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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 (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.

Apart from its role in the coagulation system, thrombin plays an important role in the inflammatory response through its protease‐activated receptors (PARs). However, the role of thrombin in the immune response is not clear.

Summary. Background:Activated protein C (APC) can regulate immune and inflammatory responses and apoptosis. Protein C transgenic mice develop less diabetic nephropathy but whether exogenous administration of APC suppresses established diabetic nephropathy is unknown.

Background:Activation of the complement system has been implicated in tumor growth. The antifibrinolytic protein, activated thrombin‐activatable fibrinolysis inhibitor (TAFIa), can modulate the activation of the complement system by inactivating the anaphylatoxins C3a and C5a. The apolipoprotein‐E (ApoE) genotype has been associated with carcinogenesis. Objective:The aim of this study was to evaluate whether TAFIa can affect the development of cancer in the ApoE‐deficient mouse model. Methods:TAFI and ApoE double‐knockout mice were generated. A group of mice was treated with the diabetogenic and carcinogenic compound streptozotocin (stz). Mice treated with saline, single knockout mice and wild‐type (wt) mice served as controls. Results:Six months after treatment with stz, mice were sacrificed. Hepatic tumors were found in male double‐knockout mice treated with stz but none was found in control animals that were not treated with stz or in single knockout of ApoE or wt animals. There was no significant difference in coagulation system activation between the groups of mice. The plasma concentrations of C5a, factor D and transforming growth factor‐β1 were increased in TAFI/ApoE double‐deficient mice treated with stz compared with the mice of the same genotype treated with saline. Conclusion:Apo‐E deficiency alone was not associated with tumors but the lack of TAFI appears to make the mice permissive for tumor formation in ApoE mice.

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.

Background: The fibrinolytic system has been implicated in the pathogenesis of pulmonary hypertension (PH). Thrombin‐activatable fibrinolysis inhibitor (TAFI) inhibits fibrinolysis and therefore its absence would be expected to increase fibrinolysis and ameliorate PH. Objective: The objective of the present study was to evaluate the effect of TAFI deficiency on pulmonary hypertension in the mouse. Methods and results: PH was induced in C57/Bl6 wild‐type (WT) or TAFI‐deficient (KO) mice by weekly subcutaneous treatment with 600 mg kg−1monocrotaline (MCT) for 8 weeks. PH was inferred from right heart hypertrophy measured using the ratio of right ventricle‐to‐left ventricle‐plus‐septum weight [RV/(LV+S)]. Pulmonary vascular remodeling was analyzed by morphometry. TAFI‐deficient MCT‐treated and wild‐type MCT‐treated mice suffered similar weight loss. TAFI‐deficient MCT‐treated mice had reduced levels of total protein and tumor necrosis factor‐alpha (TNF‐α), interleukin‐6 (IL‐6), transforming growth factor‐β (TGF‐β) and monocyte chemoattractant protein‐1 (MCP‐1) in bronchial alveolar lavage compared with wild‐type MCT‐treated mice. The ratio of RV to (LV+S) weight was significantly higher in WT/MCT than in KO/MCT mice. The pulmonary artery wall area and vascular stenosis were both greater in MCT‐treated WT mice compared with MCT‐treated TAFI‐deficient mice. Conclusions: TAFI‐deficient MCT‐treated mice had less pulmonary hypertension, vascular remodeling and reduced levels of cytokines compared with MCT‐treated WT animals, possibly as a result of reduced coagulation activation.

Objective:Protein S may exert an anticoagulant activity by enhancing the anticoagulant activity of activated protein C and/or by directly inhibiting the prothrombinase complex. Protein S itself may also directly regulate inflammatory responses and apoptosis. The role of protein S in acute lung injury (ALI) was unknown. This study evaluated the effect of protein S on ALI in the mouse. Methods:Animal ALI was induced in C57/BL6 mice by intratracheal instillation of lipopolysaccharide (LPS). Mice were treated with protein S or saline by intraperitoneal injection 1 h before LPS instillation. Results:Activated protein or protein S alone and combined activated protein C + protein S therapy decreased inflammatory markers and cytokines in mice with acute lung injury. In LPS‐treated mice compared with controls ALI was induced as shown by significantly increased levels of total protein, tumor necrosis factor‐α, interleukin‐6 and monocyte chemoattractant protein‐1 in the bronchoalveolar lavage fluid. Mice with ALI treated with protein S had significantly decreased concentrations of tumor necrosis factor‐α and interleukin‐6 in the lung compared with untreated animals. Thrombin‐antithrombin III, a marker of the activity of the coagulation cascade, was unchanged. Protein S inhibited the expression of cytokines in vitroand increased activation of the Axl tyrosine kinase pathway in A549 epithelial cells. Conclusion:Protein S protects against LPS‐induced ALI, possibly by directly inhibiting the local expression of inflammatory cytokines without affecting coagulation.

