Angiostatic effects of NK cell-derived IFN-γ counteracted by tumour cell Bcl-xL expression.

Anti-apoptotic proteins that block death receptor-mediated apoptosis favour tumour evasion of the immune system, leading to enhanced tumour progression. However, it is unclear whether blocking the mitochondrial pathway of apoptosis will protect tumours from immune cell attack. Here, we report that the anti-apoptotic protein Bcl-xL , known for its ability to block the mitochondrial pathway of apoptosis, exerted tumour-progressive activity in a murine lymphoma model. Bcl-xL overexpressing tumours exhibited a more aggressive development than control tumours. Surprisingly, Bcl-xL protection of tumours from NK cell-mediated attack did not involve protection from NK cell-mediated cytotoxicity. Instead, Bcl-xL -blocked apoptosis resulting from hypoxia and/or nutrient loss associated with the inhibition of angiogenesis caused by NK cell-secreted IFN-γ. These results support the notion that NK cells may inhibit tumour growth also by mechanisms other than direct cytotoxicity. Hence, the present results unravel a pathway by which tumours with a block in the mitochondrial pathway of apoptosis can evade the immune system.

Biosynthesis of the vitamin K-dependent matrix Gla protein (MGP) in chondrocytes: a fetuin-MGP protein complex is assembled in vesicles shed from normal but not from osteoarthritic chondrocytes.

OBJECTIVE: Mineralization has been observed in osteoarthritic cartilage but the mechanisms are incompletely understood. Vitamin K is an essential cofactor in post-translational modification of proteins where specific Glu residues become modified to Ca(++) binding gamma-carboxyglutamic acid residues (Gla). One such protein, matrix Gla protein (MGP), is a known mineralization inhibitor. This study determined if synthesis of MGP and formation of a fetuin-MGP protein complex was altered in chondrocytes and vesicles from osteoarthritis (OA) cartilage. METHODS: Chondrocytes and vesicles were isolated from normal and OA human articular cartilage and lysates prepared. Specific antibodies were used in immunoblotting to detect the mature fully gamma-carboxylated form of MGP (cMGP) and non-gamma-carboxylated MGP (ucMGP) as well as fetuin and MGP-fetuin complexes. gamma-carboxylase activity was measured by (14)CO(2) incorporation into the carboxylase peptide substrate FLEEL. Immunocytochemistry was used to examine fetuin in cartilage sections and uptake of biotin-labeled fetuin by isolated chondrocytes. RESULTS: Chondrocytes and vesicles from osteoarthritic tissue produced significantly less cMGP compared to those from normal cartilage. This correlated with significantly less vitamin K-dependent gamma-carboxylase enzyme activity in OA chondrocytes. Fetuin was found to be present in articular cartilage and cultured chondrocytes were capable of fetuin uptake. A fetuin-MGP complex was identified in normal chondrocytes and in vesicles shed from these cells but not in OA cells or vesicles. CONCLUSIONS: The absence of cMGP and of the cMGP-fetuin complex in OA cells and OA vesicles may be an important mechanism for increased mineralization of osteoarthritic cartilage.

Vitamin K-dependent carboxylation and vitamin K metabolism in liver. Effects of warfarin.

The systems involved in vitamin K-dependent carboxylation and vitamin K metabolism have been extensively studied in rat liver. To determine how clinically applicable this information is, similar in vitro studies were completed using human liver. One major difference exists in the pathways that provide reduced vitamin K1 cofactor for the carboxylation reaction. The coumarin-sensitive DT-diaphorase (EC.1.6.99.2) in human liver appears to play a relatively minor role in the dehydrogenase pathway. However, similar to rat liver, the human liver contains a warfarin-insensitive enzyme in this dehydrogenase pathway. The data suggest that this enzyme is responsible for the antidotic effect of vitamin K1 in cases of coumarin intoxication. Human vitamin K epoxide reductase, which constitutes the other pathway for vitamin K1 reduction, has kinetic and enzymological characteristics that are very similar to the rat enzyme. This enzyme exhibited similar activity in rat and human microsomes. Initial velocities for vitamin K1 epoxide reduction in rat and human microsomes were 20 and 32 pmol/mg X min, respectively. The human enzyme is highly sensitive to warfarin inhibition. The mechanism for this inhibition appears to be similar to what has been proposed for the rat enzyme. Also, a vitamin K-dependent carboxylation system is described that allows both pathways to support the carboxylation reaction with reduced vitamin K1 cofactor. The effect of warfarin on this in vitro system is consistent with the current model for the mechanism of action of coumarin anticoagulant drugs in the rat.

