A mechanism for NaCl inhibition of Reactive Blue 19 decolorization and ABTS oxidation by laccase.

Laccases produced by white rot fungi have been extensively evaluated for their potential to decolorize textile wastewaters which contain salts like sodium chloride and sodium sulfate. The effect of sodium chloride and sodium sulfate on Trametes versicolor laccase during the decolorization of an anthraquinone dye (Reactive Blue 19) and the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were evaluated by steady-state kinetic analysis. The results showed that, while sodium sulfate did not affect laccase activity, sodium chloride inhibited both ABTS oxidation and dye decolorization. However, the type of inhibition was substrate-dependent: it was hyperbolic, noncompetitive with ABTS and parabolic, noncompetitive with Reactive Blue 19. Furthermore, the results suggested that two chlorides may bind to laccase in the presence of the dye unlike recent inhibition models which suggest that there is only one inhibition site. This investigation is the first to provide evidence for and to propose a two-site model of laccase inhibition, providing new insight into NaCl inhibition of laccase. The proposed model is also useful to predict decolorization rates in the presence of sodium chloride and to determine operating conditions that will minimize inhibition.

Thrombin activatable fibrinolysis inhibitor activation and bleeding in haemophilia A.

Individuals with haemophilia A exhibit bleeding tendencies that are not always predicted by their factor (F)VIII level. It has been suggested that bleeding in haemophilia is due not only to defective prothrombin activation but also aberrant fibrinolysis. Thrombin activatable fibrinolysis inhibitor (TAFI) activation was measured in tissue factor (TF)-initiated blood coagulation in blood samples of 28 haemophiliacs and five controls. Reactions were quenched over time with FPRck and citrate and assayed for TAFIa and thrombin-antithrombin (TAT). The TAFIa potential (TP), TAFI activation rate and the TAFIa level at 20 min (TAFIa(20 min)) was extracted from the TAFI activation progress curve. In general, the time course of TAFI activation follows thrombin generation regardless of FVIII activity and as expected the rate of TAFI activation and TP decreases as FVIII decreases. The magnitude of TP was similar among the control subjects and subjects with <11% FVIII. In severe subjects with <1% FVIII at the time of blood collection, the TAFIa(20 min) was inversely and significantly correlated with haemarthrosis (-0.77, P = 0.03) and total bleeds (-0.75, P = 0.03). In all cases, TAFIa(20 min) was more strongly correlated with bleeding than TAT levels at 20 min. Overall, this study shows that TAFI activation in whole blood can be quantified and related to the clinical bleeding phenotype. Measuring TAFIa along with thrombin generation can potentially be useful to evaluate the differential bleeding phenotype in haemophilia A.

The potential for grass-fed livestock: resource constraints.

Using pasture and grazed forest-range for a system of producing live-stock by feeding grass alone reduces the inputs of energy about 60 percent and land resources about 8 percent, but also reduces by about half the production of animal protein in the United States. Under a system in which only grass was fed, livestock would be restricted to beef, milk, and lamb production. The amount of grain fed to U.S. livestock is about 135 million tons (metric) or about ten times the amount consumed by the U.S. population.

Studies of Factors V and VIII:C in an animal model of disseminated intravascular coagulation.

