PURPOSE: To evaluate the efficacy and complication of autologous plasmin (AP) injected before vitrectomy for rhegmatogenous retinal detachment (RRD). METHODS: Intravitreal AP injection (0.2 ml) was performed on the eyes without posterior vitreous detachment (PVD) 20 minutes before the vitrectomy for RRD. The extent of PVD was evaluated intraoperatively. Surgical PVD induction was performed and the ease of the procedure was graded. The extent of PVD, ease of PVD induction, and complications (including incidence of iatrogenic retinal break) were compared to those of the control eyes. In order to evaluate complications and measure activated partial thromboplastin time, a microbial culture of injected AP was performed and the rate of postoperative intraocular hemorrhage was investigated. Change in visual acuity and the rate of retinal reattachment were compared in order to evaluate the long-term surgical outcome. RESULTS: The extent of PVD was greater in the AP group than in the control group, and vitreal separation was facilitated by intravitreal AP injection. However, ease of PVD induction and frequency of iatrogenic retinal break found were not significantly different between cases and controls. Neither postoperative intraocular hemorrhage nor systemic coagulation abnormality occurred. Postoperative endophthalmitis and positive microbial culture of the AP solution were also not reported. There was no significant difference in the change in visual acuity and the rate of retinal reattachment between the two groups. CONCLUSIONS: Intravitreal AP injection can facilitate vitrectomy for RRD and has no effect on the rate of retinal reattachment.
Endophthalmitis ; Eye ; Fibrinolysin ; Hemorrhage ; Incidence ; Partial Thromboplastin Time ; Retinal Detachment ; Retinal Perforations ; Retinaldehyde ; Visual Acuity ; Vitrectomy ; Vitreous Detachment

OBJECTIVETo obtain the cDNA sequence encoding fibrinolytic enzyme from Eupolyphaga sinensis and express it in prokaryotic and eukaryotic expression system.

METHODThe primers were designed according to the cDNA of other animals'fibrinolytic enzyme. The cDNA sequence was cloned by RT-PCR and 3 RACE.

RESULTSequence analysis revealed that the length of the cDNA fragment was 672 bp and encoded a protein of 224 amino acid residues, the N end amino acid sequence residues was IVGG in accordance with other fibrinolytic enzyme. The cDNA sequence was expressed in E. coli, inactive protein was obtained. While expressed in Pichia pastoris, recombinant protein had fibrinolytic activity.

CONCLUSIONThe cDNA sequence of fibrinolytic enzyme from E. sinensis Walker was cloned and expressed for the first time and it proved a good basis for further functional study of the enzyme.

Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; Cockroaches ; chemistry ; enzymology ; genetics ; DNA, Complementary ; chemistry ; genetics ; Fibrinolysin ; chemistry ; genetics ; metabolism ; Gene Expression ; Insect Proteins ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; Sequence Alignment

BACKGROUND: In sepsis, large scale inflammatory responses can cause extensive collateral damage to the vasculature, because both coagulation and fibrinolysis are activated unevenly. Thrombin-activatable fibrinolysis inhibitor (TAFI) plays a role in modulating fibrinolysis. Since TAFI can be activated by both thrombin and plasmin, it is thought to be affected in sepsis. Hence, activated and inactivated TAFI (TAFIa/ai) may be used to monitor changes in sepsis. METHODS: TAFIa/ai-specific in-house ELISA can detect only the TAFIa/ai form, because the ELISA capture agent is potato tuber carboxypeptidase inhibitor (PTCI), which has selective affinity towards only the TAFIa and TAFIai isoforms. TAFIa/ai levels in plasma from 25 patients with sepsis and 19 healthy volunteers were quantitated with the in-house ELISA. RESULTS: We observed increased TAFIa/ai levels in samples from patients with sepsis (48.7+/-9.3 ng/mL) than in samples from healthy individuals (10.5+/-5.9 ng/mL). In contrast, no difference in total TAFI concentration was obtained between sepsis patients and healthy controls. The results suggest that TAFI zymogen was activated and that TAFIa/ai accumulated in sepsis. CONCLUSION: The detection of TAFIa/ai in plasma could provide a useful and simple diagnostic tool for sepsis. Uneven activation of both coagulation and fibrinolysis in sepsis could be caused by the activation of TAFI zymogen and elevation of TAFIa/ai. TAFIa/ai could be a novel marker to monitor sepsis and other blood-related disturbances.
Carboxypeptidase U ; Enzyme-Linked Immunosorbent Assay ; Fibrinolysin ; Fibrinolysis ; Humans ; Organothiophosphorus Compounds ; Plasma ; Protein Isoforms ; Sepsis ; Solanum tuberosum ; Thrombin

