient in CPB2, CPN or both (CpB2-/- , Cpn-/- and CpB2-/- /Cpn-/- ) were compared to wild type mice (WT) in a model of vascular leakage caused by skin irritation. In some experiments mice were pretreated with antibodies that prevent activation of proCPB2. RESULTS Skin irritation increased vascular leakage most in CpB2-/- /Cpn-/- , less in CpB2-/- and Cpn-/- and least in WT mice. There was no difference in vascular leakage without the challenge. Antibodies inhibiting activation of proCPB2 by plasmin, but not by the thrombin/thrombomodulin (TM) complex, increased vascular leakage to the level seen in CpB2-/- mice. There was no change in levels of markers of coagulation and fibrinolysis. CONCLUSIONS BK is inactivated by both CPB2 and CPN independently. Plasmin is the activator of proCPB2 in this model. Mice lacking both plasma Carboxypeptidases have more vascular leak than ones lacking either alone. Although BK levels were not determined, BK is the likely substrate for CPB2 and CPN in this model.
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Carboxypeptidase B2 and Carboxypeptidase n in the crosstalk between coagulation thrombosis inflammation and innate immunity
Journal of Thrombosis and Haemostasis, 2018Co-Authors: Lawrence L K Leung, John MorserAbstract:: Two basic Carboxypeptidases, Carboxypeptidase B2 (CPB2) and Carboxypeptidase N (CPN) are present in plasma. CPN is constitutively active, whereas CPB2 circulates as a precursor, proCarboxypeptidase B2 (proCPB2), that needs to be activated by the thrombin-thrombomodulin complex or plasmin bound to glycosaminoglycans. The substrate specificities of CPB2 and CPN are similar; they both remove C-terminal basic amino acids from bioactive peptides and proteins, thereby inactivating them. The complement cascade is a cascade of proteases and cofactors activated by pathogens or dead cells, divided into two phases, with the second phase only being triggered if sufficient C3b is present. Complement activation generates anaphylatoxins: C3a, which stimulates macrophages; and C5a, which is an activator and attractant for neutrophils. Pharmacological intervention with inhibitors has shown that CPB2 delays fibrinolysis, whereas CPN is responsible for systemic inactivation of C3a and C5a. Among mice genetically deficient in either CPB2 or CPN, in a model of hemolytic-uremic syndrome, CpB2-/- mice had the worst disease, followed by Cpn-/- mice, with wild-type (WT) mice being the most protected. This model is driven by C5a, and shows that CPB2 is important in inactivating C5a. In contrast, when mice were challenged acutely with cobra venom factor, the reverse phenotype was observed; Cpn-/- mice had markedly worse disease than CpB2-/- mice, and WT mice were resistant. These observations need to be confirmed in humans. Therefore, CPB2 and CPN have different roles. CPN inactivates C3a and C5a generated spontaneously, whereas proCPB2 is activated at specific sites, where it inactivates bioactive peptides that would overwhelm CPN.
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Carboxypeptidase B2 and n play different roles in regulation of activated complements c3a and c5a in mice
Journal of Thrombosis and Haemostasis, 2018Co-Authors: John Morser, J. Higgins, Zhifei Shao, Toshihiko Nishimura, Q. Zhou, Lawrence L K LeungAbstract:: Essentials Two basic Carboxypeptidases are present in plasma, B2 (CPB2) and N (CPN). CpB2-/- and Cpn-/- mice were challenged in a hemolytic uremic syndrome (HUS) model vs. wild type. CpB2-/- exacerbates HUS while Cpn-/- exacerbates cobra venom factor challenge vs. wild type mice. CPB2 and CPN have overlapping but non-redundant roles. SUMMARY: Background There are two basic Carboxypeptidases in plasma. Carboxypeptidase B2 (CPB2) is activated from a circulating zymogen, proCPB2, and Carboxypeptidase N (CPN) is constitutively active with both inactivating complement C3a and C5a. Aims To test the roles of CPB2 and CPN in complement-driven mouse models of cobra venom factor (CVF) challenge and hemolytic-uremic syndrome (HUS). Methods CpB2-/- , Cpn-/- and wild-type (WT) mice were compared in an HUS model induced by Shiga toxin and lipopolysaccharide administration and following CVF administration. Results HUS was exacerbated in CpB2-/- mice more than in Cpn-/- mice, compared with WT mice. CpB2-/- mice developed the HUS clinical triad of microangiopathic hemolytic anemia, uremia and thrombocytopenia. Treatment with anti-C5 antibody improved survival of both CpB2-/- and Cpn-/- mice. In contrast, when challenged acutely with CVF, the reverse phenotype was observed. Cpn-/- mice had markedly worse disease than CpB2-/- mice, whereas the WT mice were resistant. Conclusions CPN and CPB2 play overlapping but non-redundant roles in regulating complement activation in vivo. The constitutively active CPN is key for inactivation of systemic C5a, whereas CPB2 functions as an on-demand supplementary anaphylatoxin inhibitor in inactivating excessive C5a formed locally.