Background: Thrombin‐activatable fibrinolysis inhibitor (TAFI) has been reported to affect wound healing and fibrotic processes, but its role in renal tubulointerstitial fibrosis remains unknown. Objective: To study its potential role, we compared TAFI‐deficient and wild‐type mice for the degree of renal fibrosis caused by unilateral ureteral obstruction (UUO).Methods: The grade of tubulointerstitial fibrosis, the activity of plasmin, MMP‐2 and MMP‐9 were evaluated on days 4 and 9 after UUO. Results: The renal content of hydroxyproline and the activity of plasmin, MMP‐2 and MMP‐9 were significantly increased in kidneys with UUO from TAFI‐deficient mice compared with those from wild‐type mice. These differences disappeared when animals with UUO from both groups were treated with the plasmin inhibitor tranexamic acid. The renal concentrations of fibrogenic cytokines were also significantly elevated in kidneys with UUO from TAFI‐deficient mice compared with those from wild‐type mice. Conclusion: The results of this study suggest that increased renal activity of plasmin in TAFI‐deficient mice causes increased renal interstitial fibrosis in obstructive nephropathy.

Background/objective: The efficacy of a direct factor (F)Xa inhibitor, ZK‐807834, was compared with indirect inhibition by enoxaparin for inhibition and deaggregation of acute platelet‐rich thrombi in a well‐characterized porcine carotid injury model. Methods: A crush injury was performed on a randomly chosen carotid artery and the thrombus allowed to propagate for 30 min. Pigs then received intravenous drug for 35 min: ZK‐807834‐Dose 1 (40 µg kg−1bolus + 1.5 µg kg−1 min−1infusion, n = 6); ZK‐807834‐Dose 2 (20 µg kg−1bolus + 0.75 µg kg−1 min−1infusion; n = 6); enoxaparin (1 mg kg−1bolus; n = 6); or saline (n = 6). Five minutes after drug initiation, the contralateral artery was injured. Thrombus size was monitored by scintillation detection of autologous 111In‐platelets. Results: The prothrombin time ratio was 2.2 ± 0.1; 1.4 ± 0.3; 1.2 ± 0.9 and 1.1 ± 0.2, respectively. ZK‐807834‐Dose 1 significantly inhibited carotid platelet deposition (525 ± 226 × 106 cm−2; P = 0.008), whereas ZK‐807834‐Dose 2 (2325 ± 768) and enoxaparin (1236 ± 383) were not different from saline (2776 ± 642). Thrombus deaggregation was greatest for animals receiving ZK‐807834‐Dose 1 (473 ± 185). Neither ZK‐807834‐Dose 2 (1588 ± 480) nor enoxaparin (1618 ± 686) was different from saline control (2222 ± 598). Conclusions: Direct FXa inhibition with ZK‐807834, at a prothrombin time ratio of 2.2, effectively inhibits thrombosis and promptly deaggregates thrombi induced by arterial injury. In contrast, indirect FXa inhibition with enoxaparin was ineffective.

Inhibition of factor Xa with the small molecule inhibitor ZK-807834 (Mr 527 Da, Ki0.11 nM) attenuates progression of thrombosis, but the ED50is substantially lower for venous compared with arterial thrombosis in experimental animals. To determine whether this reflects differences in the extent of vascular injury, we compared the dose–response of ZK-807834 for inhibition of venous thrombosis induced with a cotton thread and copper wire device in the presence and absence of balloon catheter-induced injury to the vena cava in rabbits. ZK-807834 administration over 2 h (total dosages of 0.0023–2.3 µmol kg−1, n = 6/group) resulted in dose-dependent reductions in clot weight compared with vehicle controls, but the ED50was 0.03 µmol kg−1for non-injured veins and 0.42 µmol kg−1for injured veins. We conclude that vascular injury invokes a tissue factor-mediated response that increases the dose requirements for inhibition of venous thrombosis with ZK-807834.

