OBJECTIVETo construct the siRNA plasmid for mfgl2 gene, which has been reported to be involved in a variety of disease developments including fulminant viral hepatitis, acute rejection of allo/zero transplantation and fetal loss syndrome, and to investigate its inhibitory effects on mfgl2 expression in vitro.

METHODSA plasmid p-mfgl2shRNA complimentary to the sequence responsible for the functional domain of mouse fgl2 (mfgl2) was constructed. The pcDNA3.1 mfgl2 expression construct was able to show a satisfactory fgl2 protein expression. The plasmid expression pEGFP and a construct expressing irrelevant shRNA with a random combination of the p-mfgl2shRNA sequence were used as controls. A pEGFP-mfgl2 expressing mfgl2-EGFP fusion protein was also constructed for screening of the effect of p-mfgl2shRNA on the mfgl2 expression.

RESULTSCotransfection of p-mfgl2shRNA with pEGFP-mfgl2 decreased green fluorescent cells and the lightness of fluorescence within the cells at the 24 h, 48 h and 72 h post-transfection when compared with that in the control groups which were solely transfected with pEGFP-mfgl2. Furthermore the mfgl2 expression was significantly reduced when the pcDNA3.1 mfgl2 expression construct was cotransfected with p-mfgl2shRNA both at mRNA level by RT-PCR and protein level by RT-PCR, immunohistochemistry staining and FACS in both CHO cell and Hela cell lines.

CONCLUSIONSThe study demonstrated that the construct of p-mfgl2shRNA successfully interfered in the mfgl2 expression in vitro. It provides a basis for a further investigation of effect in vivo.

Animals ; Fibrinogen ; biosynthesis ; genetics ; Gene Expression ; Mice ; Plasmids ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics

OBJECTIVETo explore the relationship between disseminated intravascular coagulation (DIC) and levels of plasma thrombinogen segment 1+2 (F1+2), D-dimer (D-D), and thrombomodulin (TM) in patients with severe multiple injuries.

METHODSIn this study, 66 patients (49 males and 17 females, aged 15-74 years, mean=38.4 years) with multiple injuries, who were admitted to our hospital within 24 hours after injury with no personal or family history of hematopathy or coagulopathy, were divided into a minor injury group (ISS<16, n=21) and a major injury group (ISS>or=16, n=45) according to the injury severity. The patients in the major injury group were divided into a subgroup complicated with DIC (DIC subgroup, n=12) and a subgroup complicated with no DIC (non-DIC subgroup, n=33). Ten healthy people (7 males and 3 females, aged 22-61 years, mean=36.5 years+/-9.0 years), who received somatoscopy and diagnosed as healthy, served as the control group. Venous blood samples were collected once in the control group and 1, 3 and 7 days after trauma in the injury groups. The F1+2 and TM concentrations were determined by enzyme linked immunosorbent assay (ELISA), and D-D concentrations were measured by automated latex enhanced immunoassay.

RESULTSF1+2, D-D and TM levels were higher in the minor and major injury groups than in the control group. They were markedly higher in the major injury group than in the minor injury group. In the non-DIC subgroup, F1+2 levels declined gradually while D-D and TM levels declined continuously. In the DIC subgroup, F1+2 and D-D levels remained elevated while TM levels exhibited an early rise and subsequent decrease. Plasma F1+2, D-D and TM levels were higher in the DIC patients than in the non-DIC patients. Injury-induced increases in F1+2, D-D and TM plasma levels had significant positive correlation with each other at each time point.

CONCLUSIONSBesides being related to trauma severity, F1+2, D-D and TM levels correlate closely with the occurrence of posttraumatic DIC. Therefore, changes in plasma F1+2, D-D and TM levels may predict the occurrence of DIC.

Adolescent ; Adult ; Aged ; Disseminated Intravascular Coagulation ; blood ; Female ; Fibrin Fibrinogen Degradation Products ; analysis ; Humans ; Male ; Middle Aged ; Multiple Trauma ; blood ; Thrombin ; biosynthesis ; Thrombomodulin ; blood

OBJECTIVEViral hepatitis remains a major public health problem and the most common type of liver disease worldwide. There are an increasing number of patients with chronic hepatitis B who develop acute hepatitis on chronic condition (AOC) and die of acute hepatic failure both as a result of lack of understanding of the pathogenesis of the disease and lack of effective treatment. The hallmark of AOC is the extreme rapidity of the necromicroinflammatory process resulting in widespread or total hepatocellular necrosis in weeks or even days. Our previous studies have shown in an experimental animal model of fulminant viral hepatitis caused by murine hepatitis virus strain 3, the importance of macrophage activation, and expression of a unique gene mfgl 2 which encodes a serine protease capable of directly cleaving prothrombin to thrombin, resulting in widespread fibrin deposition within the liver and hepatocyte necrosis. The undergoing study in this report is designed to identify the role of hfgl 2 (human fibrinogen like protein 2) /fibroleukin in patients with viral hepatitis.