An experimental animal model of disseminated intravascular coagulation (DIC) induced by the co-infusion of coagulant-active phospholipid and activated Factor X (Factor Xa) is described. The infusion of Factor Xa at a dose of 6.6 X 10(-12) mol/kg with phosphatidylcholine/phosphatidylserine (PCPS) lipid vesicles at a dose of 4.0 X 10(-8) mol/kg was associated with significant falls in the levels of fibrinogen and Factors V and VIII, and a bleeding diathesis developed. Assays of Factors V and VIII were performed by a one-stage prothrombin time and activated partial thrombin time system, respectively. In additional experiments, the effect of the same dose combination of Factor Xa/PCPS on Factor V kinetics was studied by preinfusing 125I-labeled Factor V. After Factor Xa/PCPS infusion, Factors VIII and V were reduced at 2 min by 90 and 50% of the preinfusion levels, respectively, and at 1 h by 80 and 75%, respectively. During the same period, there was little change in the total circulating radioactivity. Autoradiography indicated small but detectable levels of circulating proteolytic products of Factor V that comigrated with peptides obtained by the incubation of Factor V with Factor Xa and activated protein C. The majority of radioactivity remained associated with the intact single-chain precursor Factor V. These observations suggested maintenance of the precursor pool after the onset of DIC. This was confirmed by performing two-stage assays of Factors V and VIII, whereby each was completely converted to the active cofactor, i.e., Va and VIII:Ca, by preincubation of the test sample with thrombin before assaying in a one-stage system as before. The Factor V levels assayed by the two-stage procedure did not change appreciably over 1 h. The Factor VIII levels fell but corrected within 1 h at a time when the level measured by a one-stage assay remained depressed. These results indicate that in the dog, infusion of Factor Xa/PCPS induces changes characteristic of DIC, and this is associated with the appearance of Factor V peptides characteristic of the expression of Factor Xa and activated protein C-like activities. The differences noted between the one-stage and two-stage assays suggest that the one-stage assay is measuring the activated fraction of each cofactor and not the total level of the available precursor for each activated species. The results suggest a close correlation between the activated fraction of both cofactors and the hemostatic abnormality that occurs in DIC.

Circulating heparan sulfate proteoglycan anticoagulant from a patient with a plasma cell disorder.

A woman, aged 68, with multiple myeloma (immunoglobulin[Ig]A kappa type) developed an anticoagulant with properties suggestive of heparin. The anticoagulant prolonged the thrombin time but not the reptilase time and was resistant to boiling, proteolytic enzyme digestion, and trichloracetic acid precipitation. The thrombin time was corrected by the addition (in vitro) of protamine sulfate or the addition of purified platelet Factor 4 (PF4) to the plasma. The anticoagulant was isolated by PF4-Sepharose affinity chromatography and analyzed in terms of its molecular weight, uronic acid, and amino acid composition. The proteoglycan isolated had a mol wt of 116,000 and appears to consist of two 38,000 dalton polysaccharide units interconnected by peptide material totaling 39,000 daltons. Electrophoretic analysis of the pronase digested peptidoglycan using the lithium acetate-agarose technique suggested the material was of the heparan sulfate type. The peptidoglycan had about one-tenth the specific activity of commercially available heparin on a weight basis. The isolated proteoglycan was indistinguishable from commercial heparin when analyzed in terms of its ability to act as a cofactor in the antithrombin III inhibition of thrombin.

Identification of a congenital dysthrombin, thrombin Quick.

A dysprothrombin designated prothrombin Quick, is isolated from the plasma of an individual with < 2% of normal functional prothrombin activity and 34% of the normal prothrombin level by immunologic assay. With Factor Xa or taipan snake venom as activators, a fragmentation pattern identical to that of normal prothrombin is observed on gel electrophoresis in dodecylsulfate. This evidence combined with the observed barium citrate adsorption of prothrombin Quick and the low activity suggests that the defect in prothrombin Quick is in the thrombin portion of the molecule. Thrombin Quick is isolated and comigrates with thrombin on dodecyl sulfate gel electrophoresis, either reduced or nonreduced. The activity of thrombin Quick on several biological substrates of thrombin is investigated. Relative to normal thrombin, thrombin Quick is 1/200 as active on fibrinogen and 1/20-1/50 as effective in activating Factors V and VIII and aggregating platelets. A complex with antithrombin III is detected by dodecyl sulfate gel electrophoresis. Further investigation with the active site titrant, dansylanginine-N-(3-ethyl-1,5-pentanediyl)amide showed that the thrombin Quick preparation has the same affinity for the titrant as thrombin, but apparently only 40% active sites per mole protein are titrable.

