Fibrinogen mRNA Research Articles

GRSF1 antagonizes age-associated hypercoagulability via modulation of fibrinogen mRNA stability

Age-associated hypercoagulability is accompanied by the increase of plasma levels of some coagulation factors including fibrinogen which may contribute to the increased risk of cardiovascular, cerebrovascular, and thrombotic diseases in elderly people. However, the underlying mechanism of increased plasma fibrinogen concentration during aging is still elusive. GRSF1 belongs to the heterogeneous nuclear ribonucleoproteins F/H (hnRNP F/H) subfamily. Here, we report that GRSF1 attenuates hypercoagulability via negative modulation of fibrinogen expression. We demonstrated that GRSF1 negatively regulated fibrinogen expression at both mRNA and protein levels. GRSF1 directly interacted with the coding region (CDS) of FGA, FGB, and FGG mRNAs, and decreased their stability thus mitigating fibrinogen expression. We further identified that only a few G-tracts within the Fib C domain of FGA, FGB, and FGG CDS and the qRRM2 domain of GRSF1 were required for their interaction. Moreover, we confirmed hypercoagulability and the decrease of GRSF1 expression level during mice aging. Functionally, GRSF1 overexpression in old mice liver decreased fibrinogen plasma level, reduced hypercoagulability, and mitigated blood coagulation activity, whereas GRSF1 knockdown in young mice liver increased fibrinogen plasma level and promoted blood coagulation activity. Collectively, our findings unveil a novel posttranscriptional regulation of fibrinogen by GRSF1 and uncover a critical role of GRSF1 in regulating blood coagulation activity.

Hepatocyte Nuclear Factor-1α Increases Fibrinogen Gene Expression in Liver and Plasma Fibrinogen Concentration in Rats with Experimental Chronic Renal Failure

Chronic kidney disease (CKD) is associated with elevated plasma fibrinogen concentration. However, the underlying molecular mechanism for elevated plasma fibrinogen concentration in CKD patients has not yet been clarified. We recently found that HNF1α was significantly upregulated in the liver of chronic renal failure (CRF) rats, an experimental model of CKD in patients. Given that the promoter region of the fibrinogen gene possesses potential binding sites for HNF1α, we hypothesized that the upregulation of HNF1α can increase fibrinogen gene expression and consequently plasma fibrinogen concentration in the experimental model of CKD. Here, we found the coordinated upregulation of Aα-chain fibrinogen and Hnfα gene expression in the liver and elevated plasma fibrinogen concentrations in CRF rats, compared with pair-fed and control animals. Liver Aα-chain fibrinogen and HNF1α mRNAs levels correlated positively with (a) liver and plasma fibrinogen levels and (b) liver HNF1α protein levels. The positive correlation between (a) liver Aα-chain fibrinogen mRNA level, (b) liver Aα-chain fibrinogen level, and (c) serum markers of renal function suggest that fibrinogen gene transcription is closely related to the progression of kidney disease. Knockdown of Hnfα in the HepG2 cell line by small interfering RNA (siRNA) led to a decrease in fibrinogen mRNA levels. Clofibrate, an anti-lipidemic drug that reduces plasma fibrinogen concentration in humans, decreased both HNF1α and Aα-chain fibrinogen mRNAs levels in (a) the liver of CRF rats and (b) HepG2 cells. The obtained results suggest that (a) an elevated level of liver HNF1α can play an important role in the upregulation of fibrinogen gene expression in the liver of CRF rats, leading to an elevated concentration of plasma fibrinogen, a protein related to the risk of cardiovascular disease in CKD patients, and (b) fibrates can decrease plasma fibrinogen concentration through inhibition of HNF1α gene expression.