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Carboxypeptidase B2 deficiency reveals opposite effects of complement c3a and c5a in a murine polymicrobial sepsis model
Journal of Thrombosis and Haemostasis, 2015Co-Authors: Zhifei Shao, Lawrence L K Leung, Toshihiko Nishimura, John MorserAbstract:Background and Objectives Carboxypeptidase B2 (CPB2) is a basic Carboxypeptidase with fibrin and complement C3a and C5a as physiological substrates. We hypothesized that in polymicrobial sepsis, CPB2-deficient mice would have sustained C5a activity, leading to disease exacerbation.
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Carboxypeptidase B2 is protective in a mouse model of shiga toxin induced hemolytic uremic syndrome
Blood, 2014Co-Authors: Zhifei Shao, Lawrence L K Leung, John Morser, Toshihiko NishimuraAbstract:Hemolytic uremic syndrome (HUS) is characterized by a triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. The most common cause of HUS is Shiga toxin (STX)-producing E. coli, and eculizumab, a monoclonal antibody against complement C5, has shown clinical efficacy in some patients. Carboxypeptidase B2 (CPB2) is a metalloprotease activated by the thrombin/thrombomodulin complex that inactivates a number of inflammatory mediators, including complement C3a, and C5a by removing their C-terminal arginine. We hypothesized that in a murine model of STX-induced HUS, CpB2-/- mice would have exacerbated disease compared to wild type (WT) mice due to excessive C3a and/or C5a in the absence of CPB2. A mouse model of STX-induced HUS was established by giving STX and LPS toxins intraperitoneally. CpB2-/- mice had worse survival than WT (37% survival vs. 87% at 48h, p=0.0156). At 48h, severe thrombocytopenia developed in both WT and CpB2-/- mice (WT: 0.096x106/μL; CpB2-/- : 0.054x106/μL) compared to controls (1.2x106/μL; p>0.0001 vs. either WT or CpB2-/- ), with CpB2-/- mice showing worse thrombocytopenia. Renal insufficiency was worse in CpB2-/- mice than WT mice (BUN at 48h: 85 mg/dL vs. 37 mg/dL, p=0.0074; creatinine: 1.33 mg/dL vs. 0.23 mg/dL; p=0.0112, for CpB2-/- and WT mice respectively, compared with normal baseline BUN and creatinine of 19 mg/dL and 0.1 mg/dL). CpB2-/- mice developed worse anemia than WT (hemoglobin 9.8 g/dL vs. 12.4 g/dL, p=0.001 in CpB2-/- vs. WT mice respectively). At 48h, liver function was worse in CpB2-/- mice than WT mice, while plasma LDH was increased in CpB2-/- mice more than WT mice. Using a standardized health score, the CpB2-/- mice were worse than WT mice at all time points. Thus this model recapitulates STX-induced HUS with the CpB2-/- mice having worse disease than WT. If the animals were treated with STX alone, there were no deaths in either genotype at 48h and only 37.5% mortality in CpB2-/- mice by 60h compared with no deaths in WT mice. BUN, creatinine, liver enzymes and LDH were increased in both genotypes treated with STX alone compared to untreated mice, but there was no significant difference between the genotypes. Treatment with LPS alone caused thrombocytopenia in both WT and CpB2-/- mice and LDH, BUN and creatinine levels were higher in CpB2-/- mice than in WT mice, but there was no death at 48h and no drop in hemoglobin. Thus while either STX alone or LPS alone caused pathological conditions in the mice, the typical triad of HUS was only present when STX and LPS were given in combination. The CpB2-/- mice had worse disease than WT mice consistent with our hypothesis on the role of CPB2 in inactivating C3a and/or C5a in STX-induced HUS. The potential efficacy of C3a and/or C5a blockade and anti-thrombotic agents will be tested in this model. Disclosures No relevant conflicts of interest to declare.
