The ethanol extract of Zizyphus jujuba var. spinosa seeds ameliorates the memory deficits in Alzheimer's disease model mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The seeds of Zizyphus jujuba var. spinosa (Bunge) Hu ex H.F. Chow (Rhamnaceae) have long been treated as hypnotic agent for sleep disturbances in traditional Chinese and Korean medicine and many previous studies have focused on its effect in central nervous system. AIMS OF STUDY: The present study aimed to provide evidence showing that the ethanol extract of Zizyphus jujuba var. spinosa seeds (EEZS), which may regulate plasmin activity, has the potential to serve as a therapeutic agent for AD. MATERIALS AND METHODS: Synaptic function was determined by measuring long-term potentiation (LTP) in Shaffer-collateral pathway of the hippocampus. Protein levels of plasmin or plasminogen were examined using western blotting. Plasmin activity was measured using ELISA. Cognitive functions were measured using passive avoidance and object recognition tests in the 5XFAD mice. RESULTS: Our in vitro analysis revealed that EEZS-treated hippocampal slices from 5XFAD mice, a mouse model of AD, showed significantly higher long-term potentiation levels than did vehicle-treated hippocampal slices from 5XFAD mice (P < 0.05). Additionally, EEZS significantly elevated the plasmin level and activity in the hippocampal slices from 5XFAD mice (P < 0.05). Co-treating the slices with EEZS and 6-aminocaproic acid, a plasmin inhibitor, blocked the ameliorating effects of EEZS on the synaptic deficits that were present in 5XFAD mice. Compatible with the in vitro study, the results of our in vivo investigation showed that administering EEZS orally to 5XFAD mice ameliorated their memory impairments. Orally administered EEZS also elevated the plasmin level and activity in the hippocampus of 5XFAD mice. CONCLUSIONS: Collectively, our findings suggest that EEZS alleviates the AD-like symptoms in 5XFAD mice by regulating of plasmin activity and EEZS may be a suitable treatment for AD.

Liquid chromatographic nanofractionation with parallel mass spectrometric detection for the screening of plasmin inhibitors and (metallo)proteinases in snake venoms.

To better understand envenoming and to facilitate the development of new therapies for snakebite victims, rapid, sensitive, and robust methods for assessing the toxicity of individual venom proteins are required. Metalloproteinases comprise a major protein family responsible for many aspects of venom-induced haemotoxicity including coagulopathy, one of the most devastating effects of snake envenomation, and is characterized by fibrinogen depletion. Snake venoms are also known to contain anti-fibrinolytic agents with therapeutic potential, which makes them a good source of new plasmin inhibitors. The protease plasmin degrades fibrin clots, and changes in its activity can lead to life-threatening levels of fibrinolysis. Here, we present a methodology for the screening of plasmin inhibitors in snake venoms and the simultaneous assessment of general venom protease activity. Venom is first chromatographically separated followed by column effluent collection onto a 384-well plate using nanofractionation. Via a post-column split, mass spectrometry (MS) analysis of the effluent is performed in parallel. The nanofractionated venoms are exposed to a plasmin bioassay, and the resulting bioassay activity chromatograms are correlated to the MS data. To study observed proteolytic activity of venoms in more detail, venom fractions were exposed to variants of the plasmin bioassay in which the assay mixture was enriched with zinc or calcium ions, or the chelating agents EDTA or 1,10-phenanthroline were added. The plasmin activity screening system was applied to snake venoms and successfully detected compounds exhibiting antiplasmin (anti-fibrinolytic) activities in the venom of Daboia russelii, and metal-dependent proteases in the venom of Crotalus basiliscus. Graphical abstract ᅟ.

The Seed of Zizyphus jujuba var. spinosa Attenuates Alzheimer's Disease-Associated Hippocampal Synaptic Deficits through BDNF/TrkB Signaling.

Ziziphus jujuba is a plant, which bears fruits and seeds that are used for medicinal purposes in Traditional oriental medicine. The seed of Zizyphus jujuba var. spinosa (EZJ) has been also traditionally used for psychiatric disorders in Chinese and Korean medicines. Recent findings have indicated that EZJ improves memory impairment, a common symptom of various neurological diseases. However, the effects of EZJ on amyloid ß (Aß) toxicity, which is a main cause of Alzheimer's disease (AD), remain to be elucidated. To illuminate the potential anti-AD effect and mechanism in the mouse hippocampal tissue, we examined the effect of standardized EZJ on Aß-induced synaptic long-term potentiation (LTP) deficit in the hippocampal tissue. EZJ blocked Aß-induced LTP deficits in a concentration-dependent manner. Moreover, EZJ increased brain-derived neurotrophic factor (BDNF) level in naïve hippocampal slices. The finding that the blockade of BDNF receptor reduced the effect of EZJ suggests that EZJ ameliorates the Aß-induced LTP deficit through BDNF/topomyosin receptor kinase B (TrkB) signaling. However, transcription or translation inhibitors failed to block the effect of EZJ, suggesting that BDNF synthesis is not required for the action of EZJ on LTP. Finally, we found that EZJ stimulates plasmin activity. In contrast, plasmin inhibitor blocked the effect of EZJ on the Aß-induced LTP deficit. Our findings indicate that EZJ ameliorates Aß-induced LTP deficits through BDNF/TrkB signaling. This phenomenon is induced by a regulatory effect of EZJ on the post-translation modification of BDNF.