Studies of thrombin-induced proteoglycan release in the degradation of human and bovine cartilage.

Because fibrin is commonly observed within arthritic joints, studies were undertaken to determine whether purified coagulation and fibrinolytic proteases degrade cartilage in vitro and to seek evidence for the activation of coagulation in arthritic joints through measurements of the levels of inhibitor-enzyme complexes and several other proteins associated with coagulation and fibrinolysis. The concentrations of 13 plasma proteins and complexes of thrombin and Factor Xa with antithrombin III were measured in synovial fluids recovered at the time of knee replacement surgery. All zymogens necessary to constitute the coagulation cascade were present. Thrombin and the combination of prothrombin plus prothrombinase induced proteoglycan release from both normal and arthritic cartilages. Factor Xa and plasmin induced release from diseased cartilage only, and urokinase, tissue plasminogen activator, and activated protein C were without effect at the levels used. At saturating levels of thrombin (> or = 2.0 microM) 80% of the proteoglycan content of normal cartilage was released within 24 h. Thrombin, which is cationic, reversibly binds cartilage with Kd = 7.0 +/- 1.0 microM and Bmax = 820 +/- 70 ng/mg of human cartilage. Levels of thrombin-antithrombin III complexes in synovial fluids and arthritis were 4-fold higher in osteo (OA) and 43-fold higher in rheumatoid (RA) than in controls (0.98 nM). Factor Xa-antithrombin III complex levels were threefold lower in OA and fivefold higher in RA than in controls (0.24 nM). These elevated levels of enzyme-inhibitor complexes imply a history of activation of coagulation within the joint, especially in RA. Since thrombin degrades cartilage in vitro and had been generated in vivo, as inferred by the existence of thrombin-antithrombin III complexes, intraarticular activation of coagulation may both contribute to the pathology of arthritis and comprise a target for therapy and diagnosis.

Thrombin-mediated activation of factor XI results in a thrombin-activatable fibrinolysis inhibitor-dependent inhibition of fibrinolysis.

Recently, it has been shown that Factor XI can be activated by thrombin, and that Factor XIa significantly contributes to the generation of thrombin via the intrinsic pathway after the clot has been formed. This additional thrombin, generated inside the clot, was found to protect the clot from fibrinolysis. A plausible mechanism for this inhibitory effect of thrombin involves TAFI (thrombin-activatable fibrinolysis inhibitor, procarboxypeptidase B) which, upon activation, may inhibit fibrinolysis by removing carboxy-terminal lysines from fibrin. We studied the role of Factor XI and TAFI in fibrinolysis using a clot lysis assay. The lysis time was decreased twofold when TAFI was absent, when TAFI activation was inhibited by anti-TAFI antibodies, or when activated TAFI was inhibited by the competitive inhibitor (2-guanidinoethylmercapto)succinic acid. Inhibition of either TAFI activation or Factor XIa exhibited equivalent profibrinolytic effects. In the absence of TAFI, no additional effect of anti-Factor XI was observed on the rate of clot lysis. We conclude that the mechanism of Factor XI-dependent inhibition of fibrinolysis is through the generation of thrombin via the intrinsic pathway, and is dependent upon TAFI. This pathway may play a role in determining the fate of in vivo formed clots.

Isolation and study of an acquired inhibitor of human coagulation factor V.