John Morser - One of the best experts on this subject based on the ideXlab platform.
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both plasma basic Carboxypeptidases Carboxypeptidase B2 and Carboxypeptidase n regulate vascular leakage activity in mice
Journal of Thrombosis and Haemostasis, 2021Co-Authors: Zhifei Shao, Lawrence L K Leung, Qin Zhou, Lei Zhao, Paul Declerck, John MorserAbstract:BACKGROUND Kallikrein is generated when the contact system is activated, subsequently cleaving high molecular weight kininogen to bradykinin (BK). BK binds to bradykinin receptor 2 (B2R) causing vascular leakage. BK is inactivated by proteolysis by the plasma Carboxypeptidase B2 and N (CPB2 and CPN). CPN is constitutively active but CPB2 is generated from its zymogen, proCPB2. OBJECTIVES Determine the role of CPB2 and CPN in the regulation of vascular leakage. METHODS Mice deficient in CPB2, CPN or both (CpB2-/- , Cpn-/- and CpB2-/- /Cpn-/- ) were compared to wild type mice (WT) in a model of vascular leakage caused by skin irritation. In some experiments mice were pretreated with antibodies that prevent activation of proCPB2. RESULTS Skin irritation increased vascular leakage most in CpB2-/- /Cpn-/- , less in CpB2-/- and Cpn-/- and least in WT mice. There was no difference in vascular leakage without the challenge. Antibodies inhibiting activation of proCPB2 by plasmin, but not by the thrombin/thrombomodulin (TM) complex, increased vascular leakage to the level seen in CpB2-/- mice. There was no change in levels of markers of coagulation and fibrinolysis. CONCLUSIONS BK is inactivated by both CPB2 and CPN independently. Plasmin is the activator of proCPB2 in this model. Mice lacking both plasma Carboxypeptidases have more vascular leak than ones lacking either alone. Although BK levels were not determined, BK is the likely substrate for CPB2 and CPN in this model.
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Carboxypeptidase B2 and Carboxypeptidase n in the crosstalk between coagulation thrombosis inflammation and innate immunity
Journal of Thrombosis and Haemostasis, 2018Co-Authors: Lawrence L K Leung, John MorserAbstract:: Two basic Carboxypeptidases, Carboxypeptidase B2 (CPB2) and Carboxypeptidase N (CPN) are present in plasma. CPN is constitutively active, whereas CPB2 circulates as a precursor, proCarboxypeptidase B2 (proCPB2), that needs to be activated by the thrombin-thrombomodulin complex or plasmin bound to glycosaminoglycans. The substrate specificities of CPB2 and CPN are similar; they both remove C-terminal basic amino acids from bioactive peptides and proteins, thereby inactivating them. The complement cascade is a cascade of proteases and cofactors activated by pathogens or dead cells, divided into two phases, with the second phase only being triggered if sufficient C3b is present. Complement activation generates anaphylatoxins: C3a, which stimulates macrophages; and C5a, which is an activator and attractant for neutrophils. Pharmacological intervention with inhibitors has shown that CPB2 delays fibrinolysis, whereas CPN is responsible for systemic inactivation of C3a and C5a. Among mice genetically deficient in either CPB2 or CPN, in a model of hemolytic-uremic syndrome, CpB2-/- mice had the worst disease, followed by Cpn-/- mice, with wild-type (WT) mice being the most protected. This model is driven by C5a, and shows that CPB2 is important in inactivating C5a. In contrast, when mice were challenged acutely with cobra venom factor, the reverse phenotype was observed; Cpn-/- mice had markedly worse disease than CpB2-/- mice, and WT mice were resistant. These observations need to be confirmed in humans. Therefore, CPB2 and CPN have different roles. CPN inactivates C3a and C5a generated spontaneously, whereas proCPB2 is activated at specific sites, where it inactivates bioactive peptides that would overwhelm CPN.