The N-terminal activation fragment of bovine prothrombin. Immunological studies leading to a one step purification.

Some immunological studies on prothrombin fragment 1 from bovine prothrombin and its warfarin-induced precursor acarboxyprothrombin are reported. Based on the results, a rapid and simple immunoadsorption method for the isolation of prothrombin fragment 1 in good yield has been established. The method exploits the conformational change induced in the fragment by removal of Ca2+. The principle may be applicable to other gamma-carboxyglutamyl-containing proteins or fragments therof.

Structural requirements for microvascular permeability-increasing ability of peptides. Studies on analogues of a fibrinogen pentapeptide fragment.

A pentapeptide, Ala-Arg-Pro-Ala-Lys, liberated from fibrinogen during plasmin-mediated fibrinolysis, was shown earlier to increase microvascular permeability in rat and human skin. Eighteen new analogues have now been synthesized in addition to the 15 previously prepared and examined for their effect on permeability. The old concept that a tetrapeptide with basic amino acids at both ends and a proline residue adjacent to the N-terminal amino acid is essential for high activity on permeability, has now been challenged. The results obtained with several of the new analogues strengthen this concept. More interestingly, however, the third amino acid, which was found in earlier studies to be less sensitive to exchange, has now been deleted as well as duplicated with only a modest loss of activity of the peptide. The chirality of the C-terminal amino acid, most surprisingly, does not seem to be crucial for peptide activity. Slightly superpotent analogues were obtained on amidation of the C-terminus. In addition, a few naturally occurring peptides, namely tuftsin, substance P, neurotensin and bradykinin, the amino acid sequences of which all exhibit characteristic features of some of our active peptide analogues were investigated in the same test system. Tuftsin displayed a potency equal to that of the pentapeptide. The other three peptides were all highly superpotent in this assay system.

Structure-activity studies on synthetic analogs to vasoactive peptides derived from human fibrinogen.

Counterparts to two vasoactive peptides previously isolated from fibrin(ogen) degraded by plasmin (EC 3.4.21.7) were synthesized by the solid phase procedure. The synthetic undecapeptide (Ser-Gln-Leu-Gln-Lys-Val-Pro-Pro-Glu-Trp-Lys) was isolated in a homogeneous state by chromatography on Sephadex G-25 and DEAE-Sepharose CL-6B and the pentapeptide (Ala-Arg-Pro-Ala-Lys) by chromatography on BioGel P-6 and column zone electrophoresis. The effect of these two peptides and of fifteen analogs to the pentapeptide on microvascular permeability in rat skin was investigated. The two synthetic counterparts were as potent as the natural peptides. With respect to the analogs, the influence of different functional groups was first studied. This was followed by attempts to minimize the active structure, induce or relieve rigidity of the peptide back-bone or otherwise accomplish modifications by a change in chirality at critical positions. Our results show that the tetrapeptide Arg-Pro-Ala-Lys has the same effect on microvascular permeability as the pentapeptide in the assay system used. Basic amino acids at both ends, as well as a proline residue adjacent to the N-terminal amino acid appear important for full or essentially full activity. On the other hand, substitution of the Ala at position 4 with several other amino acids did not result in a significant loss in biological potency.

A novel protein kinase target for the lipid second messenger phosphatidic acid.

Activation of phospholipase D occurs in response to a wide variety of hormones, growth factors, and other extracellular signals. The initial product of phospholipase D, phosphatidic acid (PA), is thought to serve a signaling function, but the intracellular targets for this lipid second messenger are not clearly identified. The production of PA in human neutrophils is closely correlated with the activation of NADPH oxidase, the enzyme responsible for the respiratory burst. We have developed a cell-free system, in which the activation of NADPH oxidase is induced by the addition of PA. Characterization of this system revealed that a multi-functional cytosolic protein kinase was a target for PA, and that two NADPH oxidase components were substrates for the enzyme. Partial purification of the PA-activated protein kinase separated the enzyme from known protein kinase targets of PA. The partially purified enzyme was selectively activated by PA, compared to other phospholipids, and phosphorylated the oxidase component p47-phox on both serine and tyrosine residues. PA-activated protein kinase activity was present in a variety of hematopoietic cells and cell lines and in rat brain, suggesting it has widespread distribution. We conclude that this protein kinase may be a novel target for the second messenger function of PA.