Chemical Modulators of Fibrinogen Production and Their Impact on Venous Thrombosis

Thrombosis is a leading cause of morbidity and mortality. Fibrinogen, the soluble substrate for fibrin-based clotting, has a central role in haemostasis and thrombosis and its plasma concentration correlates with cardiovascular disease event risk and a prothrombotic state in experimental models. We aimed to identify chemical entities capable of changing fibrinogen production and test their impact on experimental thrombosis. A total of 1,280 bioactive compounds were screened for their ability to alter fibrinogen production by hepatocyte-derived cancer cells and a selected panel was tested in zebrafish larvae. Anthralin and all-trans retinoic acid (RA) were identified as fibrinogen-lowering and fibrinogen-increasing moieties, respectively. In zebrafish larvae, anthralin prolonged laser-induced venous- occlusion times and reduced thrombocyte accumulation at injury sites. RA had opposite effects. Treatment with RA, a nuclear receptor ligand, increased fibrinogen mRNA levels. Using an antisense morpholino oligonucleotide to deplete zebrafish fibrinogen, we correlated a shortening of laser-induced venous thrombosis times with RA treatment and fibrinogen protein levels. Anthralin had little effect on fibrinogen mRNA in zebrafish larvae, despite leading to lower detectable fibrinogen. Therefore, we made a proteomic scan of anthralin-treated cells and larvae. A reduced representation of proteins linked to the canonical secretory pathway was detected, suggesting that anthralin affects protein secretion. In summary, we found that chemical modulation of fibrinogen levels correlates with measured effects on experimental venous thrombosis and could be investigated as a therapeutic avenue for thrombosis prevention.

Abstract LB-105: Plasma exposure-target engagement biomarker analysis for danvatirsen after multiple dosing schedules and route of administrations in patients with advanced solid tumors

Abstract Background: Danvatirsen (Da) is a generation 2.5 antisense oligonucleotide targeting STAT3 mRNA under clinical development in combination with Durvalumab (NCT03421353). The recommended Phase II dose is intravenous (IV) 200 mg weekly (QW, A). However, alternative 400 mg IV every two weeks (Q2W, B) and subcutaneous 200 mg QW (C) are also under investigation. The JAK-STAT signaling pathway is involved in immunity, cell division, cell death, and tumor formation. Additionally, activation of hepatic STAT3 results in increased secretion of C-reactive protein (CRP) and fibrinogen into blood. Therefore, target engagement in patients may be better understood by analyzing Da exposure and related biomarkers in the blood/plasma. We investigated Da exposure and subsequent impact on target engagement biomarkers such as CRP, platelets, fibrinogen, neutrophils and STAT3 mRNA levels. Methods: Extensive pharmacokinetics (PK) samples were collected at lead-in monotherapy and at steady state (SS) in combination. Plasma biomarker samples were collected at baseline and every two weeks thereafter. Whole blood samples for the analysis of STAT3 mRNA were collected at baseline, after lead-in monotherapy, and every four weeks thereafter. The PK parameters were estimated by non-compartmental analysis by WinNonlin. The biomarkers were analyzed by standard clinical methods and STAT3 mRNA level was measured by NanoString technology. Results: The area under plasma concentration-time curve of Da at SS within dosing interval (AUC0-τ, ng.h/ml) for A, B, and C were 69110 (24.64%), 101600 (22.11%), and 44600 (30.25%), respectively. Additionally, SS Ctrough (ng/ml) levels for A, B, and C were 28.53 (48.86%), 14.56 (36.14%), and 27.66 (49.22%), respectively. Maximum median % decrease from baseline in CRP levels was observed at week 4; A (-91%), B (-94%), C (-82%). Additionally, maximum median % changes from baseline for fibrinogen were -60%, -40%, -53%, in A, B, and C at week 4, respectively. Maximum % decrease from baseline in platelets was observed at week 8 in A (-42%), B (-41%), and C (-69%). In contrast, maximum % change from baseline in neutrophil count was observed at week 6; -37%, -31%, and -32% in A, B, and C, respectively. Additionally, the median maximum change from baseline for STAT3 mRNA was -42.4% and -33.6% for A and C, respectively. Conclusion: Cumulative AUC in A was very similar to B, whereas, AUC in C was 30% lower as compared to A or B. The SS Ctrough values in A and C were similar, although Ctrough value in B was 50% lower. Maximum % decreases in CRP levels were similar across all the doses. Moreover, route of administration did not appear to impact level of STAT3 knockdown. In summary, a similar degree of changes in pharmacodynamic biomarkers were achieved at all three dose levels regardless of route of administration, despite differences in plasma exposure. Citation Format: Kowser Miah, Patricia McCoon, Deanna Russell, Patrick Mitchell, Martin Scott, Amy Rosenfeld, Li Zhang, Pablo Martinez Rodriguez, Alicia Savage, Ganesh Mugundu. Plasma exposure-target engagement biomarker analysis for danvatirsen after multiple dosing schedules and route of administrations in patients with advanced solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-105.