A coagulation Factor V inhibitor developed in a man 75 yr of age in association with an anaplastic malignancy and drug treatment (including the aminoglycoside antibiotic, gentamicin). The patient did not bleed abnormally, despite both surgical challenge and plasma Factor V activity of less than 1%. The inhibited plasma had grossly prolonged prothrombin and activated partial thromboplastin times, but a normal thrombin time. Mixing studies indicated progressive coagulation inhibition with normal plasma, but not with Factor V-deficient plasma, and reversal of coagulation inhibition by the addition of bovine Factor V to the patient's plasma. 1 ml of patient plasma inhibited the Factor V activity of 90 ml of normal human plasma. The inhibitor was isolated by sequential affinity chromatography on protein A-Sepharose and Factor V-Sepharose. The IgG isolate markedly inhibits the activity of prothrombinase assembled from purified Factors Xa and Va, calcium ion, and phospholipid vesicles, and partially inhibits prothrombinase assembled from purified Factor Xa, calcium ion, and normal platelets. The Factor V of platelets, however, appears relatively inaccessible to the antibody, inasmuch as platelets isolated from whole blood supplemented for 8 h with the antibody functioned normally with respect to platelet Factor V-mediated prothrombinase function. The absence of obvious hemorrhagic difficulties in the patient, the total inhibition of plasma Factor V by the inhibitor, and the apparent inaccessibility of platelet Factor V to the inhibitor specifically implicate platelet Factor V in the maintenance of hemostasis.

The relative kinetics of clotting and lysis provide a biochemical rationale for the correlation between elevated fibrinogen and cardiovascular disease.

BACKGROUND: Elevated plasma fibrinogen is a well known risk factor for cardiovascular disease. The mechanistic rationale for this is not known. OBJECTIVES: These studies were carried out to determine the fibrinogen concentration dependencies of clotting and lysis times and thereby determine whether these times rationalize the correlation between an increased risk of cardiovascular disease and elevated plasma fibrinogen. METHODS: The time courses of clot formation and lysis were measured by turbidity in systems comprising a) fibrinogen, thrombin and plasmin, or b) fibrinogen, thrombin, plasminogen and t-PA, or c) plasma, thrombin and t-PA. From the lysis times, k(cat) and K(m) values for plasmin action on fibrin were determined. RESULTS: The time to clot increased linearly from 2.9 to 5.6 minutes as the fibrinogen concentration increased from 1 to 9 microM and did not increase further as the fibrinogen concentration was raised to 20 microM. In contrast, the clot lysis time increased linearly over the input fibrinogen concentration range of 2 to 20 microM. A similar linear trend was found in the two systems with t-PA and plasminogen. Apparent K(m) and k(cat) values for plasmin were 1.1 +/- 0.6 microM and 28 +/- 2 min(-1), respectively. K(m) values for plasmin in experiments initiated with t-PA and plasminogen were 1.6 +/- 0.2 microM in the purified system and 2.1 +/- 0.9 microM in plasma. CONCLUSION: As the concentration of fibrinogen increases, especially above physiologic level, the balance between fibrinolysis and clotting shifts toward the latter, providing a rationale for the increased risk of cardiovascular disease associated with elevated fibrinogen.

Long-term effects of estrogen on avian liver: estrogen-inducible switch in expression of nuclear, hormone-binding proteins.

The stimulation of chicks or embryos with estrogen results in transient, hepatic expression of the vitellogenin gene, as well as long-term, propagatable alterations in the rapidity with which the gene can be reactivated. We examined the possibility that nuclear, type II estrogen-binding sites are involved in this long-term change in response characteristics. We demonstrate that the primary induction kinetics of type II sites in embryos and chicks correlated with the expression of the vitellogenin gene and that once their induction was triggered by estrogen, they accumulated, were propagated, and persisted for months after withdrawal of the hormone. We also show that their accumulation in the embryo was accompanied by prolonged expression of both the vitellogenin and very low-density apolipoprotein II genes, in the absence of elevated levels of type I receptor, and that the type II sites, like the classical receptor, appear to be preferentially associated with active or potentially active chromatin. Finally, we describe a regulatory mechanism, tested by computer modelling, that simulated the behavioral characteristics of these nuclear estrogen-binding sites and which may explain their role in mediating the long-term effects of estrogen.

Intracellular localization of arginase in chick kidney.