Increased neutrophil elastase release in unstable angina pectoris and acute myocardial infarction.

Neutrophils, a source of proteolytic enzymes and oxygen free radicals, have been shown to participate in animal models of myocardial ischemic injury. To characterize neutrophil activation in human ischemic heart disease, a specific neutrophil elastase-derived fibrinopeptide in plasma was measured in 25 patients with stable angina pectoris, 29 patients with unstable angina pectoris, 17 patients with acute myocardial infarction and 22 control subjects. Mean plasma levels (+/- standard error) of a neutrophil elastase-derived fibrinopeptide (B beta 30-43) measured by a specific radioimmunoassay were fivefold higher in patients with acute myocardial infarction (877 +/- 337 pmol/liter, p less than 0.02) and 13-fold higher in patients with unstable angina (2,277 +/- 613 pmol/liter, p less than 0.006) as compared with control subjects (172 +/- 74 pmol/liter). Mean plasma levels of peptide B beta 30-43 in patients with stable angina (676 +/- 334 pmol/liter), although higher than in control subjects, were not significantly increased (p = 0.64). Total leukocyte counts were 11.0 +/- 0.6 x 10(6)/ml in those with acute myocardial infarction, 9.2 +/- 0.7 x 10(6)/ml in those with unstable angina, 7.1 +/- 0.3 x 10(6)/ml in those with stable angina and 7.7 +/- 0.4 x 10(6)/ml in control subjects. Although total leukocyte counts in patients with unstable angina pectoris and acute myocardial infarction were higher (p less than 0.01) than in patients with stable angina or in control subjects, elevations in peptide B beta 30-43 levels were independent of the differences in both leukocyte count and absolute neutrophil count as well as in history of smoking, hypertension, diabetes mellitus or treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

A molecular mechanism for genetic warfarin resistance in the rat.

Warfarin targets vitamin K 2,3-epoxide reductase (VKOR), the enzyme that produces reduced vitamin K, a required cofactor for g-carboxylation of vitamin K-dependent proteins. To identify VKOR, we used 4'-azido-warfarin-3H-alcohol as an affinity label. When added to a partially purified preparation of VKOR, two proteins were identified by mass spectrometry as calumenin and cytochrome B5. Rat calumenin was cloned and sequenced and the recombinant protein was produced. When added to an in vitro test system, the 47 kDa recombinant protein was found to inhibit VKOR activity and to protect the enzyme from warfarin inhibition. Calumenin was also shown to inhibit the overall activity of the complete vitamin K-dependent g-carboxylation system. The results were repeated in COS-1 cells overexpressing recombinant calumenin. By comparing calumenin mRNA levels in various tissues from normal rats and warfarin-resistant rats, only the livers from resistant rats were different from normal rats by showing increased levels. Partially purified VKOR from resistant and normal rat livers showed no differences in Km-values, specific activity, and sensitivity to warfarin. A novel model for genetic warfarin resistance in the rat is proposed, whereby the concentration of calumenin in liver determines resistance.

cAMP-elevating agents suppress dendritic cell function.

The administration of cAMP-elevating agents affects a number of autoimmune and inflammatory conditions. Because dendritic cells (DCs) play a pivotal role in autoimmunity and inflammation, the isolated effects of cAMP-elevating agents on the function of DCs was examined. In a dose-dependent manner, 8-Bromo cAMP, prostaglandin E(2), and 3-isobutyl-1-methylxanthine inhibited tumor necrosis factor alpha release and suppressed antigen presentation by DCs. The same effect was observed with rolipram, a specific inhibitor of phosphodiesterase type 4, but not with inhibitors of other phosphodiesterases. The decreased antigen presentation by DCs was associated with an enhanced production of interleukin (IL)-10 and with lower major histocompatibility complex type II (MHC II) expression. Furthermore, the inhibition of antigen presentation and MHC II expression was significantly reversed by treatment of DCs with neutralizing antibody against IL-10, suggesting the involvement of an IL-10-dependent mechanism. Taken together, these results might explain why certain cAMP-elevating agents such as rolipram are effective in blocking autoimmunity and inflammation.