Excessive extracellular matrix (ECM) accumulation is the main feature of chronic renal disease including diabetic nephropathy. Plasminogen activator inhibitor (PAI)-1 is known to play an important role in renal ECM accumulation in part through suppression of plasmin generation and matrix metalloproteinase (MMP) activation. The present study examined the effect of PAI-1 antisense oligodeoxynucleotide (ODN) on fibronectin upregulation and plasmin/MMP suppression in primary mesangial cells cultured under high glucose (HG) or transforming growth factor (TGF)-beta1, major mediators of diabetic renal ECM accumulation. Growth arrested and synchronized rat primary mesangial cells were transfected with 1 microM phosphorothioate-modified antisense or control mis-match ODN for 24 hours with cationic liposome and then stimulated with 30 mM D-glucose or 2 ng/ml TGF-beta1. PAI-1 or fibronectin protein was measured by Western blot analysis. Plasmin activity was determined using a synthetic fluorometric plasmin substrate and MMP-2 activity analyzed using zymography. HG and TGF-beta1 significantly increased PAI-1 and fibronectin protein expression as well as decreased plasmin and MMP-2 activity. Transient transfection of mesangial cells with PAI-1 antisense ODN, but not mis-match ODN, effectively reversed basal as well as HG- and TGF-beta1-induced suppression of plasmin and MMP-2 activity. Both basal and upregulated fibronectin secretion were also inhibited by PAI-1 antisense ODN. These data confirm that PAI-1 plays an important role in ECM accumulation in diabetic mesangium through suppression of protease activity and suggest that PAI-1 antisense ODN would be an effective therapeutic strategy for prevention of renal fibrosis including diabetic nephropathy.
Animals ; Blotting, Western ; Diabetic Nephropathies ; Extracellular Matrix ; Fibrinolysin ; Fibronectins ; Fibrosis ; Glucose ; Liposomes ; Mesangial Cells ; Oligodeoxyribonucleotides ; Plasminogen ; Plasminogen Activator Inhibitor 1 ; Plasminogen Activators ; Rats ; Renal Insufficiency, Chronic ; Transfection ; Transforming Growth Factor beta1 ; Transforming Growth Factors ; Up-Regulation

PURPOSE: The aim of the present study was to quantify and compare the vitreolytic effect of plasmin, hyaluronidase, and a combination of the two. METHODS: Thirty-six rabbits were randomized into 3 groups: (A) twelve rabbits had an intravitreal injection of plasmin 1 U with hyaluronidase 10 U/0.1 mL into the right eye, (B) twelve rabbits had an injection of plasmin alone (1 U/0.1 mL), and (C) twelve rabbits had an injection of hyaluronidase alone (10 U/0.1 mL). The left eye of each rabbit was used as control, which was injected with 0.1 mL phosphate buffered saline (PBS). The eyes were enucleated 1 hour and 24 hours after injection. The volume of fluid-type vitreous and gel-type vitreous was measured with a micropipette using the melting point as the difference. Statistical analysis was performed and light microscopy was used to assess potential damage to the retinal tissue. RESULTS: The volume of remaining gel-type vitreous was measured as 52.5%, 60.3%, 59.2%, and 76.5% after 1 hour enucleation and as 44.6%, 56.7%, 56.1%, and 74.7%, after 24 hours enucleation in group A, B, C, and control group, respectively. Group A, B, and C showed statistically significant differences against the control group. Group A (plasmin with hyaluronidase) showed less remaining gel-type vitreous volume than a single injection of plasmin or hyaluronidase alone. CONCLUSIONS: Intravitreal injection of plasmin with hyaluronidase showed more vitreolytic effect than a single injection of plasmin or hyaluronidase alone. The enzyme may be useful in liquefying the vitreous, and may be a useful biochemical adjunct to vitrectomy.
Eye ; Fibrinolysin ; Freezing ; Hyaluronoglucosaminidase ; Intravitreal Injections ; Light ; Microscopy ; Rabbits ; Retinaldehyde ; Tissue Plasminogen Activator ; Vitrectomy