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Carboxypeptidase B2 and n play different roles in regulation of activated complements c3a and c5a in mice
Journal of Thrombosis and Haemostasis, 2018Co-Authors: John Morser, J. Higgins, Zhifei Shao, Toshihiko Nishimura, Q. Zhou, Lawrence L K LeungAbstract:: Essentials Two basic Carboxypeptidases are present in plasma, B2 (CPB2) and N (CPN). CpB2-/- and Cpn-/- mice were challenged in a hemolytic uremic syndrome (HUS) model vs. wild type. CpB2-/- exacerbates HUS while Cpn-/- exacerbates cobra venom factor challenge vs. wild type mice. CPB2 and CPN have overlapping but non-redundant roles. SUMMARY: Background There are two basic Carboxypeptidases in plasma. Carboxypeptidase B2 (CPB2) is activated from a circulating zymogen, proCPB2, and Carboxypeptidase N (CPN) is constitutively active with both inactivating complement C3a and C5a. Aims To test the roles of CPB2 and CPN in complement-driven mouse models of cobra venom factor (CVF) challenge and hemolytic-uremic syndrome (HUS). Methods CpB2-/- , Cpn-/- and wild-type (WT) mice were compared in an HUS model induced by Shiga toxin and lipopolysaccharide administration and following CVF administration. Results HUS was exacerbated in CpB2-/- mice more than in Cpn-/- mice, compared with WT mice. CpB2-/- mice developed the HUS clinical triad of microangiopathic hemolytic anemia, uremia and thrombocytopenia. Treatment with anti-C5 antibody improved survival of both CpB2-/- and Cpn-/- mice. In contrast, when challenged acutely with CVF, the reverse phenotype was observed. Cpn-/- mice had markedly worse disease than CpB2-/- mice, whereas the WT mice were resistant. Conclusions CPN and CPB2 play overlapping but non-redundant roles in regulating complement activation in vivo. The constitutively active CPN is key for inactivation of systemic C5a, whereas CPB2 functions as an on-demand supplementary anaphylatoxin inhibitor in inactivating excessive C5a formed locally.
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Carboxypeptidase B2 deficiency reveals opposite effects of complement c3a and c5a in a murine polymicrobial sepsis model
Journal of Thrombosis and Haemostasis, 2015Co-Authors: Zhifei Shao, Lawrence L K Leung, Toshihiko Nishimura, John MorserAbstract:Background and Objectives Carboxypeptidase B2 (CPB2) is a basic Carboxypeptidase with fibrin and complement C3a and C5a as physiological substrates. We hypothesized that in polymicrobial sepsis, CPB2-deficient mice would have sustained C5a activity, leading to disease exacerbation.
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Carboxypeptidase B2 is protective in a mouse model of shiga toxin induced hemolytic uremic syndrome
Blood, 2014Co-Authors: Zhifei Shao, Lawrence L K Leung, John Morser, Toshihiko NishimuraAbstract:Hemolytic uremic syndrome (HUS) is characterized by a triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. The most common cause of HUS is Shiga toxin (STX)-producing E. coli, and eculizumab, a monoclonal antibody against complement C5, has shown clinical efficacy in some patients. Carboxypeptidase B2 (CPB2) is a metalloprotease activated by the thrombin/thrombomodulin complex that inactivates a number of inflammatory mediators, including complement C3a, and C5a by removing their C-terminal arginine. We hypothesized that in a murine model of STX-induced HUS, CpB2-/- mice would have exacerbated disease compared to wild type (WT) mice due to excessive C3a and/or C5a in the absence of CPB2. A mouse model of STX-induced HUS was established by giving STX and LPS toxins intraperitoneally. CpB2-/- mice had worse survival than WT (37% survival vs. 87% at 48h, p=0.0156). At 48h, severe thrombocytopenia developed in both WT and CpB2-/- mice (WT: 0.096x106/μL; CpB2-/- : 0.054x106/μL) compared to controls (1.2x106/μL; p>0.0001 vs. either WT or CpB2-/- ), with CpB2-/- mice showing worse thrombocytopenia. Renal insufficiency was worse in CpB2-/- mice than WT mice (BUN at 48h: 85 mg/dL vs. 37 