Thrombin-activatable procarboxypeptidase B regulates activated complement C5a in vivo

Abstract

Plasma procarboxypeptidase B (proCPB)is activated by the endothelial thrombinprothrombomodulin complex. Activated (CPB) functions as a fibrinolysis inhibitor, but it may play a broader role by inactivating inflammatory mediators. To test this hypothesis, C5a-induced alveolitis was studied in wild-type (WT) and proCPBdeficient mice (proCPB ؊/؊ ). C5a-induced alveolitis, as measured by cell counts and total protein contents in bronchoalveolar lavage fluids, was markedly enhanced in the proCPB ؊/؊ mice. E229K thrombin, a thrombin mutant with minimal clotting activity but retaining its ability to activate protein C and proCPB, attenuated C5a-induced alveolitis in WT but not in proCPB ؊/؊ mice, indicating that its beneficial effect is mediated primarily by its activation of proCPB. Lung tissue histology confirmed these cellular inflammatory responses. Delayed administration of E229K thrombin after the C5a instillation was ineffective in reducing alveolitis in WT mice, suggesting that the beneficial effect of E229K thrombin is due to the direct inhibition of C5a by CPB. Our studies show that thrombin-activatable proCPB, in addition to its role in fibrinolysis, has intrinsic anti-inflammatory functions. Its activation, along with protein C, by the endothelial thrombin-TM complex represents a homeostatic response to counteract the inflammatory mediators generated at the site of vascular injury. IntroductionThe complement system is part of the innate immune system and plays a major role in the host defense against pyrogenic bacterial infection. 1,2 It is also involved in a wide variety of acute and chronic inflammatory disorders, including sepsis, acute respiratory distress syndrome, rheumatoid arthritis, and glomerulonephritis. The complement system consists of multiple proteins in plasma and on cell surfaces and can be activated by 3 distinct pathways, all converging on the cleavage of C3 into C3a and C3b. C3b in turn becomes an essential component of the C5 convertase enzyme complex, leading to cleavage of C5 into C5a and C5b. Both C3a and C5a are strong chemoattractants. 1,2 They cause oxidative burst and enhance phagocytosis and granule enzyme release in neutrophils. They are vasodilators at sites of inflammation. C3a and C5a exert their effects through binding to cellular C3a receptors (C3aR) and C5a receptors (C5aR), respectively. C5aR are found on circulating myeloid cells, including neutrophils, monocytes, eosinophils, and basophils, as well as nonmyeloid cells in many organs, especially in the lung and the liver. 2-4 Widespread up-regulation of the C5aR occurs during sepsis, and mice deficient in C5aR succumbed to bacterial pneumonias in animal models, indicating that C5a/ C5aR signaling is essential in host defenses in the lung and other organs. 5,6 Both C3a and C5a are inhibited by carboxypeptidase N (CPN), a constitutively active plasma carboxypeptidase.Thrombin-activatable procarboxypeptidase B (proCPB), also known as procarboxypeptidase U, procarboxypeptidase R, and thrombin-activatable fibrinolysis inhib...

Plasma carboxypeptidase B downregulates inflammatory responses in autoimmune arthritis

“…The protective effect of anti-C5 antibody on CAIA in Cpb2 -/-mice ( Figure 2C) was remarkable, given that C5-deficient mice developed mild CAIA (Figure 2A). The influence of mouse genetic background on CAIA severity is well established (24), and the C5-deficient mice used in these experiments were of a mixed genetic background derived from C57BL/10 and DBA/2 (25), whereas the Cpb2 -/-mice were on the C57BL/6 background (26). Furthermore, the C5-deficient mice received 4 mg of anti-collagen antibodies, whereas the Cpb2 -/-mice received only 2 mg of anti-collagen antibodies.…”

International Union of Basic and Clinical Pharmacology. LXXXVII. Complement Peptide C5a, C4a, and C3a Receptors

“…The zinc metalloprotease carboxypeptidase N (CPN) is released in an active form (Levin et al, 1982), and carboxypeptidase R (CPR) is an acute-phase protein, up-regulated in inflammation and secreted in an inactive form, proCPR (reviewed in Campbell et al, 2001). ProCPR is bound to plasminogen and activated by plasmin or thrombin (Wang et al, 1994;Sato et al, 2000;Nishimura et al, 2007;Leung et al, 2008). It preferentially degrades C5a over C3a (Campbell et al, 2002) and can remove Lys residues from fibrin clots, preventing plasminogen binding (Bajzar et al, 1995).…”

Activated Protein C Mutant with Minimal Anticoagulant Activity, Normal Cytoprotective Activity, and Preservation of Thrombin Activable Fibrinolysis Inhibitor-dependent Cytoprotective Functions

“…Activated TAFI (TAFIa (9) also named carboxypeptidase U (10), carboxypeptidase R (11) and plasma carboxypeptidase B (12)) inhibits fibrinolysis by abrogating the fibrin cofactor function of tissue-type plasminogen activator (tPA)-mediated plasminogen activation (6,9). In addition to anti-fibrinolytic actions, TAFIa mediates anti-inflammatory effects via inactivation of bradykinin (BK) and the complement anaphylatoxins C3a and C5a (13)(14)(15)(16).…”