PURPOSE: The purposes of this study were to compare and quantify the expression of Stromelysin-1 and MT-MMP-1 in the gingival tissues of patients with type 2 diabetes mellitus(DM) and healthy adults with chronic periodontitis. MATERIALS AND METHODS: Gingival tissue samples were obtained during periodontal surgery or tooth extraction. According to the patient's systemic condition & clinical criteria of gingiva, each gingival sample was devided into three groups. Group 1 (n=8) is clinically healthy gingiva without bleeding and no evidence of bone resorption or periodontal pockets, obtained from systemically healthy 8 patients. Group 2 (n=8) is inflammed gingiva from patients with chronic periodontitis. Group 3 (n=8) is inflammed gingiva from patients with chronic periodontitis associated with type 2 DM. Tissue samples were prepared and analyzed by Western blotting. The quantification of Stromelysin-1 and MT-MMP-1 were performed using a densitometer and statistically analyzed by one-way ANOVA followed by Tukey test. RESULTS: In the analysis of expression levels, Stromelysin-1 and MT-MMP-1 expressions were similar in group 1 and 2. Stromelysin-1 and MT-MMP-1 expressions was more increased in group 3 than group 1, 2. The difference between group 3 and group 1, 2 was statistically significant. Also, in the interrelationship of Stromelysin-1 and MT-MMP-1 expressions, expressions of Stromelysin-1 and MT-MMP-1 showed increasing tendency in chronic periodontitis associated with type 2 DM and it seems that the MT-MMP-1 expressions were increasing in proportion to Stromelysin-1 expressions. CONCLUSION: It is suggested that Stromelysin-1 and MT-MMP-1 may be partly involved in the progression of periodontal inflammation associated with type 2 DM, as related to a metabolism of other factors, such as AGE, plasmin and other inflammatory mediators. Therefore, the expression levels of Stromelysin-1 and MT-MMP-1 can be inflammatory markers of periodontal inflammed tissue with type 2 DM.
Adult ; Blotting, Western ; Bone Resorption ; Chronic Periodontitis ; Diabetes Mellitus, Type 2 ; Fibrinolysin ; Gingiva ; Hemorrhage ; Humans ; Inflammation ; Matrix Metalloproteinase 14 ; Membranes ; Periodontal Pocket ; Tooth Extraction

PURPOSE:Vascular endothelial growth factor (VEGF) is known to play an important role in the process of angiogenesis and chronic inflammation. Plasminogen activator inhibitor (PAI)-1 and tissue plasminogen activator (tPA) are main regulators of the plasmin system. The functions of these components are shown to be closely associated and recent studies have reported their potential roles in the asthmatic airways. We determined plasma levels of soluble VEGF (sVEGF), PAI-1, tPA and endothelin (ET)-1 in children with recurrent early wheeze. Our purpose was to examine whether there would be any difference in these biomarkers in relation to the relapse rate of wheezing. METHODS:Fifty-eight children aged 2-6 years who were admitted with acute wheezing were enrolled. They were divided into two groups: patients with more than three relapses of wheezing (group 1, n=34) and those with less than one relapse (group 2, n=24). Plasma levels of sVEGF, PAI-1, ET-1 and tPA on admission were measured using ELISA in the two patient groups and controls (n=16). RESULTS:PAI-1, sVEGF and tPA significantly increased during acute wheezing episode. The levels of these biomarkers were significantly higher in group 1 than in group 2 (P<0.01). ET-1 showed no significant difference between the patient groups and controls. CONCLUSION:Our study showed significantly elevated plasma levels of sVEGF and plasmin system regulators in children with recurrent early wheeze, which was even higher in the group with more frequent relapses. Our results suggest that these biomarkers may be associated with airway inflammation and may contribute to the later development of asthma in these children.
Asthma ; Biomarkers ; Child ; Endothelial Growth Factors ; Endothelins ; Enzyme-Linked Immunosorbent Assay ; Fibrinolysin* ; Humans ; Inflammation ; Plasma* ; Plasminogen Activator Inhibitor 1 ; Plasminogen Activators ; Recurrence ; Respiratory Sounds ; Tissue Plasminogen Activator ; Vascular Endothelial Growth Factor A*

PURPOSE:Vascular endothelial growth factor (VEGF) is known to play an important role in the process of angiogenesis and chronic inflammation. Plasminogen activator inhibitor (PAI)-1 and tissue plasminogen activator (tPA) are main regulators of the plasmin system. The functions of these components are shown to be closely associated and recent studies have reported their potential roles in the asthmatic airways. We determined plasma levels of soluble VEGF (sVEGF), PAI-1, tPA and endothelin (ET)-1 in children with recurrent early wheeze. Our purpose was to examine whether there would be any difference in these biomarkers in relation to the relapse rate of wheezing. METHODS:Fifty-eight children aged 2-6 years who were admitted with acute wheezing were enrolled. They were divided into two groups: patients with more than three relapses of wheezing (group 1, n=34) and those with less than one relapse (group 2, n=24). Plasma levels of sVEGF, PAI-1, ET-1 and tPA on admission were measured using ELISA in the two patient groups and controls (n=16). RESULTS:PAI-1, sVEGF and tPA significantly increased during acute wheezing episode. The levels of these biomarkers were significantly higher in group 1 than in group 2 (P<0.01). ET-1 showed no significant difference between the patient groups and controls. CONCLUSION:Our study showed significantly elevated plasma levels of sVEGF and plasmin system regulators in children with recurrent early wheeze, which was even higher in the group with more frequent relapses. Our results suggest that these biomarkers may be associated with airway inflammation and may contribute to the later development of asthma in these children.
Asthma ; Biomarkers ; Child ; Endothelial Growth Factors ; Endothelins ; Enzyme-Linked Immunosorbent Assay ; Fibrinolysin* ; Humans ; Inflammation ; Plasma* ; Plasminogen Activator Inhibitor 1 ; Plasminogen Activators ; Recurrence ; Respiratory Sounds ; Tissue Plasminogen Activator ; Vascular Endothelial Growth Factor A*