METHODSLiver tissues were obtained from 23 patients with AOC hepatitis B, and from 13 patients with inactive chronic hepatitis B (CHB) and 14 patients with chronic hepatitis B with cirrhosis during the year of 1995 to the end of 2001. Liver biopsies were performed within 30 min after the patients were diagnosed with death as a result of acute hepatic failure. Liver samples were also obtained from 4 liver donors as normal controls. In addition, peripheral blood mononuclear cells (PBMC) were isolated from 30 patients (unpaired) with AOC hepatitis B and 10 patients with CHB during the May of 2001 to March of 2002 and 10 healthy volunteer as negative control. PBMCs were freshly isolated and smeared on slides and kept at -80 degree C for further use. Histological sections were stained with hemotoxylin and eosin. A 169 bp of hfgl 2 cDNA probe and a polyclonal or monoclonal antibody against hfgl 2 were used to detect the expression of hfgl 2 mRNA and protein in liver samples as well as PBMC by immune histochemistry separately.

RESULTSLiver tissues from the patients with acute on chronic hepatitis had classical pathological features of acute necroinflammation. Hfgl 2 was detected by immune histochemistry in 21 of 23 patients (91.3%) in liver sections from patients with acute on CHB, while only 1 of 13 patients (7.7%) with CHB and cirrhosis and no evidence of active disease had hfgl 2 mRNA or protein expression. 28 of 30 patients (93.3%) with acute on CHB and 1 of 10 with CHB were detected with hfgl 2 expression in PBMC. There was no hfgl 2 expression in either the liver tissue or the PBMC from the normal donors. There was positive correlation of hfgl 2 expression and the severity of the disease displayed by the value of bilirubin and PT.

CONCLUSIONThe molecular and cellular results reported here in patients with acute on chronic hepatitis and who died of acute hepatic failure correlates with previous report in 8 patients with fulminant hepatic failure (FHF) and mimic closely the changes observed in the murine model of fulminant viral hepatitis in which the pathogenesis of the disease has been studied in a stepwise fashion. This study further suggests that virus induced hfgl 2 prothrombinase/fibroleukin expression and the potent function of the protein it encodes plays a pivotal role in initiating acute severe hepatitis on the baseline of chronic hepatitis. The measurement of hfgl 2/fibroleukin expression in PBMC may serve as a useful marker to monitor the severity of disease in patients with the AOC hepatitis B and a target for therapeutic intervention.

Adolescent ; Adult ; Child ; Child, Preschool ; Female ; Fibrinogen ; biosynthesis ; genetics ; Hepatitis B, Chronic ; metabolism ; pathology ; Humans ; Infant ; Leukocytes, Mononuclear ; metabolism ; Liver ; metabolism ; pathology ; Male ; Middle Aged ; Prognosis ; RNA, Messenger ; biosynthesis ; genetics ; Severity of Illness Index ; T-Lymphocytes ; metabolism

The aim of the present study was to evaluate plasma total homocysteine (Hcys) and serum fibrinogen concentrations in subclinical hypothyroid (SH) and overt hypothyroid patients before and after L-thyroxine (LT4) replacement and to compare them in euthyroid subjects. Fifteen SH and 20 hypothyroid premenopausal women were recruited in the study. We measured fasting plasma levels of Hcys and serum levels of free thyroxine (fT4), free triiodothyronine (fT3), thyrotropin (TSH), folate, vitamin B12, fibrinogen, renal functions, and lipid profiles in patients with SH and overt hypothyroid patients before and after LT4 treatment. Eleven healthy women were included in the study as a control group. Pretreatment Hcys levels were similar in SH and control subjects, whereas mean fibrinogen level of SH patients was higher than that of control subjects (p<0.05). Baseline Hcys (p<0.01) and fibrinogen (p<0.001) levels of the overt hypothyroid patients were significantly higher than those of the healthy subjects, and the pretreatment Hcys levels decreased with LT4 treatment (p<0.001). In conclusion, our data support that SH is not associated with hyperhomocysteinemia and Hcys does not appear to contribute to the increased risk for atherosclerotic disease in patients with SH.
Adult ; Case-Control Studies ; Female ; Fibrinogen/biosynthesis ; Folic Acid/blood ; Homocysteine/*blood ; Humans ; Hypothyroidism/*blood/*diagnosis ; Kidney/metabolism ; Middle Aged ; Thyrotropin/blood ; Thyroxine/*blood ; Triiodothyronine/blood ; Vitamin B 12/blood