mg/dL, p=0.0074; creatinine: 1.33 mg/dL vs. 0.23 mg/dL; p=0.0112, for CpB2-/- and WT mice respectively, compared with normal baseline BUN and creatinine of 19 mg/dL and 0.1 mg/dL). CpB2-/- mice developed worse anemia than WT (hemoglobin 9.8 g/dL vs. 12.4 g/dL, p=0.001 in CpB2-/- vs. WT mice respectively). At 48h, liver function was worse in CpB2-/- mice than WT mice, while plasma LDH was increased in CpB2-/- mice more than WT mice. Using a standardized health score, the CpB2-/- mice were worse than WT mice at all time points. Thus this model recapitulates STX-induced HUS with the CpB2-/- mice having worse disease than WT. If the animals were treated with STX alone, there were no deaths in either genotype at 48h and only 37.5% mortality in CpB2-/- mice by 60h compared with no deaths in WT mice. BUN, creatinine, liver enzymes and LDH were increased in both genotypes treated with STX alone compared to untreated mice, but there was no significant difference between the genotypes. Treatment with LPS alone caused thrombocytopenia in both WT and CpB2-/- mice and LDH, BUN and creatinine levels were higher in CpB2-/- mice than in WT mice, but there was no death at 48h and no drop in hemoglobin. Thus while either STX alone or LPS alone caused pathological conditions in the mice, the typical triad of HUS was only present when STX and LPS were given in combination. The CpB2-/- mice had worse disease than WT mice consistent with our hypothesis on the role of CPB2 in inactivating C3a and/or C5a in STX-induced HUS. The potential efficacy of C3a and/or C5a blockade and anti-thrombotic agents will be tested in this model. Disclosures No relevant conflicts of interest to declare.
Adelina E Acostamartin - One of the best experts on this subject based on the ideXlab platform.
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quantitative mass spectrometry analysis using pacific for the identification of plasma diagnostic biomarkers for abdominal aortic aneurysm
PLOS ONE, 2011Co-Authors: Maggy Chwastyniak, Annabelle Dupont, Francis Juthier, Adelina E Acostamartin, Alexandre Panchaud, Corinne GautierAbstract:Background Abdominal aortic aneurysm (AAA) is characterized by increased aortic vessel wall diameter (>1.5 times normal) and loss of parallelism. This disease is responsible for 1–4% mortality occurring on rupture in males older than 65 years. Due to its asymptomatic nature, proteomic techniques were used to search for diagnostic biomarkers that might allow surgical intervention under nonlife threatening conditions. Methodology/Principal Findings Pooled human plasma samples of 17 AAA and 17 control patients were depleted of the most abundant proteins and compared using a data-independent shotgun proteomic strategy, Precursor Acquisition Independent From Ion Count (PAcIFIC), combined with spectral counting and isobaric tandem mass tags. Both quantitative methods collectively identified 80 proteins as statistically differentially abundant between AAA and control patients. Among differentially abundant proteins, a subgroup of 19 was selected according to Gene Ontology classification and implication in AAA for verification by Western blot (WB) in the same 34 individual plasma samples that comprised the pools. From the 19 proteins, 12 were detected by WB. Five of them were verified to be differentially up-regulated in individual plasma of AAA patients: adiponectin, extracellular superoxide dismutase, protein AMBP, kallistatin and Carboxypeptidase B2. Conclusions/Significance Plasma depletion of high abundance proteins combined with quantitative PAcIFIC analysis offered an efficient and sensitive tool for the screening of new potential biomarkers of AAA. However, WB analysis to verify the 19 PAcIFIC identified proteins of interest proved inconclusive save for five proteins. We discuss these five in terms of their potential relevance as biological markers for use in AAA screening of population at risk.
Zhifei Shao - One of the best experts on this subject based on the ideXlab platform.