The effect of recombinant microplasmin (micro-plasmin) on acute cerebral infarction was evaluated in rats, and compared with recombinant tissue plasminogen activator (rt-PA). After the model of middle cerebral artery occlusion (MCAO) was established by autologous blood clots, different doses of micro-plasmin (2.5, 5, and 10 mg x kg(-1)) were administered into the thrombus intra-arterial. Twelve hours after administration of micro-plasmin, the neurological deficit score of rats was recorded and the infarct volumes were determined. Bleeding time (BT), fibrin degradation product (FDP) concentration in serum and thrombin time (TT), prothrombin time (PT) and fibrinogen (FIB) concentration in plasma were tested after administration. Intra-arterial administration of micro-plasmin could reduce significantly neurological deficit score and infarct volumes in MCAO rats. FDP concentration increased significantly as compared with model group. There were no significant differences in TT, PT and BT. FIB concentration reduced markedly as compared with model group, but had no significant difference as compared with sham group. The results suggest that micro-plasmin is effective in treatment of rat acute cerebral infarction, and has no significant influence on fibrinolytic system and blood clotting system, indicating that micro-plasmin may be useful for treatment of acute cerebral infarction, and not lead to hemorrhage. Micro-plasmin seems to be distinguished from clinical used rt-PA by its no hemorrhage effect.
Animals ; Bleeding Time ; Brain ; pathology ; Cerebral Hemorrhage ; etiology ; metabolism ; pathology ; Cerebral Infarction ; drug therapy ; pathology ; Fibrin Fibrinogen Degradation Products ; metabolism ; Fibrinogen ; metabolism ; Fibrinolysin ; pharmacology ; Infarction, Middle Cerebral Artery ; complications ; Male ; Peptide Fragments ; pharmacology ; Prothrombin Time ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; pharmacology ; Thrombin Time ; Tissue Plasminogen Activator ; pharmacology

OBJECTIVEThe purpose of this work was to investigate the distribution pattern of fibrinolytic factors and their inhibitors in rabbit tissues.

METHODSThe components of the fibrinolytic system in extracts from a variety of rabbit tissues, including tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), plasminogen (Plg), plasmin (Pl) and alpha(2) plasmin inhibitor (alpha(2)PI), were determined by colorimetric assay.

RESULTSThe tissue extracts in renal, small intestine, lung, brain and spleen demonstrated strong fibrinolytic function, in which high activity of tPA, Plg and Pl was manifested; whereas in skeletal muscle, tongue and stomach, higher activity of PAI-1 and alpha(2)PI showed obviously. Also excellent linear correlations were found between levels of tPA and PAI-1, Pl and alpha(2)PI, Plg and Pl. In related tissues, renal cortex and renal marrow showed distinctly higher activity of tPA and lower activity of PAI-1, with the levels of Plg and Pl in renal cortex being higher than those in renal marrow, where the alpha(2)PI level was higher than that in renal cortex. Similarly, the levels of tPA, Plg and Pl in small intestine were higher than those in large intestine, but with respect to PAI-1 and alpha(2)PI, the matter was reverse. In addition, the fibrinolytic activity in muscle tissue was lower, however, the levels of tPA, Plg, and Pl in cardiac muscle were obviously higher than those in skeletal muscles, and the levels of PAI-1 and alpha(2)PI were significantly lower than those in skeletal muscle.

CONCLUSIONOur data demonstrate that a remarkable difference of the fibrinolytic patterns exists in rabbit tissues, which has probable profound significance in understanding the relationship between the function of haemostasis or thrombosis and the physiologic function in tissues.

Animals ; Female ; Fibrinolysin ; metabolism ; Fibrinolysis ; Gastric Mucosa ; metabolism ; Gastrointestinal Tract ; metabolism ; Intestinal Mucosa ; metabolism ; Male ; Organ Specificity ; Plasminogen ; metabolism ; Plasminogen Activator Inhibitor 1 ; metabolism ; Rabbits ; Tissue Extracts ; metabolism ; Tissue Plasminogen Activator ; metabolism ; alpha-2-Antiplasmin ; metabolism