Thrombin-activatable fibrinolysis inhibitor (TAFI) is enhanced in major trauma patients without infectious complications.

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.

High incidence of tumors in diabetic thrombin activatable fibrinolysis inhibitor and apolipoprotein E double-deficient mice.

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-ß1 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.

Pulmonary hypertension is ameliorated in mice deficient in thrombin-activatable fibrinolysis inhibitor.

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.

Direct effects of protein S in ameliorating acute lung injury.

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.

Angiotensin II increases urokinase-type plasminogen activator expression and induces aneurysm in the abdominal aorta of apolipoprotein E-deficient mice.

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.

KIAP, a novel member of the inhibitor of apoptosis protein family.

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.

RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides.

S100/calgranulin polypeptides are present at sites of inflammation, likely released by inflammatory cells targeted to such loci by a range of environmental cues. We report here that receptor for AGE (RAGE) is a central cell surface receptor for EN-RAGE (extracellular newly identified RAGE-binding protein) and related members of the S100/calgranulin superfamily. Interaction of EN-RAGEs with cellular RAGE on endothelium, mononuclear phagocytes, and lymphocytes triggers cellular activation, with generation of key proinflammatory mediators. Blockade of EN-RAGE/RAGE quenches delayed-type hypersensitivity and inflammatory colitis in murine models by arresting activation of central signaling pathways and expression of inflammatory gene mediators. These data highlight a novel paradigm in inflammation and identify roles for EN-RAGEs and RAGE in chronic cellular activation and tissue injury.

Corin, a mosaic transmembrane serine protease encoded by a novel cDNA from human heart.

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.

RAGE and amyloid-beta peptide neurotoxicity in Alzheimer's disease.

Amyloid-beta peptide is central to the pathology of Alzheimer's disease, because it is neurotoxic--directly by inducing oxidant stress, and indirectly by activating microglia. A specific cell-surface acceptor site that could focus its effects on target cells has been postulated but not identified. Here we present evidence that the 'receptor for advanced glycation end products' (RAGE) is such a receptor, and that it mediates effects of the peptide on neurons and microglia. Increased expressing of RAGE in Alzheimer's disease brain indicates that it is relevant to the pathogenesis of neuronal dysfunction and death.

Modulation of glycosaminoglycan addition in naturally expressed and recombinant human thrombomodulin.

The two major glycoforms of full-length human thrombomodulin (TM), one with (TM(CS+)) and one without (TM(CS-)) chondroitin sulfate (CS) were analyzed on Western blots of primary and transformed cells and in cells expressing recombinant TM. TM on the surface of Chinese hamster ovary and COS-7 cells is solely TM(CS-). Primary arterial endothelial cells (HAEC and HPAEC) express a greater fraction of TM with CS attached than venous cells (HUVEC). Human lung carcinoma cells (A549) express more TM(CS+) than primary cells and recombinant TM on human melanoma cells (CHL-1) occurs in two very high molecular weight forms of TM(CS+). We explored this variation in TM(CS+) with soluble recombinant TM in several cell lines and analyzed the ambiguous CS addition site in human TM by site-directed mutagenesis. Mutation of Ser474 to Ala blocks CS addition in Chinese hamster ovary and COS-7 cells but not CHL-1 cells which add CS to Ser472 and Ser474. Structure of the O-link domain affects partitioning into TM(CS+) since substituting with the decorin CS addition sequence, substituting all Ser and Thr except Ser474 with Ala, and deleting around the potential beta-turn all increase the ratio of TM(CS+) to TM(CS-). A combination of the decorin substitution and deletion of the remaining O-link domain yields the most TM(CS+).

Expression efficiency of the human thrombomodulin-encoding gene in various vector and host systems.