A novel cDNA has been identified from human heart that encodes an unusual mosaic serine protease, designated corin. Corin has a predicted structure of a type II transmembrane protein and contains two frizzled-like cysteine-rich motifs, seven low density lipoprotein receptor repeats, a macrophage scavenger receptor-like domain, and a trypsin-like protease domain in the extracellular region. Northern analysis showed that corin mRNA was highly expressed in the human heart. In mice, corin mRNA was detected by in situ hybridization in the cardiac myocytes of the embryonic heart as early as embryonic day (E) 9.5. By E11.5-13.5, corin mRNA was most abundant in the primary atrial septum and the trabecular ventricular compartment. Expression in the heart was maintained through the adult. In addition, mouse corin mRNA was also detected in the prehypertrophic chrondrocytes in developing bones. By fluorescent in situ hybridization analysis, the human corin gene was mapped to 4p12-13 where a congenital heart disease locus, total anomalous pulmonary venous return, had been previously localized. The unique domain structure and specific embryonic expression pattern suggest that corin may have a function in cell differentiation during development. The chromosomal localization of the human corin gene makes it an attractive candidate gene for total anomalous pulmonary venous return.

A collection of 56 purified thrombin mutants, in which 76 charged or polar surface residues on thrombin were mutated to alanine, was used to identify key residues mediating the interactions of thrombin with thrombomodulin (TM), protein C, and thrombin-activatable fibrinolysis inhibitor (TAFI). Comparison of protein C activation in the presence and absence of TM identified 11 residues mediating the thrombin-TM interaction (Lys(21), Gln(24), Arg(62), Lys(65), His(66), Arg(68), Thr(69), Tyr(71), Arg(73), Lys(77), Lys(106)). Three mutants (E25A, D51A, R89A/R93A/E94A) were found to have decreased ability to activate TAFI yet retained normal protein C activation, whereas three other mutants (R178A/R180A/D183A, E229A, R233A) had decreased ability to activate protein C but maintained normal TAFI activation. One mutant (W50A) displayed decreased activation of both substrates. Mapping of these functional residues on thrombin revealed that the 11 residues mediating the thrombin-TM interaction are all located in exosite I. Residues important in TAFI activation are located above the active-site cleft, whereas residues involved in protein C are located below the active-site cleft. In contrast to the extensive overlap of residues mediating TM binding and fibrinogen clotting, these data show that distinct domains in thrombin mediate its interactions with TM, protein C, and TAFI. These studies demonstrate that selective enzymatic properties of thrombin can be dissociated by site-directed mutagenesis.

In an attempt to isolate a cDNA encoding an opioid receptor, a cDNA library was cionstructed in the ?ZAP vector using NG108-15 mRNA as template. Using an in vitro transcription-translation assay and a sib selection strategy, a single phage was isolated. An RNA transcribed from this cDNA was able to direct in vitro translation of opioid binding sites. The insert was sequenced and comparison with data banks showed a 100% homology with theE. coli envY gene. We assume that the presence of the envY sequence in the NG108-15 cDNA libary was due to a contamination of the ?ZAP vector withE. coli DNA. A search for opioid binding sites, onE. coli strains showed that envY+ strains, but not envY- mutants were able to bind opiates. On envY+ cells, the sites are stereospecific, saturable and of high affinity for the opiate ligands. These sites bind opiate agonists and antagonists but neither µ nor d opioid peptides. In contrast, rabbit reticulocyte lysate primed with RNA transcribed in vitro from, the envY sequence elicited the synthesis of an opioid binding site with mixed µ and d properties. In addition, transfection of the envY sequence into mammalian cells resulted in the expression of opioid binding sites. Depending on the type of cells transfected, these sites were selective for either the µ or d ligands.

