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Background: Osteopontin (OPN) is a multifunctional proinflammatory matricellular protein overexpressed in multiple human cancers and associated with tumor progression and metastases. Thrombin cleavage of OPN reveals a cryptic binding site for α 4 β 1 and α 9 β 1 integrins., Methods: Thrombin cleavage-resistant OPN R153A knock-in (OPN-KI) mice were generated and compared to OPN deficient mice (OPN-KO) and wild type (WT) mice in their ability to support growth of melanoma cells. Flow cytometry was used to analyze tumor infiltrating leukocytes., Results: OPN-KI mice engineered with a thrombin cleavage-resistant OPN had reduced B16 melanoma growth and fewer pulmonary metastases than WT mice. The tumor suppression phenotype of the OPN-KI mouse was identical to that observed in OPN-KO mice and was replicated in WT mice by pharmacologic inhibition of thrombin with dabigatran. Tumors isolated from OPN-KI mice had increased tumor-associated macrophages with an altered activation phenotype. Immunodeficient OPN-KI mice (NOG-OPN-KI) or macrophage-depleted OPN-KI mice did not exhibit the tumor suppression phenotype. As B16 cells do not express OPN, thrombin-cleaved fragments of host OPN suppress host antitumor immune response by functionally modulating the tumor-associated macrophages. YUMM3.1 cells, which express OPN, showed less tumor suppression in the OPN-KI and OPN-KO mice than B16 cells, but its growth was suppressed by dabigatran similar to B16 cells., Conclusions: Thrombin cleavage of OPN, derived from the host and the tumor, initiates OPN's tumor-promoting activity in vivo., (© 2022 International Society on Thrombosis and Haemostasis.)

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, 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 with 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 complex, increased vascular leakage to the level seen in Cpb2 -/- mice. There was no change in levels of markers of coagulation and fibrinolysis., Conclusions: Bradykinin 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 those lacking either alone. Although BK levels were not determined, BK is the likely substrate for CPB2 and CPN in this model., (© 2021 International Society on Thrombosis and Haemostasis.)

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., (© 2018 International Society on Thrombosis and Haemostasis.)

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., (© 2018 International Society on Thrombosis and Haemostasis.)

Chemerin is a chemoattractant and adipokine that circulates in blood as inactive prochemerin (chem163S). Chem163S is activated by a series of C-terminal proteolytic cleavages resulting in diverse chemerin forms with different levels of activity. We screened a panel of proteases in the coagulation, fibrinolytic, and inflammatory cascades to identify those that process prochemerin in plasma. Factor XIa (FXIa) cleaved chem163S, generating a novel chemerin form, chem162R, as an intermediate product, and chem158K, as the final product. Processing at Arg 162 was not required for cleavage at Lys 158 or regulation of chemerin bioactivity. Contact phase activation of human platelet-poor plasma by kaolin led to cleavage of chem163S, which was undetectable in FXI-depleted plasma and markedly enhanced in platelet-rich plasma (PRP). Contact phase activation by polyphosphate in PRP resulted in 75% cleavage of chem163S. This cleavage was partially inhibited by hirudin, which blocks thrombin activation of FXI. After activation of plasma, levels of the most potent form of chemerin, chem157S, as well as inactive chem155A, increased. Plasma levels of chem163S in FXI-deficient patients were significantly higher compared with a matched control group (91 ± 10 ng/mL vs 58 ± 3 ng/mL, n = 8; P < .01) and inversely correlated with the plasma FXI levels. Thus FXIa, generated on contact phase activation, cleaves chem163S to generate chem158K, which can be further processed to the most active chemerin form, providing a molecular link between coagulation and inflammation.

Unlabelled: Circulating factor VIII (FVIII) is derived from liver and from extrahepatic sources probably of endothelial origin, but the vascular sites of FVIII production remain unclear. Among organs profiled, only liver and lymph nodes (LNs) show abundant expression of F8 messenger RNA (mRNA). Transcriptomic profiling of subsets of stromal cells, including endothelial cells (ECs) from mouse LNs and other tissues, showed that F8 mRNA is expressed by lymphatic ECs (LECs) but not by capillary ECs (capECs), fibroblastic reticular cells, or hematopoietic cells. Among blood ECs profiled, F8 expression was seen only in fenestrated ECs (liver sinusoidal and renal glomerular ECs) and some high endothelial venules. In contrast, von Willebrand factor mRNA was expressed in capECs but not in LECs; it was coexpressed with F8 mRNA in postcapillary high endothelial venules. Purified LECs and liver sinusoidal ECs but not capECs from LNs secrete active FVIII in culture, and human and mouse lymph contained substantial, Fviii: C activity. Our results revealed localized vascular expression of FVIII and von Willebrand factor and identified LECs as a major cellular source of FVIII in extrahepatic tissues.