Transcriptome sequencing of Eospalax fontanierii to determine hypoxia regulation of cardiac fibrinogen.

With the increase in blood viscosity, the blood circulation resistance will increase when animals are in hypoxia. However, these phenomenons do not appear in hypoxic-adapted animals. Eospalax fontanierii is a subterranean rodent and is an ideal species for research in hypoxia adaptation. Eighteen healthy adult E. fontanierii individuals were equally divided into three groups that were exposed to 21% O2 for 1week, 10.5% O2 for 44h, and 6.5% O2 for 6h, and then, the hearts were collected for transcriptome sequencing. After differentially expressed analysis, fibrinogen genes were selected for qPCR and Western blot verification. Eighteen healthy adult Sprague-Dawley rats (SD rats) were treated with the same oxygen concentrations, and their hearts were simultaneously subjected to qPCR. The quantitative real-time PCR and Western blot results were completely opposite to those of the rats. E. fontanierii fibrinogen mRNA was significantly downregulated when expressed under the conditions of 10.5% and 6.5% O2 compared with 21% O2. Correspondingly, fibrinogen mRNA in E. fontanierii was expressed at lower levels than SD rats in 10.5% and 6.5% O2. After tail-cutting experiment, the results showed that the coagulation rate of E. fontanierii was slowed down under hypoxic conditions. These results showed that E. fontanierii may downregulate the expression of fibrinogen mRNA in hypoxia to reduce the aggregation of red blood cells and platelets in plasma, which may prevent blood from becoming overly viscous, and at the same time, reduce blood circulation resistance and the probability of thrombosis in hypoxia to protect the heart.

FDP-E induces adipocyte inflammation and suppresses insulin-stimulated glucose disposal: effect of inflammation and obesity on fibrinogen Bβ mRNA.

Obesity is associated with increased fibrinogen production and fibrin formation, which produces fibrin degradation products (FDP-E and FDP-D). Fibrin and FDPs both contribute to inflammation, which would be expected to suppress glucose uptake and insulin signaling in adipose tissue, yet the effect of FDP-E and FDP-D on adipocyte function and glucose disposal is completely unknown. We tested the effects of FDPs on inflammation in 3T3-L1 adipocytes and primary macrophages and adipocyte glucose uptake in vitro. High-fat-fed mice increased hepatic fibrinogen mRNA expression ninefold over chow-fed mice, with concomitant increases in plasma fibrinogen protein levels. Obese mice also displayed increased fibrinogen content of epididymal fat pads. We treated cultured 3T3-L1 adipocytes and primary macrophages with FDP-E, FDP-D, or fibrinogen degradation products (FgnDP-E). FDP-D and FgnDP-E had no effect on inflammation or glucose uptake. Cytokine mRNA expression in RAW264.7 macrophage-like cells and 3T3-L1 adipocytes treated with FDP-E induced inflammation with maximal effects at 100 nM and 6 h. Insulin-stimulated 2-deoxy-d-[(3)H]glucose uptake was reduced by 71% in adipocytes treated with FDP-E. FDP-E, but not FDP-D or FgnDP-E, induces inflammation in macrophages and adipocytes and decreases glucose uptake in vitro. FDP-E may contribute toward obesity-associated acute inflammation and glucose intolerance, although its chronic role in obesity remains to be elucidated.