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both plasma basic Carboxypeptidases Carboxypeptidase B2 and Carboxypeptidase n regulate vascular leakage activity in mice
Journal of Thrombosis and Haemostasis, 2021Co-Authors: Zhifei Shao, Lawrence L K Leung, Qin Zhou, Lei Zhao, Paul Declerck, John MorserAbstract:BACKGROUND Kallikrein is generated when the contact system is activated, subsequently cleaving high molecular weight kininogen to bradykinin (BK). BK binds to bradykinin receptor 2 (B2R) causing vascular leakage. BK is inactivated by proteolysis by the plasma Carboxypeptidase B2 and N (CPB2 and CPN). CPN is constitutively active but CPB2 is generated from its zymogen, proCPB2. OBJECTIVES Determine the role of CPB2 and CPN in the regulation of vascular leakage. METHODS Mice deficient in CPB2, CPN or both (CpB2-/- , Cpn-/- and CpB2-/- /Cpn-/- ) were compared to wild type mice (WT) in a model of vascular leakage caused by skin irritation. In some experiments mice were pretreated with antibodies that prevent activation of proCPB2. RESULTS Skin irritation increased vascular leakage most in CpB2-/- /Cpn-/- , less in CpB2-/- and Cpn-/- and least in WT mice. There was no difference in vascular leakage without the challenge. Antibodies inhibiting activation of proCPB2 by plasmin, but not by the thrombin/thrombomodulin (TM) complex, increased vascular leakage to the level seen in CpB2-/- mice. There was no change in levels of markers of coagulation and fibrinolysis. CONCLUSIONS BK is inactivated by both CPB2 and CPN independently. Plasmin is the activator of proCPB2 in this model. Mice lacking both plasma Carboxypeptidases have more vascular leak than ones lacking either alone. Although BK levels were not determined, BK is the likely substrate for CPB2 and CPN in this model.
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Carboxypeptidase B2 and n play different roles in regulation of activated complements c3a and c5a in mice
Journal of Thrombosis and Haemostasis, 2018Co-Authors: John Morser, J. Higgins, Zhifei Shao, Toshihiko Nishimura, Q. Zhou, Lawrence L K LeungAbstract:: Essentials Two basic Carboxypeptidases are present in plasma, B2 (CPB2) and N (CPN). CpB2-/- and Cpn-/- mice were challenged in a hemolytic uremic syndrome (HUS) model vs. wild type. CpB2-/- exacerbates HUS while Cpn-/- exacerbates cobra venom factor challenge vs. wild type mice. CPB2 and CPN have overlapping but non-redundant roles. SUMMARY: Background There are two basic Carboxypeptidases in plasma. Carboxypeptidase B2 (CPB2) is activated from a circulating zymogen, proCPB2, and Carboxypeptidase N (CPN) is constitutively active with both inactivating complement C3a and C5a. Aims To test the roles of CPB2 and CPN in complement-driven mouse models of cobra venom factor (CVF) challenge and hemolytic-uremic syndrome (HUS). Methods CpB2-/- , Cpn-/- and wild-type (WT) mice were compared in an HUS model induced by Shiga toxin and lipopolysaccharide administration and following CVF administration. Results HUS was exacerbated in CpB2-/- mice more than in Cpn-/- mice, compared with WT mice. CpB2-/- mice developed the HUS clinical triad of microangiopathic hemolytic anemia, uremia and thrombocytopenia. Treatment with anti-C5 antibody improved survival of both CpB2-/- and Cpn-/- mice. In contrast, when challenged acutely with CVF, the reverse phenotype was observed. Cpn-/- mice had markedly worse disease than CpB2-/- mice, whereas the WT mice were resistant. Conclusions CPN and CPB2 play overlapping but non-redundant roles in regulating complement activation in vivo. The constitutively active CPN is key for inactivation of systemic C5a, whereas CPB2 functions as an on-demand supplementary anaphylatoxin inhibitor in inactivating excessive C5a formed locally.
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Carboxypeptidase B2 deficiency reveals opposite effects of complement c3a and c5a in a murine polymicrobial sepsis model
Journal of Thrombosis and Haemostasis, 2015Co-Authors: Zhifei Shao, Lawrence L K Leung, Toshihiko Nishimura, John MorserAbstract:Background and Objectives Carboxypeptidase B2 (CPB2) is a basic Carboxypeptidase with fibrin and complement C3a and C5a as physiological substrates. We hypothesized that in polymicrobial sepsis, CPB2-deficient mice would have sustained C5a activity, leading to disease exacerbation.