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., (© 2015 International Society on Thrombosis and Haemostasis.)

Background: Thrombomodulin treatment modulates the properties of dendritic cells (DCs) converting them from immunogenic to tolerogenic and inducing its own expression on DCs. Thrombomodulin binds to the inflammatory mediator, high mobility group protein B1 (HMGB1), antagonizing signalling through its receptor, receptor for advanced glycation end products (RAGE)., Methods: To test if soluble thrombomodulin could antagonize HMGB1 signaling via RAGE on DCs. DCs were prepared from mouse bone marrow cells or human monocytes. In some experiments dendritic cells were sorted into thrombomodulin+ and thrombomodulin- populations. Expression of surface maturation markers was determined by flow cytometry following treatment with thrombomodulin in the presence or absence of HMGB1., Results: Thrombomodulin+ dendritic cells secrete less HMGB1 into the medium. HMGB1 reduces the effects of thrombomodulin on expression of DC maturation markers. Treatment with thrombomodulin reduces the expression of maturation markers such as CD80 and CD86 and increases the expression of thrombomodulin on the DC surface. Treatment of DCs with neutralizing anti-HMGB1 antibody acted synergistically with thrombomodulin in increasing thrombomodulin expression on DCs. Treatment with thrombomodulin can still reduce the expression of surface markers on DCs derived from mice that are deficient in RAGE showing that thrombomodulin can affect DCs by an alternative mechanism., Conclusions: The results of this study show that thrombomodulin modulates DCs both by antagonizing the interaction of HMGB1 with RAGE and by an independent mechanism.

Background: 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., Objective: To evaluate whether thrombin has a modulatory role in allergic bronchial asthma., Methods: Bronchial asthma was induced in mice by intraperitoneal sensitization and inhalation challenge with ovalbumin. Thrombin or its inhibitors were administered by inhalation before each allergen challenge., Results: Mice with low but sustained coagulation activation had reduced allergic inflammation, and allergic asthma was inhibited by low doses of thrombin but worsened by high doses. Allergic asthma was worsened by antithrombin, argatroban, hirudin, and anti-thrombomodulin antibody. Mice with a higher level of an inhibitor of both thrombin and activated protein C had worse disease. Heterozygous PAR-1 mice had less allergic inflammation, but PAR-1 agonist worsened it. Allergic bronchial inflammation was worsened in mice that received adoptive transfer of PAR-1 agonist-treated Th2 cells as compared with controls. Low levels of thrombin suppressed the maturation and secretion of cytokines in dendritic cells, but high levels enhanced this., Conclusions: The effects of thrombin on allergic asthma are dose-dependent, with detrimental effects at high doses and protective effects at low doses. These data demonstrate that thrombin modulates the outcome in allergic bronchial asthma., (© 2013 International Society on Thrombosis and Haemostasis.)

Background: Infectious complications frequently occur after major trauma, leading to increased morbidity and mortality. Thrombin-activatable fibrinolysis inhibitor (TAFI), a procarboxypeptidase in plasma, plays a dual role in regulating both coagulation and inflammation. Activated TAFI (TAFIa) has broad anti-inflammatory properties due to its inactivation of active inflammatory mediators (anaphylatoxins C3a and C5a, bradykinin, osteopontin)., Objectives: The purpose of this study was to determine if TAFI plays a role in the development of inflammatory complications after major trauma., Patients/methods: Upon arrival at the emergency department (ED), plasma levels of TAFI and TAFIa were measured in 26 multiple traumatized patients for 10 consecutive days. Systemic levels of inflammatory mediators, including interleukin-6 (IL-6), procalcitonin (PCT), C-reactive protein (CRP) and leukocytes were determined., Results: Fifteen patients developed pneumonia and/or sepsis (compl) and 11 had no complications (wo compl). Overall injury severity and age were comparable in both groups. Complications occurred approximately 5 days after trauma. IL-6 increased on day 5, whereas CRP, PCT and leukocytes started to increase on day 6 in the compl-group. Upon arrival at the ED and on days 1 and 4, TAFI levels were significantly lower in the compl-group compared to the wo compl-group (p=0.0215). Similarly, TAFIa was significantly lower on day 4 in the compl-group than in the wo compl-group (p=0.049)., Conclusions: This pilot study shows that TAFI levels are inversely correlated with inflammation-associated development of complications after major trauma., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Regulatory T cells (Tregs) are a specific subset of T lymphocytes that regulate the function of other subsets of lymphocytes. Contradictory results have been reported regarding the role of Tregs in lung fibrosis. We wished to clarify the role of Tregs in the early and late stages of bleomycin-induced lung fibrosis in mice by depleting them with anti-CD25+ antibody (PC61). Mice treated with PC61 in early stages had significantly decreased number of CD4+CD25+ T cells compared to mice treated with the isotype control. The number of inflammatory cells, the concentrations of collagen, TGFβ1, the content of collagen and hydroxyproline in lung tissue were significantly reduced in PC61-treated mice compared to mice treated with the isotype control group. Pathological examination of the lung also disclosed reduced fibrotic changes and decreased fibrosis score in the PC61 group compared to control group. By contrast, mice treated with PC61 in late stages of the disease showed more infiltration of inflammatory cells and higher fibrotic score and hydroxyproline content in the lungs than mice treated with the isotype control. Our results suggest that Tregs play a detrimental role in early stages but protective role in late stages of pulmonary fibrosis in mice., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