Developmental expression and organisation of fibrinogen genes in the zebrafish

The zebrafish is a model organism for studying vertebrate development and many human diseases. Orthologues of the majority of human coagulation factors are present in zebrafish, including fibrinogen. As a first step towards using zebrafish to model human fibrinogen disorders, we cloned the zebrafish fibrinogen cDNAs and made in situ hybridisations and quantitative reverse transcription-polymerase chain reactions (qRT-PCR) to detect zebrafish fibrinogen mRNAs. Prior to liver development or blood flow we detected zebrafish fibrinogen expression in the embryonic yolk syncytial layer and then in the early cells of the developing liver. While human fibrinogen is encoded by a three-gene, 50 kilobase (kb) cluster on chromosome 4 ( FGB-FGA-FGG ), recent genome assemblies showed that the zebrafish fgg gene appears distanced from fga and fgb , which we confirmed by in situ hybridisation. The zebrafish fibrinogen Bβ and γ protein chains are conserved at over 50% of amino acid positions, compared to the human polypeptides. The zebrafish Aα chain is less conserved and its C-terminal region is nearly 200 amino acids shorter than human Aα. We generated transgenic zebrafish which express a green fluorescent protein reporter gene under the control of a 1.6 kb regulatory region from zebrafish fgg . Transgenic embryos showed strong fluorescence in the developing liver, mimicking endogenous fibrinogen expression. This regulatory sequence can now be used for overexpression of transgenes in zebrafish hepatocytes. Our study is a proof-of-concept step towards using zebrafish to model human disease linked to fibrinogen gene mutations.

Pathogenesis of severe acute respiratory syndrome

Pathogenesis of severe acute respiratory syndrome

Circadian Variations in Coagulation and Fibrinolytic Factors among Four Different Strains of Mice

This study examined circadian variation in coagulation and fibrinolytic parameters among Jcl:ICR, C3H/HeN, BALB/cA, and C57BL/6J strains of mice. Plasma plasminogen activator inhibitor 1 (PAI‐1) levels fluctuated in a circadian manner and peaked in accordance with the mRNA levels at the start of the active phase in all strains. Fibrinogen mRNA levels peaked at the start of rest periods in all strains, although plasma fibrinogen levels remained constant. Strain differences in plasma antithrombin (AT) activity and protein C (PC) levels were then identified. Plasma AT activity was circadian rhythmic only in Jcl:ICR, but not in other strains, although the mRNA levels remained constant in all strains. Levels of plasma PC and its mRNA fluctuated in a circadian manner only in Jcl:ICR mice, whereas those of plasma prothrombin, factor X, factor VII, prothrombin time (PT), and activated partial thrombin time (APTT) remained constant in all strains. These results suggest that genetic heterogeneity underlies phenotypic variations in the circadian rhythmicity of blood coagulation and fibrinolysis. The circadian onset of thrombotic events might be due in part to the rhythmic gene expression of coagulation and fibrinolytic factors. The present study provides fundamental information about mouse strains that will help to understand the circadian variation in blood coagulation and fibrinolysis.

Hepatic Cytochrome P450 Gene Regulation during Endotoxin-Induced Inflammation in Nuclear Receptor Knockout Mice