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Carboxypeptidase B2 is protective in a mouse model of shiga toxin induced hemolytic uremic syndrome
Blood, 2014Co-Authors: Zhifei Shao, Lawrence L K Leung, John Morser, Toshihiko NishimuraAbstract:Hemolytic uremic syndrome (HUS) is characterized by a triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. The most common cause of HUS is Shiga toxin (STX)-producing E. coli, and eculizumab, a monoclonal antibody against complement C5, has shown clinical efficacy in some patients. Carboxypeptidase B2 (CPB2) is a metalloprotease activated by the thrombin/thrombomodulin complex that inactivates a number of inflammatory mediators, including complement C3a, and C5a by removing their C-terminal arginine. We hypothesized that in a murine model of STX-induced HUS, CpB2-/- mice would have exacerbated disease compared to wild type (WT) mice due to excessive C3a and/or C5a in the absence of CPB2. A mouse model of STX-induced HUS was established by giving STX and LPS toxins intraperitoneally. CpB2-/- mice had worse survival than WT (37% survival vs. 87% at 48h, p=0.0156). At 48h, severe thrombocytopenia developed in both WT and CpB2-/- mice (WT: 0.096x106/μL; CpB2-/- : 0.054x106/μL) compared to controls (1.2x106/μL; p>0.0001 vs. either WT or CpB2-/- ), with CpB2-/- mice showing worse thrombocytopenia. Renal insufficiency was worse in CpB2-/- mice than WT mice (BUN at 48h: 85 mg/dL vs. 37 mg/dL, p=0.0074; creatinine: 1.33 mg/dL vs. 0.23 mg/dL; p=0.0112, for CpB2-/- and WT mice respectively, compared with normal baseline BUN and creatinine of 19 mg/dL and 0.1 mg/dL). CpB2-/- mice developed worse anemia than WT (hemoglobin 9.8 g/dL vs. 12.4 g/dL, p=0.001 in CpB2-/- vs. WT mice respectively). At 48h, liver function was worse in CpB2-/- mice than WT mice, while plasma LDH was increased in CpB2-/- mice more than WT mice. Using a standardized health score, the CpB2-/- mice were worse than WT mice at all time points. Thus this model recapitulates STX-induced HUS with the CpB2-/- mice having worse disease than WT. If the animals were treated with STX alone, there were no deaths in either genotype at 48h and only 37.5% mortality in CpB2-/- mice by 60h compared with no deaths in WT mice. BUN, creatinine, liver enzymes and LDH were increased in both genotypes treated with STX alone compared to untreated mice, but there was no significant difference between the genotypes. Treatment with LPS alone caused thrombocytopenia in both WT and CpB2-/- mice and LDH, BUN and creatinine levels were higher in CpB2-/- mice than in WT mice, but there was no death at 48h and no drop in hemoglobin. Thus while either STX alone or LPS alone caused pathological conditions in the mice, the typical triad of HUS was only present when STX and LPS were given in combination. The CpB2-/- mice had worse disease than WT mice consistent with our hypothesis on the role of CPB2 in inactivating C3a and/or C5a in STX-induced HUS. The potential efficacy of C3a and/or C5a blockade and anti-thrombotic agents will be tested in this model. Disclosures No relevant conflicts of interest to declare.
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thrombin cleavage of osteopontin disrupts a pro chemotactic sequence for dendritic cells which is compensated by the release of its pro chemotactic c terminal fragment
Journal of Biological Chemistry, 2014Co-Authors: Zhifei Shao, John Morser, Lawrence L K LeungAbstract:Thrombin cleavage alters the function of osteopontin (OPN) by exposing an integrin binding site and releasing a chemotactic C-terminal fragment. Here, we examined thrombin cleavage of OPN in the context of dendritic cell (DC) migration to define its functional domains. Full-length OPN (OPN-FL), thrombin-cleaved N-terminal fragment (OPN-R), thrombin- and Carboxypeptidase B2-double-cleaved N-terminal fragment (OPN-L), and C-terminal fragment (OPN-CTF) did not have intrinsic chemotactic activity, but all potentiated CCL21-induced DC migration. OPN-FL possessed the highest potency, whereas OPNRAA-FL had substantially less activity, indicating the importance of RGD. We identified a conserved 168RSKSKKFRR176 sequence on OPN-FL that spans the thrombin cleavage site, and it demonstrated potent pro-chemotactic effects on CCL21-induced DC migration. OPN-FLR168A had reduced activity, and the double mutant OPNRAA-FLR168A had even lower activity, indicating that these functional domains accounted for most of the pro-chemotactic activity of OPN-FL. OPN-CTF also possessed substantial pro-chemotactic activity, which was fully expressed upon thrombin cleavage and its release from the intact protein, because OPN-CTF was substantially more active than OPNRAA-FLR168A containing the OPN-CTF sequence within the intact protein. OPN-R and OPN-L possessed similar potency, indicating that the newly exposed C-terminal SVVYGLR sequence in OPN-R was not involved in the pro-chemotactic effect. OPN-FL and OPN-CTF did not directly bind to the CD44 standard form or CD44v6. In conclusion, thrombin cleavage of OPN disrupts a pro-chemotactic sequence in intact OPN, and its loss of pro-chemotactic activity is compensated by the release of OPN-CTF, which assumes a new conformation and possesses substantial activity in enhancing chemokine-induced migration of DCs.