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., Objectives: We investigated the therapeutic potential of APC in mice with streptozotocin-induced diabetic nephropathy., Methods: Diabetes was induced in unilaterally nephrectomized C57/Bl6 mice using intraperitoneal (i.p.) injection of streptozotocin. Four weeks later, the mice were treated with i.p. exogenous APC every other day for 1 month., Results: APC-treated mice had a significantly improved blood nitrogen urea-to-creatinine ratio, urine total protein to creatinine ratio and proteinuria, and had significantly less renal fibrosis as measured by the levels of collagen and hydroxyproline. The renal tissue concentration of monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF) and the RNA expression of platelet-derived growth factor (PDGF), transforming growth factor-β1 and connective tissue growth factor (CTGF) were significantly lower in APC-treated mice than in untreated animals. The percentage of apoptotic cells was reduced and the expression of podocin, nephrin and WT-1 in the glomeruli was significantly improved in mice treated with APC compared with untreated mice. The levels of coagulation markers were not affected by APC treatment., Conclusion: Exogenous APC improves renal function and mitigates pathological changes in mice with diabetic nephropathy by suppressing the expression of fibrogenic cytokines, growth factors and apoptosis, suggesting its potential usefulness for the therapy of this disease., (© 2012 International Society on Thrombosis and Haemostasis.)

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., (© 2010 International Society on Thrombosis and Haemostasis.)

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.

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(-1) monocrotaline (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-alpha), interleukin-6 (IL-6), transforming growth factor-beta (TGF-beta) 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.

The rapid increase in the elderly population is leading to a corresponding increase in the number of people requiring medical care. To date no comparative study between community-acquired pneumonia (CAP) and nursing home-acquired pneumonia (NHAP) has been reported in the very elderly non-intubated patients. The present study was undertaken to compare the clinical characteristics and microbial etiology between CAP and NHAP in elderly patients >/=85-years old. There were 54 patients with NHAP and 47 with CAP. Performance status was significantly worse in the NHAP than in the CAP group. Among all patients, the most frequent pathogens were Chlamydophilia pneumoniae followed by Streptococcus pneumoniae, Mycoplasma pneumoniae influenza virus and Staphylococcus aureus. The frequency of S. peumoniae was significantly higher in NHAP patients than in CAP patients after adjusting for age and sex. Physical activity, nutrition status and dehydration were significant prognostic factors of pneumonia among all patients. In-hospital mortality was significantly higher in NHAP than in CAP after adjusting for age and sex. This study demonstrated that the etiology and clinical outcome differ between CAP and NHAP patients in the very elderly non-intubated population., (Copyright 2010 Elsevier Ltd. All rights reserved.)

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-alpha, 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-alpha 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 vitro and 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.

Decreased fibrinolytic function favors the development of pulmonary fibrosis. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a strong suppressor of fibrinolysis, but its role in lung fibrosis is unknown. Therefore, we compared bleomycin-induced lung fibrosis in TAFI-deficient, heterozygous, and wild-type mice. The animals were sacrificed 21 days after bleomycin administration, and markers of lung fibrosis and inflammation were measured. The bronchoalveolar lavage fluid levels of total protein, neutrophil proteases (elastase, myeloperoxidase), cytokines (tumor necrosis factor-alpha, interleukin-13), chemokine (monocyte chemoattractant protein-1), coagulation activation marker (thrombin-antithrombin complex), total soluble collagen, and growth factors (platelet-derived growth factor, transforming growth factor-beta1, granulocytic-macrophage growth factor) were significantly decreased in knockout mice compared to wild-type mice. Further, histological findings of fibrosis, fibrin deposition, and hydroxyproline and collagen content in the lung were significantly decreased in knockout mice compared to wild-type mice. Depletion of fibrinogen by ancrod treatment led to equalization in the amount of fibrosis and collagen deposition in the lungs of knockout and wild-type mice. No difference was detected in body temperature or arterial pressure between the different mouse phenotypes. These results suggest that the anti-fibrinolytic activity of TAFI promotes lung fibrosis by hindering the rate at which fibrin is degraded.

