Carboxypeptidase B2 and carboxypeptidase N in the crosstalk between coagulation, thrombosis, inflammation, and innate immunity.

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.

Carboxypeptidase B2 and N play different roles in regulation of activated complements C3a and C5a in mice.

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.

Carboxypeptidase B2 deficiency reveals opposite effects of complement C3a and C5a in a murine polymicrobial sepsis model.

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. METHODS: Polymicrobial sepsis was induced by cecal ligation and puncture (CLP). RESULTS: Contrary to our hypothesis, Cpb2(-/-) mice had significantly improved survival, with reduced lung edema, less liver and kidney damage, and less disseminated intravascular coagulation. Hepatic pro-CPB2 was induced by CLP, leading to increased pro-CPB2 levels. Thrombomodulin present on mesothelium supported thrombin activation of pro-CPB2. Both wild-type and Cpb2(-/-) animals treated with a C5a receptor antagonist had improved survival, demonstrating that C5a was detrimental in this model. Treatment with a fibrinolysis inhibitor, tranexamic acid, caused a decrease in survival in both genotypes; however, the Cpb2(-/-) animals retained their survival advantage. Administration of a C3a receptor antagonist exacerbated the disease in both wild-type and Cpb2(-/-) mice and eliminated the survival advantage of Cpb2(-/-) mice. C5a receptor is expressed in both peritoneal macrophages and neutrophils; in contrast, C3a receptor expression is restricted to peritoneal macrophages, and C3a induced signaling in macrophages but not neutrophils. CONCLUSIONS: While C5a exacerbates the peritonitis, resulting in a deleterious generalized inflammatory state, C3a activation of peritoneal macrophages may limit the initial infection following CLP, thereby playing a diametrically opposing protective role in this polymicrobial sepsis model.

What has been learnt from the thrombin-activatable fibrinolysis inhibitor-deficient mouse?

SUMMARY: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a circulating zymogen that is activated physiologically by the thrombin/thrombomodulin complex to activated TAFI (TAFIa) which is a basic carboxypeptidase. Substrates include fibrin, leading to a reduction in rate of plasmin generation, and several proinflammatory mediators such as bradykinin, thrombin-cleaved osteopontin and complement factor C5a. TAFI-deficient mice have no phenotype without being challenged and TAFIa appears to play a limited role in physiological fibrinolysis in vivo. In several disease models, the TAFI-deficient mice have different outcomes from the wild type (WT), but whether the difference is beneficial or an exacerbation of the disease depends on the model. The consequences of TAFI deficiency include increased plasmin as a result of enhanced incorporation of plasminogen and tissue plasminogen activator into the fibrin clot, but also loss of its ability to degrade other substrates, with the resultant up-regulation of several proinflammatory mediators, including C5a. Criteria are recommended to demonstrate that a substrate is a physiological substrate of TAFIa.

Polyphosphate binds with high affinity to exosite II of thrombin.

BACKGROUND: Polyphosphate (a linear polymer of inorganic phosphate) is secreted from platelet dense granules, and we recently showed that it accelerates factor V activation by thrombin. OBJECTIVE: To examine the interaction of polyphosphate with thrombin. METHODS AND RESULTS: Thrombin, but not prothrombin, altered the electrophoretic migration of polyphosphate in gel mobility assays. Thrombin binding to polyphosphate was influenced by ionic strength, and was evident even in plasma. Two positively charged exosites on thrombin mediate its interactions with other proteins and accessory molecules: exosite I (mainly with thrombin substrates), and exosite II (mainly with certain anionic polymers). Free thrombin, thrombin in complex with hirudin's C-terminal dodecapeptide and gamma-thrombin all bound polyphosphate similarly, excluding exosite I involvement. Mutations within exosite II, but not within exosite I, the Na(+)-binding site or hydrophobic pocket, weakened thrombin binding to polyphosphate as revealed by NaCl dependence. Surface plasmon resonance demonstrated tight interaction of polyphosphate with thrombin (K(d) approximately 5 nm) but reduced interaction with a thrombin exosite II mutant. Certain glycosaminoglycans, including heparin, only partially competed with polyphosphate for binding to thrombin, and polyphosphate did not reduce heparin-catalyzed inactivation of thrombin by antithrombin. CONCLUSION: Polyphosphate interacts with thrombin's exosite II at a site that partially overlaps with, but is not identical to, the heparin-binding site. Polyphosphate interactions with thrombin may be physiologically relevant, as the polyphosphate concentrations achievable following platelet activation are far above the approximately 5 nM K(d) for the polyphosphate-thrombin interaction.

Essential thrombin residues for inhibition by protein C inhibitor with the cofactors heparin and thrombomodulin.

BACKGROUND: Protein C inhibitor (PCI) and antithrombin (AT) are serine protease inhibitors (serpins) that inhibit a wide array of blood coagulation serine proteases including thrombin. OBJECTIVE: Fifty-five Ala-scanned recombinant thrombin mutants were used to determine thrombin residues important for inhibition by PCI with and without the cofactors heparin and thrombomodulin (TM) and compared with the prototypical serpin, AT. RESULTS: Residues around the active site (Tyr50 and Glu202) and the sodium-binding site (Glu229 and Arg233) were required for thrombin inhibition by PCI with and without cofactors. Exosite-2 residues (Arg89, Arg93, Glu94, Arg98, Arg245, Arg248, and Gln251) were critical for heparin-accelerated inhibition of thrombin by PCI. Exosite-1 residues (especially Lys65 and Tyr71) were required for enhanced PCI inhibition of thrombin-TM. Interestingly, we also found that the TM chondroitin sulfate moiety is not required for the approximately 150-fold enhanced rate of thrombin inhibition by PCI. Using the aforementioned thrombin exosite-2 mutants that were essential for heparin-catalyzed PCI-thrombin inhibition reactions we found no change in PCI inhibition rates for thrombin-TM. CONCLUSIONS: Collectively, these results show that (i) similar thrombin exosite-2 residues are critical for the heparin-catalyzed inhibition by PCI and AT, (ii) PCI and AT are different in their thrombin-TM inhibition properties, and (iii) PCI has a distinct advantage over AT in the regulation of the activity of thrombin-TM.