Aspirin does not potentiate effect of suboptimal dose of the thrombin inhibitor inogatran during coronary thrombolysis.

OBJECTIVES: Coronary artery often reoccludes after thrombolytic therapy with recombinant tissue-plasminogen activator (rt-PA). This reocclusion is thought to be due to in situ platelet activation mediated by thromboxane (Tx) A2 and thrombin; hence, aspirin and thrombin inhibitors are often used in patients with acute myocardial infarction. This study was designed to examine the modulation of coronary artery reocclusion by a novel low molecular weight direct thrombin inhibitor inogatran with or without aspirin. METHODS: 22 dogs with electrically-induced occlusive intracoronary thrombus were treated with saline (n = 7, group A), or high dose inogatran (0.25 mg/kg bolus followed by 0.6 mg/kg per h for 2 h, n = 5, group B), or low dose inogatran (0.125 mg/kg bolus followed by 0.3 mg/kg per h for 2 h, n = 5, group C), or aspirin+low dose inogatran (n = 5, Group D). Recombinant tissue-plasminogen activator (rt-PA) was infused for 20 min starting 2 min after the bolus in all dogs. Coronary artery blood flow was monitored for 120 min after rt-PA administration. RESULTS: Reperfusion rates were similar in all groups, but the time to reperfusion was shortest in group B dogs (18 +/- 2 min vs. 32 +/- 7 min in group A dogs, P < 0.05). Reocclusion rates were 80%, 0%, 50%, and 60% in groups A, B, C, and D dogs, respectively. The restored blood flow persisted for 19 +/- 10, > 120 min, 71 +/- 30 and 54 +/- 26 min in groups A, B, C, and D dogs, respectively. At the end of rt-PA infusion, prothrombin time (PT) and activated partial thromboplastin time (APTT) were increased 1.3-2 times the control value, and the changes in PT and APTT were similar in all groups. Thrombin generation and activity, assessed by rise in thrombin-antithrombin complex and fibrinopeptide A levels, and decrease in fibrinogen levels were most marked in group A dogs, and less so in group B, C and D dogs. CONCLUSIONS: These data show that high dose of direct thrombin inhibitor inogatran shortens time to reflow and abolishes coronary artery reocclusion. However, aspirin does not potentiate the effect of suboptimal doses of inogatran.

Processing and expression of rat and human clotting factor-X-encoding cDNAs.

The cDNA encoding clotting factor X, which participates in the middle stage of the blood coagulation cascade was cloned from a rat liver cDNA library. Sequencing of the rat factor-X-encoding cDNA revealed that this vitamin-K-dependent protein has a dibasic Arg-Arg sequence at the propeptide cleavage site, as occurs in other vitamin-K-dependent proteins. Although the human and rat deduced amino acid sequences are remarkably similar (76% identical), they do significantly differ in that human factor-X contains a unique Thr-Arg sequence at the propeptide cleavage site [Fung et al., Proc. Natl. Acad. Sci. USA 82 (1985) 3591-3595], where a dibasic sequence would normally be expected. This specific site is the recognition motif for the endoprotease, furin, which is located in the Golgi apparatus. Both rat and human cDNAs expressed in Cos-1 cells resulted in secretion of a mixture of single- and two-chain forms of factor X. The two-chain forms were devoid of the propeptide and were produced at similar rates by the transfected cells. The efficient processing of human factor X, when compared to rat factor X, may indicate that an additional protease(s), which recognizes the Thr-Arg motif, may be involved in proteolytic processing of the human enzyme.

Matrix Gla protein (MGP) and bone morphogenetic protein-2 in aortic calcified lesions of aging rats.