Inflammatory agents such as lipopolysaccharide (LPS) down-regulate the hepatic expression of many cytochrome P450 (P450) mRNAs and proteins. Previous studies suggested that suppression of some P450 mRNAs could involve the regulation or modulation of the nuclear receptors peroxisome proliferator-activated receptor alpha (PPARalpha) or pregnane X receptor (PXR). To determine the involvement of these receptors in P450 down-regulation, PPARalpha knockout (KO), PXR KO, and appropriate wild-type (WT) mice were administered either saline or 1 mg/kg LPS. Hepatic mRNA and protein expression of several P450 isoforms, interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF) alpha, alpha1-acid glycoprotein (AGP), and fibrinogen (FBG) were examined 16 h later. LPS administration significantly decreased the hepatic expression of CYP1A2, 2A5, 2C29, 2E1, 3A11, 4A10, and 4A14 mRNAs in both groups of PPARalpha and PXR mice, whereas CYP3A13 mRNA was increased slightly in PPARalpha WT and KO mice, but not in PXR mice. Effects of LPS administration on mouse hepatic P450 proteins (probed using rat P450 2C, 3A, 4A, and 2E antibodies) were consistent with mRNA results in most cases. LPS treatment significantly increased IL-1beta, IL-6, TNFalpha, AGP, and FBG mRNA in both PPARalpha and PXR mice, with the greatest effect observed with TNFalpha. Because decreases in P450 mRNA expression were essentially identical in both WT and KO mice for both nuclear receptors, these data indicate that down-regulation of P450 during inflammation does not require the nuclear receptors PPARalpha and PXR.

Effect of L-NAME on the Synthesis of Plasma Fibrinogen in Mice

The effect of nitric oxide (NO) on plasma fibrinogen was investigated by treating mice with oral NG-nitro-L-arginine-methyl ester (L-NAME), a non-selective NO synthase (NOS) inhibitor. Treatment with L-NAME significantly increased the concentration of plasma fibrinogen and hepatic expression of the α- and β-chains of fibrinogen, whereas plasma NO2− level and hepatic expression of endothelial NOS (eNOS) mRNA were significantly decreased. These results suggest that L-NAME treatment can increase plasma fibrinogen levels via an increase in expression of fibrinogen mRNA in the liver, and NO may be involved in plasma fibrinogen increase.

The septic abscess wall: a cytokine-generating organ associated with portal venous cytokinemia, hepatic outflow fibrosis, sinusoidal congestion, inflammatory cell sequestration, hepatocellular lipid deposition, and focal cell death.

An acute septic inflammatory response with access to the portal circulation was created in a rat model using an intra-abdominal abscess composed of a sterile agar pellet, or one contaminated with 102 Escherichia coli (E. coli) and 109 Bacteriodes fragilis (B. fragilis). After 3 days postimplantation, a well-formed intra-abdominal abscess occurred whose wall showed IL-6 DNA by PCR and IL-6 mRNA by in situ hybridization. Portal venous blood draining into the liver from the intra-abdominal abscess had increased levels of TNF-alpha, IL-1beta, and IL-6 in both sterile and septic groups compared with a control normal animal group. Increased levels of these cytokines were also found in suprahepatic inferior vena caval blood, but were correlated with the higher portal vein levels, suggesting a gradient from abscess wall to portal vein into the systemic circulation via the liver. Liver histology demonstrated sinusoidal congestion centering on the central vein, growing worse with progression from normal in control, to sterile, to septic. Similarly, the degree of intrahepatic myeloperoxidase-positive inflammatory cell infiltration and hepatocellular lipid deposition and apoptosis also increased from control, to sterile, to septic. Gene expression by in situ hybridization demonstrated a significant increase in IL-6 and fibrinogen mRNAs in cells surrounding the central vein in sterile and septic animals, being greatest in animals with sepsis, associated with an increased deposition of collagen in the central vein area, most prominent in the septic liver. The pericentral vein cells with IL-6 and fibrinogen mRNA increases paralleled the increases in cells containing IL-6 and fibrinogen mRNAs in the abscess walls of sterile and septic animals, respectively. The data suggest that an intra-abdominal abscess, especially when contaminated with gram-negative bacteria, induces mRNA-generated cytokine responses in the abscess wall that are related to increased portal venous levels of the inflammatory cytokines TNF-alpha, IL-1beta, and IL-6 perfusing the liver. These, in turn, induce localized production of IL-6 and fibrinogen mRNAs in cells at the central vein area with resultant outflow fibrosis and increased inflammatory cell sequestration within the liver lobular sinuses. This is associated with a generalized inflammatory response and intrahepatic portal sinusoid congestion. There is also increased hepatocellular lipid deposition and apoptosis. Thus, the cytokine production of the abscess wall itself appears to be a major mediator of the septic hepatic response.