Expression systems were developed for evaluating recombinant human thrombomodulin (TM) production in different host cell lines by investigating the performance of five mammalian expression vectors. The expression vectors were constructed so that they contain multiple monocistronic gene cassettes which include a gene encoding a dominant selectable marker, HyR (hygromycin B phosphotransferase), under the regulation of the thymidine kinase promoter, the target gene which encodes a truncated human re-TM under the regulation of various promoters, an amplifiable gene (Dhfr) encoding murine dihydrofolate reductase under the regulation of either the SV40 early or late promoter along with the SV40 enhancer and the SV40 ori. We tested the performance of the five expression vectors in human embryonic kidney cells (HEK293), baby hamster kidney cells (BHK), human melanoma cells (CHL-1) and Dhfr- Chinese hamster ovary cells (CHO/Dhfr-). We found that the efficiency of DNA uptake, transient expression and stable expression of the different expression vectors were all cell-line dependent. However, the myeloproliferative sarcoma virus (MPSV) LTR promoter consistently showed higher expression levels in all cell lines, particularly in HEK293 cells. These results were confirmed by the distribution curves of the level of expression of individual clones. Furthermore, by amplifying Dhfr in transfected CHO/Dhfr- cells with 100 nM methotrexate, we achieved a 20-fold increase in re-TM production using the SV40 late promoter to control murine Dhfr expression. Our data from DNA and mRNA analysis reveal that pMPSV-TM has a high transcription efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombomodulin activity and localization.

An overview on the properties, actions and localization of thrombomodulin (TM) in situations of tissue injury and in selected tumors is presented. The localization and activity of TM after injury to vascular endothelium shows that following balloon catheter denudation of the endothelium of the rabbit aorta, the activity and immunohistochemical staining is markedly reduced. The functional and antigenic levels approach the control levels approximately one week after the initial injury. The results suggest that the neointimal smooth muscle cells express TM. This phenotypic plasticity of the neointimal smooth muscle cells may be important in conferring thrombo-resistance to the lumenal lining cells of vessels after injury. Studies are also reviewed on the use of soluble recombinant TM to prevent thrombosis after ligature of vessels in an experimental model. Further characterization on the immunohistochemical distribution of TM in normal tissues and tumors shows that staining with a monoclonal anti TM antibody can be very useful in separating mesotheliomas from pulmonary adenocarcinomas. These studies may lead to insights concerning the role of TM in tissue-injury-repair and tissue differentiation.

Intravenous recombinant soluble human thrombomodulin prevents venous thrombosis in a rat model.

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.

Protein synthesis in human lymphoblastoid cells (Namalwa) after treatment with butyrate and 5'-bromodeoxyuridine.

The pattern of protein synthesis has been compared in Namalwa cells following treatment with butyrate and 5'-bromodeoxyuridine (BrdUrd). Although these treatments cause a substantial increase in Sendai virus-induced interferon synthesis (up to 300-fold) we observed no comparable effect on the synthesis of other cellular proteins. Using two-dimensional gel electrophoresis we have investigated the proteins synthesised before and 8 h after Sendai virus infection of treated cells. Only 2 of the 300 most abundant cellular proteins were reproducibly affected, these always showed increased rates of synthesis in butyrate-treated cells. The most significant was a 3-4-fold enhancement in synthesis of a 35,000 molecular weight protein which we have called BEP35. On individual occasions treatment caused changes in the rates of synthesis of other proteins, these were not reproducible and involved less than 4% of the proteins investigated. None of the Sendai virus structural proteins or virus-induced cellular proteins were affected by the treatment. We conclude that butyrate and BrdUrd treatments have a relatively specific effect on the synthesis of interferon in Namalwa cells, as the majority of protein synthesis remains unaffected.

The effect of hypertonic salt on interferon and interferon mRNA synthesis in human MG63 cells.

After infection with Sendai virus or Newcastle disease virus (NDV) strain F, human osteosarcoma MG63 cells produced large amounts of interferon-beta. Both interferon production and overall protein synthesis were strongly inhibited by hypertonic salt. Interferon mRNA synthesis, however, was little affected by hypertonic salt up to twice normal salt concentrations, although cellular RNA synthesis was inhibited under these conditions. The results are compared to those obtained with polyriboinosinic acid: polyribocytidylic acid copolymer [poly(rI) . poly(rC)] inductions of MG63 cells.

Polypeptides of infectious bronchitis virus. I. Polypeptides of the virion.