The vitamin K-dependent protein, matrix Gla protein (MGP) is a binding protein for bone morphogenetic protein-2 (BMP-2). Here we present additional evidence that the Ca2+-induced conformer of the vitamin K-dependent Gla region in MGP is involved in BMP-2 binding. Recombinant BMP-2 binds to the Gla-containing region of MGP in the presence of Ca2+. Immunohistochemistry showed that calcified lesions in the aortic wall of aging rats contained elevated concentrations of MGP that was poorly gamma-carboxylated and did not bind BMP-2. In contrast, we were able to identify glandular structures in the mucosa of the rat nasal septum that gave bright fluorescent signals with both antigens; confocal microscopy confirmed their colocalization. These results demonstrate that the BMP-2/MGP complex exists in vivo, consistent with a role for MGP as a BMP-2 inhibitor. Age-related arterial calcification may be a consequence of under-gamma-carboxylation of MGP, allowing unopposed BMP-2 activity.

Studies on a subcellular system for vitamin K-dependent carboxylation.

The subcellular localization of acarboxyprothrombin and the vitamin K-dependent carboxylation system has been studied using livers from vitamin K-deficient rats and found to be mainly in the membranes of the rough microsomal fraction. Bovine acarboxyprothrombin or fragment 1 thereof were inactive as substrates in the rat carboxylating system.

Synthesis of vitamin K-dependent proteins by cultured human tumor cells.

The observation that warfarin inhibits the growth and metastasis of certain types of clinical and experimental tumors suggests a role for vitamin K in tumor biology. We have investigated synthesis of vitamin K-dependent proteins in four malignant (lung epidermoid carcinoma, melanoma, colon adenocarcinoma, and breast adenocarcinoma) and three normal (colon epithelium, breast epithelium, and fibroblasts) cell lines of human origin grown in tissue cultures. Our results show the following: 1) Vitamin K-dependent carboxylase activity is present in all of the malignant and normal cell lines studied. 2) The malignant as well as normal cell lines synthesize a family of vitamin K-dependent proteins. Microsomal precursors of these proteins with apparent molecular mass of 74, 62, and 34 kDa are common to all malignant and normal cell lines whereas precursors of higher and lower molecular mass seem to be synthesized by some but not all tumor cell lines. 3) The 74 kDa precursor synthesized by colon carcinoma and breast carcinoma was positively identified as a precursor of protein S.

Purification of a fibrinolysis inhibitor in serum from post-traumatic patients.

A fibrinolysis inhibitor was purified in serum from post-traumatic patients by the use of flat bed electrofocusing of serum desalted by gel chromatography followed by affinity chromatography on a column of matrix-linked plasminogen. Disc gel electrophoresis yielded one protein band. The inhibitor protein was also found in normal serum, but in a lower concentration.

Warfarin resistance is associated with a protein component of the vitamin K 2,3-epoxide reductase enzyme complex in rat liver.

Warfarin, the most used drug in the world in long-term anticoagulation prophylaxis, targets the vitamin K 2,3-epoxide reductase (VKOR) of the vitamin K cycle in liver. Recently, the enzyme has been identified as a multicomponent lipid-protein enzyme system in the endoplasmic reticulum (ER) membrane (17). As the first step towards understanding genetic resistance to warfarin, we present in this paper data on VKOR from normal and a strain of warfarin resistant laboratory rats maintained in the United States. Metal induced in vitro assembly of the enzyme complex demonstrates that the glutathione-S-transferase (GST) enzyme component of the complex loses its GST activity upon formation of VKOR. Less VKOR activity is measured upon assembly of the complex from warfarin resistant rats. The GST activity measured in warfarin resistant rats, before assembly of the complex, is 10-fold less sensitive to warfarin inhibition than the GST activity measured in normal rats. Microsomal epoxide hydrolase (mEH) is the second component of VKOR. When incubated with the components of VKOR before assembly of the complex, antibodies raised against mEH prevented formation of the enzyme complex. Sequencing of mEH cDNAs from normal and warfarin resistant rats revealed identical sequences. The data suggest that the mutation responsible for genetic warfarin resistance is associated with the GST component of VKOR.

Matrix Gla protein synthesis and gamma-carboxylation in the aortic vessel wall and proliferating vascular smooth muscle cells--a cell system which resembles the system in bone cells.