Liver gene regulation in rats following both 70 or 90% hepatectomy and endotoxin treatment.

The metabolic state effect of liver failure on liver gene regulation was evaluated in a rat model. Following 70 or 90% hepatectomy and lipopolysaccharide or vehicle treatment at intervals up to 24 h, the liver remnants were analyzed for mRNA levels for acute-phase, liver-specific and growth-related proteins. After 70% hepatectomy mRNA for alpha 1-acid glycoprotein, alpha 2-macroglobulin, thiostatin and fibrinogen, haptoglobin increased three- to sevenfold (P < 0.05), and mRNA for cyclin D and histone 3 increased seven- and 15-fold (P < 0.05), respectively. After lipopolysaccharide injection and 70% hepatectomy were done, mRNA for acute-phase proteins raised significantly (P < 0.05), more to five to 20-fold, while mRNA for growth-related proteins raised significantly (P < 0.05) less to three- to fourfold. After 90% hepatectomy, acute-phase protein mRNA increased five- to ninefold (P < 0.05) more than after 70% hepatectomy, while mRNA for histone 3 and cyclin D did not increase within 24 h, which indicates a delayed growth after 90% hepatectomy. In 90% of hepatectomized rats treated with lipopolysaccharide, acute-phase protein mRNA raised three- to sixfold (P < 0.05) less than after vehicle treatment. In endotoxemia from liver failure, the synthesis of acute-phase proteins is upregulated by gene regulation at the expense of that for regeneration, which may be an appropriate response for immediate survival. In severe liver failure, endotoxin may interfere with the appropriate gene regulation.

Increased Hepatic Fibrinogen Bβ-gene Transcription Is not enough to Increase Plasma Fibrinogen Levels

The fibrinogen Aalpha, Bbeta, and gamma polypeptides are encoded by three separate genes, which are arranged in the order gamma-alpha-beta. In order to study the biosynthesis of fibrinogen in vivo we generated a line of transgenic mice carrying extra copies of the fibrinogen beta-gene. To clone the mouse fibrinogen Bbeta-chain gene, a mouse 129 Sv/Ev genomic cosmid library was screened, using the mouse fibrinogen Aalpha-, Bbeta-chain cDNA. A clone containing the complete fibrinogen Bbeta-chain gene including approximately 11-kb of the natural promoter region was identified and subsequently microinjected into mice. Southern blot analysis identified a founder that carried additional copies of the fibrinogen Bbeta-chain gene. Transgenic offspring of this founder were interbred and heterozygous and homozygous transgenic mice were obtained. Northern blot analysis demonstrated approximately a 3-fold increase in fibrinogen Bbeta mRNA in heterozygous mice as compared to wild-type, whereas homozygous transgenic mice showed approximately a 9-fold increase. The levels of the Aalpha and gamma mRNAs in transgenic homozygous mice were not changed as compared to those in wild-type mice. Fibrinogen levels in plasma were not significantly increased in transgenic mice as compared to wild-type mice. These results indicate that: additional copies of the fibrinogen Bbeta-chain gene lead to increased levels of the Bbeta-chain mRNA in the liver; the increased levels of Bbeta-chain mRNA in homozygous overexpression mice do not change the transcription levels of the two other fibrinogen mRNAs in vivo; the absence of an increased plasma fibrinogen level in the transgenic mice indicates that this level is not regulated solely by transcription of the Bbeta-chain gene.

Differential hepatic lobar gene expression in offspring exposed to altered maternal dietary protein intake.