TAFI (thrombin-activatable fibrinolysis inhibitor) is a recently discovered plasma protein that can be activated by thrombin-catalyzed proteolysis to a carboxypeptidase B-like enzyme that inhibits fibrinolysis. This work shows that the thrombin-thrombomodulin complex, rather than free thrombin, is the most likely physiologic activator. Thrombomodulin increases the catalytic efficiency of the reaction by a factor of 1250, an effect expressed almost exclusively through an increase in kcat. The kinetics of the reaction conform to a model whereby thrombin can interact with either TAFI (Km = 1.0 microM) or thrombomodulin (Kd = 8.6 nM), and either binary complex so formed can then interact with the third component to form the ternary thrombin-thrombomodulin-TAFI complex from which activated TAFI is produced with kcat = 1.2 s-1. This work also shows that activated TAFI down-regulates tPA-induced fibrinolysis half-maximally at a concentration of 1.0 nM in a system of purified components. This concentration of TAFI is about 2% of the level of the zymogen in plasma, which indicates that ample activated TAFI could be generated to very significantly modulate fibrinolysis in vivo. Therefore, TAFI in vitro and possibly in vivo defines an explicit molecular connection between the coagulation and fibrinolytic cascades, such that expression of activity in the former down-regulates the activity of the latter.

Recombinant human prothrombin (rII) and two mutant forms (R155A, R271A,R284A (rMZ) and R271A,R284A (rMZdesF1)) were expressed in mammalian cells. Following activation and purification, recombinant thrombin (rIIa) and stable analogues of meizothrombin (rMZa) and meizothrombin(desF1) (rMZdesF1a) were obtained. Studies of the activation of protein C in the presence of recombinant soluble thrombomodulin (TM) show TM-dependent stimulation of protein C activation by all three enzymes and, in the presence of phosphatidylserine/phosphatidylcholine phospholipid vesicles, rMZa is 6-fold more potent than rIIa. In the presence of TM, rMZa was also shown to be an effective activator of TAFI (thrombin-activatable fibrinolysis inhibitor) (Bajzar, L., Manuel, R., and Nesheim, M. E. (1995) J. Biol. Chem. 270, 14477-14484). All three enzymes were capable of inducing platelet aggregation, but 60-fold higher concentrations of rMZa and rMZdesF1a were required to achieve the effects obtained with rIIa. Second order rate constants (M-1.min-1) for inhibition by antithrombin III (AT-III) were 2.44 x 10(5) (rIIa), 6.10 x 10(4) (rMZa), and 1.05 x 10(5) (rMZdesF1a). The inhibition of rMZa and rMZdesF1a by AT-III is not affected by heparin. All three enzymes bound similarly to hirudin. The results of this and previous studies imply that full-length meizothrombin has marginal procoagulant properties compared to thrombin. However, meizothrombin has potent anticoagulant properties, expressed through TM-dependent activation of protein C, and can contribute to down-regulation of fibrinolysis through the TM-dependent activation of TAFI.

The solution structure has been determined for a 19-residue peptide that is fully folded at room temperature. The sequence of this peptide is based on the C-loop, residues 371-389, of the fourth epidermal growth factor-like domain of thrombomodulin, a protein that acts as a cofactor for the thrombin activation of protein C. Despite its small size, the peptide forms a compact structure with almost no repeating secondary structure. The results indicate the structure is held together by hydrophobic interactions, which in turn stabilize the two beta-turns in the structure. The first beta-turn in the C-loop represents a conserved motif that is found in the published structures of five other epidermal growth factor-like proteins. The critical role of Phe376 in the stabilization of the first beta-turn is consistent with mutagenesis data with soluble thrombomodulin. The results also show that a small subdomain of a larger protein can fold independently, and therefore it could act as an initiation site for further folding.