The objective of this study was to investigate the correlation between factor XIII (FXIII) activity and disseminated intravascular coagulation (DIC) parameters and also to evaluate the clinical usefulness of DIC diagnosis. Citrated plasma from eighty patients with potential DIC was analyzed for FXIII activity. The primary patient conditions (48 male and 32 female, mean age, 51 years) were malignancy (n = 29), infection (n = 25), inflammation (n = 6), heart disease (n= 3), thrombosis (n = 2), injury (n = 2), and other miscellaneous conditions (n = 13). FXIII testing was performed using the CoaLinkTM FXIII Incorporation Assay Kit (PeopleBio Inc.). Among 80 patients who were suspected to have DIC based on clinical analysis, 46 (57.5%) fulfilled the overt DIC criteria (DIC score > = 5) according to the International Society of Thrombosis and Haemostasis. FXIII levels in the plasma were significantly decreased in overt DIC compared to non-overt DIC patients (mean 75.1% and 199.7% respectively, p < 0.0001). Interestingly, we found a significant inverse correlation between DIC scores and FXIII activity. In addition, FXIII activity significantly correlated with other hemostatic markers that included platelet count, prothrombin time, activated partial thromboplastin time, fibrinogen, and D-dimer. FXIII levels were significantly lower in patients with liver or renal dysfunction. In conclusion, FXIII cross-linking activity measurements may have differential diagnostic value as well as predictive value in patients who are suspected to have DIC.
Prothrombin Time ; Platelet Count ; Partial Thromboplastin Time ; Middle Aged ; Male ; Liver Diseases/pathology ; Liver/pathology ; Kidney Diseases/pathology ; Kidney/pathology ; Inflammation ; Humans ; Hemostasis ; Fibrin Fibrinogen Degradation Products/biosynthesis ; Female ; Factor XIII/*biosynthesis ; Disseminated Intravascular Coagulation/*blood/*diagnosis ; Cross-Linking Reagents/pharmacology/therapeutic use ; Blood Coagulation Tests ; Aged ; Adult

To investigate the effect of GST-KGDX (glutathione S-transferase-Lys-Gly-Asp-X) fusion protein, GP IIb/IIIa receptor antagonist, on platelet function in vitro. The KGDX (Lys-Gly-Asp-X) gene was assembled from 2 synthetic oligonucleotides, 36 bp in length, using BamH I and Xho I restriction enzyme sites at the end of the gene for cloning into the expression vector pGEX4T-1. Expression of fusion protein was directed by the tac promoter. The Escherichia coli DH5a contained the plasmid pGEX-4T-1-KGDX was expressed by 37 degrees C heat induction. The fusion protein of KGDX with glutathione S-transferase (GST-KGDX) was purified in one step from the bacterial lysate by glutathione-agarose beads for affinity chromatography. GST-KGDX was found to be soluble and abundant, the yield of 35 mg/L of cultures was obtained. The GST-KGDX was expressed in E. coli to a level of 48.02% of total cellular protein. GST-KGDX inhibited ADP-induced human platelet aggregation stronger than GST (P < 0.05 or < 0.01). In flow cytometry assay for fibrinogen binding, both GST and GST-KGDX inhibited platelet aggregation by binding with high affinity to GPIIb/IIIa. Mean fluorescence intensity of GST-KGDX fusion protein was significantly higher than that of GST. It is concluded that the GST-KGDX fusion protein can be produced by E. coli and used as an antiplatelet agent.
Adult ; Escherichia coli ; genetics ; Female ; Fibrinogen ; metabolism ; Flow Cytometry ; Glutathione Transferase ; pharmacology ; Humans ; Male ; Oligopeptides ; pharmacology ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; isolation & purification ; pharmacology ; Platelet Glycoprotein GPIIb-IIIa Complex ; antagonists & inhibitors ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; isolation & purification ; pharmacology