Increased plasma fibrinogen concentrations are a recognized risk factor for coronary heart disease, and increased fibrinogen levels in adults are associated with parameters of reduced early growth. We studied fibrinogen gene expression in adult offspring of dams fed either a 20% (control) or an 8% protein diet [maternal low-protein (MLP) rats] during pregnancy and lactation and determined whether any effects were consistent between left and right liver lobes, since the fetal liver has a unique blood supply that produces differential stimuli to the left and right lobes. In MLP offspring, there was a reduction in all three fibrinogen mRNA copy numbers in the left liver lobe [left vs. right lobes for alpha-, beta-, and gamma-fibrinogen (x10(6) copies/ng total RNA): 8.04 vs. 23.16, P<0.001; 4.74 vs. 13.07, P<0.001; and 4.61 vs. 16.38, P = 0.007, respectively], with a parallel reduction in fibrinogen concentration in the left liver lobe (8.53+/-0.33 vs. 10.41 +/-0.65 arbitrary units, P = 0.014, left and right lobes, respectively). No such effect was observed in offspring of control dams. To investigate the underlying mechanism, glucocorticoid receptor function and mRNA levels were studied, since expression of fibrinogen genes is regulated by glucocorticoid hormones. The binding affinity of the high-affinity glucocorticoid receptor was reduced only in the left liver lobe of the MLP offspring (P = 0.02, left. vs. right), with a parallel reduction in this lobe in glucocorticoid receptor mRNA level (P = 0.006, left vs. right). In conclusion, maternal dietary protein restriction reduces fibrinogen gene expression, fibrinogen protein, and mRNA level and binding affinity of glucocorticoid receptors only in the left liver lobe of the adult offspring.

Expression of Fibrinogen mRNA in the Liver in Triton WR-1339-induced hyperlipidemic rats

Expression of Fibrinogen mRNA in the Liver in Triton WR-1339-induced hyperlipidemic rats

Changes in mRNA Levels of Fibrinogen Subunit Polypeptides in Rats Defibrinogenated with Batroxobin

Batroxobin is a snake venom that is a thrombin-like enzyme used for clinical treatment. We analyzed hepatic mRNA levels for fibrinogen subunit polypeptides and prothrombin by reverse transcription-polymerase chain reaction as well as coagulation and fibrinolysis factors in plasma 1, 3, 5 and 24 hours after Batroxobin treatment (3 BU/100 g) in rats. The mRNA levels of α- and β-chains of fibrinogen were significantly increased with decreases in plasma fibrinogen, α 2-plasmin inhibitor, and plasminogen levels, while the mRNA levels for prothrombin remained unchanged. These results suggest that fibrinogen mRNA synthesis is regulated by plasma fibrinogen levels in Batroxobin-induced defibrinogenated rats.

Coordinate augmentation in expression of genes encoding transcription factors and liver secretory proteins in hypo-oncotic states

In the nephrotic syndrome (NS) proteins of intermediate size (40 to 200 kD) are lost into the urine resulting in a decrease in plasma albumin concentration and as a consequence a reduction in plasma colloid osmotic pressure (pi). Plasma pi has also been reported to be reduced in the condition of hereditary analbuminemia. The liver, in an apparent compensatory response, increases synthesis of a group of secreted proteins defending plasma pi. Regulation of several of these proteins, including both positive and negative acute phase proteins, is at the transcriptional level. This is the only known condition in which transcription of both positive and negative acute phase proteins (APPs) are increased simultaneously. The specific transcription factor(s) that might regulate this cascade is not defined. RNA was extracted from livers of 5 rats with hereditary analbuminemia (the Nagase analbuminemic rat, NAR), 5 rats with NS induced by adriamycin (Adria), 5 rats with NS caused by passive Heymann nephritis (NS) and 5 control animals. The concentrations of mRNAs encoding four secreted proteins (albumin, transferrin, fibrinogen, and apo A-1), five transcription factors, early growth response factor 1 (EGRF-1), HNF-4, NGFI-C, EGR-3, and Krox20 relative to two housekeeping genes, beta actin and GAPDH were determined simultaneously using kinetic reverse transcriptase polymerase chain methodology (kRT-PCR). The levels of all mRNAs encoding secreted proteins except for albumin (which was reduced in NAR) were increased in NS and NAR and correlated significantly with one another. mRNA encoding EGRF 1 was increased fivefold in NS and NAR, and correlated significantly with mRNAs encoding Apo A-1, transferrin and albumin in the two NS groups. HNF-4 mRNA was increased approximately twofold in both NS groups and correlated with albumin (R = 0.881, P < 0.001), transferrin (R = 0.563, P = 0.012) and apo A-1 (R = 0.644, P = 0. 003). While fibrinogen mRNA correlated with that of each of the other secreted proteins, it did not correlate with either HNF-4 or EGRF-1 mRNA. Krox20, EGR3 and NGF1C were expressed at nearly undetectable levels. The hepatic response in conditions characterized by reduced plasma pi include increased levels of mRNAs encoding a group of secreted proteins, including the negative APPs albumin, transferrin and apo A-1, and the positive APP fibrinogen. Levels of mRNAs encoding negative APPs and fibrinogen correlate with one another, suggesting that they are coordinately controlled. Both EGRF-1 and HNF-4 may regulate the expression of the negative APPs, which have increased transcription in hypo-oncotic states.