Thrombin-activable fibrinolysis inhibitor (TAFI) is a recently described plasma zymogen that can be activated by thrombin to an enzyme with carboxypeptidase B-like activity. The enzyme, TAFIa, potently attentuates fibrinolysis. TAFI activation, like protein C activation, is augmented about 1250-fold by thrombomodulin (TM). In this work, the effects of both soluble and cellular forms of TM on TAFI activation-dependent suppression of fibrinolysis were investigated. Soluble TM included in clots formed from purified components, barium citrate-adsorbed plasma, or normal human plasma maximally increased the tissue plasminogen activator-induced lysis time 2-3-fold, with saturation occurring at 5, 10, and 1 nM TM in the three respective systems. Soluble TM did not effect lysis in the system of purified components lacking TAFI or in plasmas immunodepleted of TAFI. In addition, the antifibrinolytic effect of TM was negated by monoclonal antibodies against either TAFI or TM. The inhibition of fibrinolysis by cellular TM was assessed by forming clots in dialyzed, barium citrate-adsorbed, or normal plasma over cultured human umbilical vein endothelial cells (HUVECs). Tissue plasminogen activator-induced lysis time was increased 2-fold, with both plasmas, in the presence of HUVECs. The antifibrinolytic effect of HUVECs was abolished 66% by specific anti-TAFI or anti-TM monoclonal antibodies. A newly developed functional assay demonstrated that HUVECs potentiate the thrombin-catalyzed, TM-dependent formation of activated TAFI. Thus, endothelial cell TM, in vitro at least, appears to participate in the regulation of not only coagulation but also fibrinolysis.

Two human T-lymphoblastoid cell lines, CCRF/CEM and Molt 4, produced beta interferon (IFN-beta) upon infection with Sendai virus. Molt 4, but not CCRF/CEM, spontaneously produced up to 300 U of IFN-gamma per ml, apparently not contaminated with IFN-alpha or -beta. Phytohemagglutinin, a T-cell mitogen, did not stimulate IFN production in these lines. A third T-lymphoblastoid line, CCRF/HSB2, produced no IFN either spontaneously or after infection with Sendai virus or treatment with phytohemagglutinin. The Molt 4 cells contained an mRNA which could be translated by oocytes to give IFN-gamma. Molt 4 cells therefore provide a convenient source of human IFN-gamma and its mRNA for experimental purposes.

The proteins synthesized in vitro in response to 42S and 26S RNAs from Semliki Forest virus were labeled with formyl-[35S]methionine from initiator tRNA. One protein which comigrated with viral capsid protein was labeled under the direction of 26S RNA, and only one labeled peptide was detected after digestion with trypsin. Further digestion with pronase gave rise to the dipeptide fMet-AsN. Several labeled polypeptides were found in the 42S RNA directed product and these had molecular weights of up to 150,000. However, tryptic digestion of the product yielded only one formylmethionyl-labeled peptide, which had a different mobility from that directed by the 26S RNA. Further digestion with pronase gave a single dipeptide, fMet-Ala. This indicates that nonstructural proteins as large as 150,000 daltons are probably synthesized from one initiation site on the 42S RNA. Translation starting from the internal initiation site on the 42S RNA, which is equivalent to that on the 26S RNA, could not be detected under the conditions used. Internal initiation sites which are similarly inactive have also been detected in other viral RNAs (e.g., brome mosaic virus, tobacco mosaic virus, and polyoma 19S RNA) and this suggests that, although eukaryotic mRNAs can contain more than one initiation site for protein synthesis, only the site nearer the 5' terminus is active in vitro.

A total of 11 distinct oligonucleotide fingerprints were obtained in studies of the ribonucleic acids of 13 isolates of infectious bronchitis virus. Different serotypes had distinct fingerprints, but so did varieties within a serotype, allowing a greater degree of strain differentiation than was previously possible. Some conclusions can be drawn from the fingerprints concerning theories of origin and spread of infectious bronchitis virus.

The effects of imbalanced subunit synthesis, temperature, colchicine, and cytochalasin on the secretion from Xenopus laevis oocytes of a variety of avian and mammalian proteins were investigated; these proteins were encoded by microinjected messenger RNA. Cytochalasin and colchicine together severely reduced secretion in a temperature-independent manner, the exact reduction varying among the different proteins. In contrast cytochalasin alone had no effect, whereas colchicine alone caused a smaller, temperature-dependent reduction. The synthesis and subcellular compartmentation of these proteins were unaffected by the drug treatments; however, the proteins did not accumulate in the drug-treated oocytes but were degraded. The rate of degradation of each protein was similar to its rate of exocytosis from untreated oocytes. A similar result was obtained without recourse to drugs by studying the fate of immunoglobulin light chains trapped in oocytes by a deficiency in heavy chain synthesis. These results are discussed in terms of the disruptive effects, as revealed by electron microscopy, of the drug treatments on the cytoskeleton of the oocyte.