Although chickens are uricotelic and do not have significant urea-ornithine cycle in any tissue, the kidneys contain a high concentration of arginase which apparently functions to regulate degradation of dietary arginine. A series of investigations has been made to determine the intracellular localization of this arginase in chicken kidney. Tissue fractionation using sucrose density gradients and differential centrifugation showed as association of arginase activity with certain marker enzymes and with fractions identified as mitochondria by electron microscopy. This is consistent with the localization of the arginase in the mitochondrial matrix of chicken kidney cells. Such a finding has significance in understanding the regulation of arginine degradation in chickens.

Lactose maldigestion in Ascaris-infected preschool children.

Two studies were conducted to investigate the contribution of Ascaris lumbricoides to lactose maldigestion in preschool children in two different communities in Panama where milk is available as a source of nutrients and the prevalence of Ascaris is known to be high. Both Ascaris-infected and uninfected children were given a standard lactose load and lactose absorption was studied by measuring the rise in plasma glucose in study 1 and by determination of breath hydrogen concentrations at regular intervals after ingestion of the test dose in study 2. All children were tested before anthelmintic treatment with levamisole and 3 wk after therapy. The mean rise in blood glucose from infected (n = 13) children 40 min after the ingestion of lactose was about half of that of the controls (n = 21). After deworming, lactose digestion improved in previously infected children. In study 2, significant differences in breath hydrogen concentrations postmilk ingestion were observed between the Ascaris-infected (n = 47) and the uninfected children (n = 35) before treatment. There was a substantial reduction of breath hydrogen after milk ingestion in the previously infected children after therapy. No differences were observed in breath hydrogen content of the uninfected children during the pre- and posttreatment phases of the study in the lactose tolerance test. These studies provide evidence that infection with Ascaris lumbricoides impairs lactose digestion in preschool children.

Influences in child growth associated with poverty in the 1970's: an examination of HANESI and HANESII, cross-sectional US national surveys.

This research examines associations between various measures of child growth (height, weight, triceps skinfold thickness, subscapular skinfold thickness), dietary variables, and poverty status in a sample of 13,750 black and white children aged 1 to 17 yr. The data used in this survey were collected in the National Health and Nutrition Examination Surveys I and II (HANESI, 1971-1975, and HANESII, 1976-1980). In general, lower mean values for all the growth measures examined were found in children living below the defined poverty threshold in comparison with those above the poverty threshold. The magnitude of these poverty-associated differences tended to decrease between the times of the HANESI and HANESII surveys, though not sufficiently to be statistically significant. These differences in growth were not consistently associated with differences in dietary intake of energy between poverty groups or surveys.

Serum retinol concentrations in children are affected by food sources of beta-carotene, fat intake, and anthelmintic drug treatment.

The provision of vitamin A in food sources of beta-carotene is an alternative to the distribution of high-dose capsules. To examine factors that may influence the success of food-based programs, a study was carried out in Sumatra, Indonesia, of the effect of food sources of beta-carotene, extra dietary fat, and Ascaris lumbricoides infection on serum retinol concentrations in children. Meals and snacks with various amounts of beta-carotene and fat were fed at midday to children 3-6 y of age for 3 wk. Some groups of children were dewormed with the anthelmintic levamisole before the feeding period, whereas others remained infected. Results showed that the incorporation of beta-carotene sources (mainly in the form of red sweet potatoes) into the meal significantly increased serum retinol concentrations. The greatest rise in serum retinol occurred when meals contained added beta-carotene sources and added fat and the children were dewormed. Adding more fat to the meal and deworming the children caused a rise in serum retinol similar to that seen when feeding additional beta-carotene sources. Moreover, the effects of fat and deworming together were additive to the effects of additional beta-carotene sources. When the meal contained additional beta-carotene sources, added fat caused a further improvement in serum retinol concentrations but only if A. lumbricoides infection was low. These studies indicated that food-based interventions in vitamin A-deficient areas might be successful and that other interventions such as increasing dietary fat concentrations and anthelmintic treatment should be considered along with increasing consumption of beta-carotene-rich food.