Cytokine modulation by PX differently affects specific acute phase proteins during sepsis in rats.

To explore the regulation of the acute phase response in vivo, the effects of pentoxifylline (PX) treatment (100 mg/kg ip 1 h before infection) were investigated in infected and pair-fed rats 2 and 6 days after an intravenous injection of live bacteria (Escherichia coli). PX treatment prevented the increase in plasma tumor necrosis factor (TNF)-alpha (peak 1.5 h after the infection) and resulted in an 84 and 61% inhibition of plasma interleukin (IL)-1beta and IL-6, respectively (peaks at 3 h). Plasma corticosterone kinetics were not modified by the treatment. Infection increased alpha1-acid glycoprotein (AGP), alpha2-macroglobulin (A2M), and fibrinogen plasma concentrations and decreased albumin levels. PX significantly reduced AGP plasma concentration as early as day 2 in infected animals but reduced A2M and fibrinogen plasma levels only at day 6. The treatment had no effect on the albumin plasma concentration. Hepatic AGP and fibrinogen mRNA levels increased in infected rats, whereas those of A2M were unchanged and those of albumin were decreased. Two days after infection, AGP and fibrinogen mRNA levels were reduced in treated infected animals. PX was ineffective in modifying those of A2M and albumin. These data demonstrate, in vivo, that different acute phase proteins are individually regulated in sepsis. The in vivo effects of PX treatment support the hypothesis that TNF-alpha plays an important role in the regulation of AGP production, whereas other factors seem to be involved in the regulation of A2M, fibrinogen, and albumin expression.

C-PKC-dependent modulation of plasma fibrinogen levels during the acute-phase response in young and old rats

The expression and subcellular distribution of liver cPKC α and β, nPKC δ and aPKC ζ isoenzymes and the plasma levels of fibrinogen were measured in young, 2- and 6-month-old, and aged, 24-month-old, normal and turpentine-treated rats, to induce an aseptic inflammatory condition and the acute-phase response. In young and old rats a down-regulated expression of cPKC α and, to a lesser extent, β isoenzymes, was observed 8 h after turpentine administration, i.e. at times preceding the maximal expression of fibrinogen mRNA. Under these conditions, the plasma fibrinogen levels peaked by 24 h in young animals, being up to 7-fold over the values of untreated controls at 72 h. By contrast, old untreated control rats showed 4-fold increases of basal plasma fibrinogen levels compared with young controls, with down-regulated expression of cPKC α. In old rats, treatment with turpentine increased up to 1.9-fold over the basal control values the fibrinogen concentration within 72 h. Levels similar to those of young turpentine-treated animals were reached at this time. The results of this study suggest a prominent role for cPKC α in eliciting the synthesis of fibrinogen after inducing an acute-phase response with turpentine administration in young as well as old rats. This isoform may act by regulating the serine phosphorylation of Stat3 transcription factor, whose activation is under IL-6 control, a multifunctional cytokine that is proving to be a major contributor to the acute-phase response. No evidence for a role of aPKC ζ or of nPKC δ was demonstrated under these conditions.