Infectious bronchitis virus (IBV), strain Beaudette, grown in cultured cells contained five structural proteins with apparent mol. wt. of 170 000 (p170), 94 000 (gp94), 50 000 (pp50) 30 000 (gp30) and 26 000 (p26). Both gp94 and gp30 are glycopeptides since they were labelled with [3H]glucosamine. The only phosphorylated polypeptide was pp50, and both it and gp94 were occasionally resolved into two bands. Two other polypeptides with mol. wt. of 28 000 (p28) and 14 000 (p14) were sometimes associated with the virus. In egg-grown virus two additional proteins were found with mol. wt. of 110 000 (p110) and 75 000 (gp75). The cell protein, actin, was also found in highly purified IBV virions. Different serotypes of either tissue culture-grown or egg-grown virus showed one of the two distinct polypeptide patterns of IBV described by Nagy & Lomniczi (1979) and Collins & Alexander (1980 a, b). Strain Beaudette gave a pattern characteristic of the M type, while strain Connecticut gave a pattern characteristic of the C type. The polypeptides present in Connecticut virus were p170, gp98, pp50, gp28 and p26. Thus, the differences between the two pattern involve the mobility of both the large (gp94/gp98) and small (gp30/gp28) glycopeptides..

Production and screening of cell hybrids producing a monoclonal antibody to human interferon-alpha.

Mice and rats were immunized with human lymphoblastoid interferon, a mixture of human leukocyte (interferon-alpha) and human fibroblast (interferon-beta) interferons, and their serum anti-interferon levels measured. Anti-interferon activity was detected in all animals, although the mouse sera had higher titers than the rat sera. The spleens of two mice were removed and used to prepare hybrid myelomas. Anti-interferon activity in tissue culture supernatants was measured using a direct neutralization assay and a clone (NK2) producing antibody to human interferon-alpha (leukocyte) was isolated. The anti-interferon activity of this monoclonal antibody was confirmed using a new indirect immunoprecipitation (IIP) assay, which shows some advantages over the direct neutralization assay. The antibody did not neutralize human interferon-beta (fibroblast) or mouse interferon.

Post-translational events in the production of human lymphoblastoid interferon.

Newly synthesized interferon and its mRNA were membrane-associated in a human lymphoblastoid cell (Namalwa) that had been induced with Sendai virus. Treatment with zinc, which acts as an inhibitor of proteolytic cleavage, prevented interferon production. When cytoskeletal function was disrupted by adding both colchicine and cytochalasin B to the induced Namalwa cells, secretion of interferon was inhibited. It is concluded that after translation of the interferon mRNA, the newly synthesized interferon polypeptide is discharged into the lumen of the endoplasmic reticulum, and undergoes a proteolytic cleavage before it is secreted by a process involving the cytoskeleton.

The Xenopus oocyte as a surrogate secretory system. The specificity of protein export.

Combining messenger RNA from one kind of secretory cell with the cytoplasm of another such cell can reveal the nature and specificity of protein export mechanisms. We show that messenger RNAs from secretory cells of chickens, rats, mice, frogs, guinea-pigs, locusts and barley plants, when injected into Xenopus oocytes, direct the synthesis and export of proteins. Chicken ovalbumin, Xenopus albumin, mouse thyroid-stimulating hormone, locust vitellin and guinea-pig milk proteins were identified using specific antibodies, whilst chicken lysozyme and ovomucoid, rat albumin, Xenopus vitellogenin and rat seminal vesicle basic proteins were identified provisionally from their molecular weights. Certain endogenous proteins are sequestered and secreted although most oocyte proteins are not exported. Similarly the major polyoma viral protein and the simian virus 40 and polyoma tumour antigens are retained within the oocyte. Radioactive proteins exported by oocytes programmed with chicken oviduct or Xenopus liver RNA are not re-exported in detectable amounts when injected into fresh oocytes, nor is there secretion of chicken oviduct or guinea-pig mammary gland primary translation products prepared using wheat germ extracts. Thus the export of secretory proteins from oocytes cannot be explained by leakage and may require a cotranslational event. The secretory system of the oocyte is neither cell-type nor species-specific yet is highly selective. We suggest that the oocyte can be used as a general surrogate system for the study of gene expression, from transcription through translation to the final subcellular or extracellular destination of the processed protein.