Relationships between Ascaris infection and growth of malnourished preschool children in Kenya.

A longitudinal study in Ascaris-infected and noninfected children was conducted in two Kenyan villages. Anthropometric, clinical, and stool exams were performed three times at 14-week intervals. All children received an anthelmintic drug (levamisole) at the second examination. In the 14 weeks before deworming, children with Ascaris (n = 61) did not differ from controls (n = 125) in percentage expected weight gain. In the 14 weeks after deworming, previously infected children showed higher percentage expected weight gain than controls. Before deworming, there was a statistically significant (P less than 0.0005) decrease in triceps skinfold thickness in Ascaris-infected children versus controls. After deworming, skinfold increased significantly (P less than 0.0005) in previously infected children versus controls. Multiple regression analysis showed that Ascaris infection was by far the most important variable of those studied explaining decrease in skinfold thickness before and increase after deworming. It appears that even light Ascaris infections might adversely influence nutritional status, and deworming might enhance growth.

Reversible inhibitors of TAFIa can both promote and inhibit fibrinolysis.

The plasma carboxypeptidase activated thrombin-activable fibrinolysis inhibitor (TAFIa), is thermally unstable at 37 degrees C, with a half-life of 8 or 15 min depending on the isoform. The arginine analog, 2-guanidinoethylmercaptosuccinate (GEMSA), not only inhibits TAFIa but also slows the spontaneous inactivation of the enzyme, thereby reducing the activity of TAFIa, while extending its apparent half-life. Because, as shown in previous work, the ability of TAFIa to prolong clot lysis can be more dependent on its half-life than its concentration, in this study we determined whether reversible inhibitors of TAFIa could paradoxically prolong clot lysis. Potato tuber carboxypeptidase inhibitor (PTCI) or GEMSA were titrated into normal pooled human plasma, in the presence of soluble thrombomodulin. Both inhibitors mediate a biphasic antifibrinolytic effect, prolonging clot lysis at lower concentrations and enhancing clot lysis at higher concentrations. The antifibrinolytic effect of GEMSA is maximized at 1 mmol L-1, increasing clot lysis time from 100 min to 350 min. The antifibrinolytic effect of PTCI is maximized at 100 nmol L-1, increasing clot lysis time from 100 min to 240 min. To further characterize the nature of this biphasic effect, TAFI at various concentrations was added to TAFI-immunodepleted human plasma in the presence of PTCI or GEMSA. The magnitude of the effect depends on the concentration of TAFIa, the concentration of inhibitor, and the potency of the inhibitor. We propose that the biphasic antifibrinolytic effect is mediated by the dynamic equilibrium of free TAFIa that inactivates quickly, and TAFIa bound to inhibitor that inactivates slowly. TAFIa inhibitors used as therapeutic agents might not only enhance lysis at higher concentrations, but also stabilize fibrin clots at intermediate concentrations.

Factor Xa is highly protected from antithrombin-fondaparinux and antithrombin-enoxaparin when incorporated into the prothrombinase complex.

Antithrombin and its cofactor, heparin, target both the product of prothrombin activation by prothrombinase, thrombin, as well as the enzyme responsible for the reaction, factor (F)Xa. These studies were carried out to quantify the effects of each of the prothrombinase components on the half-life of FXa in the presence of antithrombin and the low-molecular-weight heparins (enoxaparin, Aventis, Laval, Quebec, Canada) or the heparin pentasaccharide (fondaparinux, Organon Sanofi-Synthelabo, Cypress, TX, USA). Experiments were carried out using a recombinant form of prothrombin in which the active site serine has been mutated to cysteine and subsequently labeled with fluorescein. This mutant allowed calculation of the second order rate constant for inhibition of FXa by antithrombin in such a way that competition for antithrombin by thrombin is eliminated and competition for FXa by prothrombin is accounted for. Intrinsic rate constants for the inhibition of FXa by antithrombin-enoxaparin and antithrombin-fondaparinux, in the presence of the various prothrombinase components, were calculated. Addition of phospholipid had no significant effect on the second order rate constant for inhibition of FXa by antithrombin, while addition of FVa appeared to be mildly protective. Further addition of prothrombin however, caused profound protection of FXa, increasing its half-life from 1.1 to 353 s in the case of fondaparinux, and from 0.4 to 42 s in the case of enoxaparin. Similar results were reported for unfractionated heparin previously [1]. Therefore, in the presence of unfractionated heparin, fondaparinux, or enoxaparin, prothrombinase is profoundly protected from antithrombin.