Maggy Chwastyniak - One of the best experts on this subject based on the ideXlab platform.
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Quantitative Mass Spectrometry Analysis Using PAcIFIC for the Identification of Plasma Diagnostic Biomarkers for Abdominal Aortic Aneurysm
2016Co-Authors: Adelina E. Acosta-martin, Re Panchaud, Maggy Chwastyniak, Annabelle Dupont, Francis Juthier, Corinne Gautier, Brigitte Jude, David R. GoodlettAbstract:Background: Abdominal aortic aneurysm (AAA) is characterized by increased aortic vessel wall diameter (.1.5 times normal) and loss of parallelism. This disease is responsible for 1–4 % mortality occurring on rupture in males older than 65 years. Due to its asymptomatic nature, proteomic techniques were used to search for diagnostic biomarkers that might allow surgical intervention under nonlife threatening conditions. Methodology/Principal Findings: Pooled human plasma samples of 17 AAA and 17 control patients were depleted of the most abundant proteins and compared using a data-independent shotgun proteomic strategy, Precursor Acquisition Independent From Ion Count (PAcIFIC), combined with spectral counting and isobaric tandem mass tags. Both quantitative methods collectively identified 80 proteins as statistically differentially abundant between AAA and control patients. Among differentially abundant proteins, a subgroup of 19 was selected according to Gene Ontology classification and implication in AAA for verification by Western blot (WB) in the same 34 individual plasma samples that comprised the pools. From the 19 proteins, 12 were detected by WB. Five of them were verified to be differentially up-regulated in individual plasma of AAA patients: adiponectin, extracellular superoxide dismutase, protein AMBP, kallistatin and Carboxypeptidase B2. Conclusions/Significance: Plasma depletion of high abundance proteins combined with quantitative PAcIFIC analysis offered an efficient and sensitive tool for the screening of new potential biomarkers of AAA. However, WB analysis to verif
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quantitative mass spectrometry analysis using pacific for the identification of plasma diagnostic biomarkers for abdominal aortic aneurysm
PLOS ONE, 2011Co-Authors: Maggy Chwastyniak, Annabelle Dupont, Francis Juthier, Adelina E Acostamartin, Alexandre Panchaud, Corinne GautierAbstract:Background Abdominal aortic aneurysm (AAA) is characterized by increased aortic vessel wall diameter (>1.5 times normal) and loss of parallelism. This disease is responsible for 1–4% mortality occurring on rupture in males older than 65 years. Due to its asymptomatic nature, proteomic techniques were used to search for diagnostic biomarkers that might allow surgical intervention under nonlife threatening conditions. Methodology/Principal Findings Pooled human plasma samples of 17 AAA and 17 control patients were depleted of the most abundant proteins and compared using a data-independent shotgun proteomic strategy, Precursor Acquisition Independent From Ion Count (PAcIFIC), combined with spectral counting and isobaric tandem mass tags. Both quantitative methods collectively identified 80 proteins as statistically differentially abundant between AAA and control patients. Among differentially abundant proteins, a subgroup of 19 was selected according to Gene Ontology classification and implication in AAA for verification by Western blot (WB) in the same 34 individual plasma samples that comprised the pools. From the 19 proteins, 12 were detected by WB. Five of them were verified to be differentially up-regulated in individual plasma of AAA patients: adiponectin, extracellular superoxide dismutase, protein AMBP, kallistatin and Carboxypeptidase B2. Conclusions/Significance Plasma depletion of high abundance proteins combined with quantitative PAcIFIC analysis offered an efficient and sensitive tool for the screening of new potential biomarkers of AAA. However, WB analysis to verify the 19 PAcIFIC identified proteins of interest proved inconclusive save for five proteins. We discuss these five in terms of their potential relevance as biological markers for use in AAA screening of population at risk.