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 microg kg(-1) bolus + 1.5 microg kg(-1) min(-1) infusion, n=6); ZK-807834-Dose 2 (20 microg kg(-1) bolus + 0.75 microg kg(-1) min(-1) infusion; n=6); enoxaparin (1 mg kg(-1) bolus; 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 x 10(6) 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, Ki 0.11 nM) attenuates progression of thrombosis, but the ED50 is 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 micro mol kg-1, n = 6/group) resulted in dose-dependent reductions in clot weight compared with vehicle controls, but the ED50 was 0.03 micro mol kg-1 for non-injured veins and 0.42 micro mol kg-1 for 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.

Urokinase-type plasminogen activator (uPA) is increased in human abdominal aortic aneurysm (AAA). Chronic infusion of angiotensin II (Ang II) results in AAA in apolipoprotein E-deficient mice. We tested the hypothesis that Ang II infusion results in an elevation of uPA expression contributing to aneurysm formation. Ang II or vehicle was infused by osmotic pumps into apoE-KO mice. All mice treated with Ang II developed a localized expansion of the suprarenal aorta (75% increase in outer diameter), accompanied by an elevation of blood pressure (22 mmHg), compared to the vehicle-treated group. Histological examination of the dilated aortic segment revealed similarities to human AAA including focal elastin fragmentation, macrophage infiltration, and intravascular hemorrhage. Ang II treatment resulted in a 13-fold increase in the expression of uPA mRNA in the AAA segment in contrast to a twofold increase in the atherosclerotic aortic arch. Increased uPA protein was detected in the abdominal aorta as early as 10 days after Ang II infusion before significant aorta expansion. Thus, Ang II infusion results in macrophage infiltration, increased uPA activity, and aneurysm formation in the abdominal aorta of apoE-KO mice. These data are consistent with a causal role for uPA in the pathogenesis of AAA.

We have identified a novel human gene, kiap (kidney inhibitor of apoptosis protein) that encodes a single BIR domain and a RING zinc finger domain. kiap has been assigned to the q13.3 region of human chromosome 20 by fluorescent in situ hybridization analysis. Northern blot analysis indicates that KIAP is expressed mainly in placenta, lymph node and fetal kidney. In this report, we show that overexpression of KIAP blocks apoptosis induced by menadione or by overexpression of BAX. In addition, we show that overexpression of KIAP enhances apoptosis induced by etoposide, and, that KIAP fails to block apoptosis induced by overexpression of Fas. Thus, KIAP, a new member of the inhibitor of apoptosis protein (IAP) family, has pleiotropic effects on apoptosis induced by various stimuli.

Structure-function relationships in the 6 epidermal growth factor-like domains of human thrombomodulin (TME, residues 227-462) were studied by deletion mutagenesis. Purified and characterised proteins were used for kinetic studies. Deletion of EGF1, EGF2 and residues 310-332 in EGF3 had no effect on thrombin binding (Kd) or on kcat/KM for protein C activation by the thrombin-thrombomodulin complex. Deletion of the rest of EGF3 and the interdomain loop between EGF3 and EGF4 had no effect on Kd but decreased kcat/KM to 10% of TME. Deletion of residues 447-462 of EGF6 had no effect on kcat/KM but increased Kd for thrombin approximately 6-fold. Thus, the region 333-350 in EGF3-4 is critical for protein C activation by the thrombin-thrombomodulin complex and the region 447-462 in EGF6 is critical for thrombin binding.

Thrombomodulin is an endothelial surface thrombin receptor. Thrombin bound to thrombomodulin loses all procoagulant activity and instead activates the protein C anticoagulant pathway. We developed a recombinant thrombomodulin analog and compared the effects of recombinant thrombomodulin (100 micrograms/ea), saline (controls), recombinant hirudin (1.0 mg/kg), and heparin (100 units/kg) on thrombus formation, activated partial thromboplastin time, and tail transection bleeding time in a rat model of stasis-induced venous thrombosis. Results showed that thrombus was detected in the vena cava in six of the six rats treated with saline solution, in zero of the six rats treated with recombinant thrombomodulin (p less than 0.05), in one of six rats treated with recombinant hirudin (p less than 0.05), and in zero of six rats treated with heparin (p less than 0.05). The activated partial thromboplastin time in rats receiving recombinant thrombomodulin was slightly longer than controls (22 +/- 8 vs 37 +/- 6, p less than 0.05). The bleeding times in rats receiving recombinant thrombomodulin were approximately twice as long as controls (215 +/- 68 vs 545 +/- 173, p = 0.037). In all rats treated with recombinant hirudin or heparin, activated partial thromboplastin times were greater than 120 seconds and bleeding times were greater than 1200 seconds. We conclude that recombinant thrombomodulin inhibits venous thrombosis in a rat model with less prolongation of activated partial thromboplastin time and bleeding time than heparin or hirudin.