Temporal changes in factors associated with neutrophil elastase and coagulation in intensive care patients with a biphasic waveform and disseminated intravascular coagulation.

Summary. The biphasic waveform is an early marker of disseminated intravascular coagulation (DIC). Neutrophil elastase (NE) cleaves coagulation factors; thus, elevated elastase levels or its dysregulation by alpha-1-protease inhibitor (Alpha1PI) may be linked to DIC. Time courses over a period were determined for factors associated with NE and coagulation in 14 Intensive Care Unit patients with a biphasic waveform who developed DIC. The data were analyzed using a random coefficient linear regression model to predict the variables' mean values on day 0 and their mean rates of change over the period in which the biphasic waveform appeared. The biphasic waveform was normal on day 0, maximized on day 1, and approached normal again by day 4. Alpha1PI/NE complex levels were 2.5-fold greater than normal for the entire period. The A1PI activity, antigen, and specific activity levels were normal on day 0 and increased thereafter by 21.0, 10.5, and 8.9% of normal per day, respectively. Factor II, V, VII, IX, and X activity levels were, respectively, 57, 46, 46, 77, and 46% of normal on day 0, whereas factor VIII and fibrinogen levels were normal. All coagulation factor levels trended upward with time but not significantly. The prothrombin time, but not the activated partial thromboplastin time, was prolonged, and the platelet counts and hematocrits were below normal on day 0 and remained so thereafter. We conclude that events associated with neutrophil activation, elastase release, and perturbations of coagulation precede both the appearance of the biphasic waveform and the diagnosis of DIC in these patients.

Role of mRNA transcript stability in modulation of expression of the gene encoding thrombin activable fibrinolysis inhibitor.

Regulation of mRNA stability has emerged as a major control point in eukaryotic gene expression. The abundance of a particular mRNA can be rapidly regulated in response to a stimulus by altering the stability of existing translatable transcripts rather than by altering the rate of transcription initiation. Alternative polyadenylation of transcripts during mRNA processing can be important in determining transcript abundance if the different forms of mRNA possess different stabilities or translatability. The mRNA transcript encoding thrombin activable fibrinolysis inhibitor (TAFI) is an attractive candidate for regulation of mRNA stability because of the relatively long length of its 3'-untranslated region and because the transcript can be polyadenylated at three different sites. As well, we have previously reported that treatment of HepG2 cells with interleukins (IL) - 1beta and - 6 destabilizes the endogenous TAFI mRNA expressed in this cell line. In the current study, we report that the TAFI 3'-untranslated region contains cis-acting instability element(s) and that these elements in fact determine the intrinsic stability of the TAFI transcript. Moreover, we found that the three different polyadenylated mRNA forms have different intrinsic stabilities, with the mRNA half-life increasing from the longest to the shortest transcript. Interestingly, treatment with IL-1beta plus IL-6 not only resulted in a 2-fold decrease in stability of the transcript produced using the 3'-most polyadenylation site but also resulted in profound shifts in the relative abundances of the respective polyadenylated forms through changes in the frequency of utilization of the three polyadenylation sites. As such, in the presence of IL-1beta and IL-6, the longest transcript is over a thousand times more abundant than the two shorter transcripts whereas in the absence of the stimulus it comprises only 1% of the total TAFI transcripts.