Matrix GLA protein (MGP) is an inhibitor of calcification in the arterial wall and its activity is dependent upon vitamin K-dependent gamma-carboxylation. This modification is carried out by a warfarin sensitive enzyme system that converts specific Glu residues to gamma-carboxyglutamic acid (GLA) residues. Recent studies have demonstrated that the gamma-carboxylation system in the arterial wall, in contrast to that in the liver, is unable to use vitamin K as an antidote to warfarin. By use of immunohistochemistry we demonstrate that MGP is expressed in the arterial wall and immunocytochemistry localized the MGP precursors to the endoplasmic reticulum in vascular smooth muscle cells. Resting smooth vascular muscle cells in the aortic wall and proliferating cells from explants of the aorta have all the enzymes needed for gamma-carboxylation of MGP. However, when compared to the liver system, expression of the enzymes of the gamma-carboxylation system in vascular smooth muscle cells is different. Of particular interest is the finding that the specific activity of the warfarin sensitive enzyme vitamin K epoxide reductase is 3-fold higher in vascular smooth muscle cells than in liver. DT-diaphorase, which catalyses the antidotal pathway for vitamin K reduction in liver, is 100-fold less active in resting vascular smooth muscle cells than in liver. Data obtained from an in vitro gamma-carboxylation system suggest that the antidotal pathway catalyzed by DT-diaphorase in the vessel wall is unable to provide the carboxylase with enough reduced vitamin K to trigger gamma-carboxylation of MGP. This finding provides an explanation to the inability of vitamin K to work as an antidote to warfarin intoxication of the arterial wall. Therefore the vitamin K dependent gamma-carboxylation system in the arterial wall share a common feature with the system in bone cells by being unable to utilize vitamin K as an antidote.

Vitamin K antagonism of coumarin intoxication in the rat.

An in vitro system which expresses all enzyme activities related to vitamin K-dependent carboxylation of blood clotting factors was prepared from livers of rats overdosed with warfarin, difenacoum and dicumarol respectively. In this system, the activities of the two pathways that are known to produce active reduced vitamin K1 cofactor for the carboxylation reaction were measured. Also the ability of high concentrations of vitamin K1 to overcome inhibition of clotting factor synthesis was studied. In the systems prepared from livers of warfarin and difenacoum intoxicated rats, pathway I was inactive. Vitamin K epoxide reductase was also inactive which strongly suggests that this enzyme catalyzes the activity of pathway I in vivo. Reduction of vitamin K1 by pathway II bypassed the inactive pathway I and resulted in carboxylation activity. This pathway therefore mediates the antidotic effect of vitamin K1 in the coumarin intoxicated liver. In the in vitro system prepared from dicumarol intoxicated livers the activity of pathway I was not significantly affected. Dicumarol however was a strong inhibitor when added to liver microsomes in vitro.

Identification of mRNA coding for factor VII protein in human alveolar macrophages--coagulant expression may be limited due to deficient postribosomal processing.

Clotting factors synthesized by monocytes and macrophages may initiate coagulation reactions during inflammation. Functional vitamin K-dependent coagulation factors have been found to be associated with human monocytes/macrophages, but there are no reports identifying mRNA coding for vitamin K-dependent proteins in these cells. In the present studies, factor VII mRNA was found in total RNA extracted from freshly isolated human alveolar macrophages using hybridization with a complementary DNA probe. On the other hand, vitamin K-dependent carboxylase activity which is required for postribosomal modification of the protein, was not detectable in the macrophages before or after culture, and human blood mononuclear leukocytes also lacked this enzyme activity. Control human and rat hepatoma cells exhibited high levels of carboxylase activity within the same experiments. Using sensitive kinetic assays, no increase in factor VII activity was detected during culture of alveolar macrophages under conditions promoting 1.78 +/- .24 (n = 8) fold increases of tissue factor activity. These findings with freshly isolated cells demonstrate that alveolar macrophages synthesize factor VII mRNA in vivo. However, the mRNA was found in the absence of evidence for gamma-carboxylase activity or processing of the factor into a functional clotting enzyme. The results imply that functional expression of any synthesized coagulation factor VII in alveolar macrophages may be limited or prevented due to a cellular deficiency at the level of postribosomal processing.