Using a Radial Diffusion Method to Investigate the Role of Plasmin Degradation of Fibrin in a Physical Model of an Early-phase Wound.

BACKGROUND: Fibrin clot formation, which acts to stabilize a wound following injury, is among the key early aspects of dermal wound healing. This preliminary matrix is eventually degraded via a process known as fibrinolysis and replaced with a collagen-rich matrix that continues to be remodeled to minimize scarring. Disruptions in these carefully coordinated events lead to certain undesirable conditions such as fibrosis and the formation of abnormal scars that are associated with excess amounts of collagen. The hypothesis proposed herein is that the presence of collagen (and potentially other molecules) in an early-phase model of healing alters fibrinolysis and that this effect can be attenuated with mediators of the process. MATERIALS AND METHODS: Laboratory in vitro experiments were conducted using agarose-fibrin gel systems with and without collagen to study fibrinolysis caused by plasmin (a serine protease that degrades fibrin) and the effects of aprotinin (a serine protease inhibitor) and bromelain (an extract from pineapple) on fibrin clot breakdown. The extent of fibrinolysis was monitored at various times (0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours) by measuring the size of rings of fibrinolysis following the diffusion of plasmin. The data obtained at 0.5, 12, and 24-hour time points were considered (because there was no difference found in the data collected for closer intermediates nor for the longer times beyond 24 hours) and were compared using the nonparametric Mann-Whitney U statistical significance test. RESULTS: The results obtained showed aprotinin significantly inhibited fibrinolysis in systems containing collagen, while bromelain improved fibrinolysis. In general, the presence of increasing amounts of collagen in the system decreased the extent of fibrinolysis. CONCLUSIONS: These findings support the notion that early-phase deposition of collagen contributes to disrupted fibrinolysis, which could lead to impaired healing as well as potentially facilitate control of fibrinolysis.

Characterization of a Novel Fibrinolytic Enzyme, BsfA, from Bacillus subtilis ZA400 in Kimchi Reveals Its Pertinence to Thrombosis Treatment.

Recently, the cardiovascular disease has been widely problematic in humans probably due to fibrin formation via the unbalanced Western style diet. Although direct (human plasmin) and indirect methods (plasminogen activators) have been available, bacterial enzyme methods have been studied because of their cheap and mass production. To detect a novel bacterial fibrinolytic enzyme, 111 bacterial strains with fibrinolytic activity were selected from kimchi. Among them, 14 strains were selected because of their stronger activity than 0.02 U of plasmin. Their 16S rRNA sequence analysis revealed that they belong to Bacillus, Leuconostoc, Propionibacterium, Weissella, Staphylococcus, and Bifidobacterium. The strain B. subtilis ZA400, with the highest fibrinolytic activity, was selected and the gene encoding fibrinolytic enzyme (bsfA) was cloned and expressed in the E. coli overexpression system. The purified enzyme was analyzed with SDS-PAGE, western blot, and MALDI-TOF analyses, showing to be 28.4 kDa. Subsequently, the BsfA was characterized to be stable under various stress conditions such as temperature (4-40°C), metal ions (Mn(2+), Ca(2+), K(2+), and Mg(2+)), and inhibitors (EDTA and SDS), suggesting that BsfA could be a good candidate for development of a novel fibrinolytic enzyme for thrombosis treatment and may even be useful as a new bacterial starter for manufacturing functional fermented foods.

Plasmin regulation through allosteric, sulfated, small molecules.

Plasmin, a key serine protease, plays a major role in clot lysis and extracellular matrix remodeling. Heparin, a natural polydisperse sulfated glycosaminoglycan, is known to allosterically modulate plasmin activity. No small allosteric inhibitor of plasmin has been discovered to date. We screened an in-house library of 55 sulfated, small glycosaminoglycan mimetics based on nine distinct scaffolds and varying number and positions of sulfate groups to discover several promising hits. Of these, a pentasulfated flavonoid-quinazolinone dimer 32 was found to be the most potent sulfated small inhibitor of plasmin (IC50 = 45 µM, efficacy = 100%). Michaelis-Menten kinetic studies revealed an allosteric inhibition of plasmin by these inhibitors. Studies also indicated that the most potent inhibitors are selective for plasmin over thrombin and factor Xa, two serine proteases in coagulation cascade. Interestingly, different inhibitors exhibited different levels of efficacy (40%-100%), an observation alluding to the unique advantage offered by an allosteric process. Overall, our work presents the first small, synthetic allosteric plasmin inhibitors for further rational design.

Inhibition of Thrombin-Activated Fibrinolysis Inhibitor Increases Survival in Experimental Kidney Fibrosis.

Uncontrolled diabetes, inflammation, and hypertension are key contributors to progressive renal fibrosis and subsequent loss of renal function. Reduced fibrinolysis appears to be a feature of ESRD, but its contribution to the fibrotic program has not been extensively studied. Here, we show that in patients with CKD, the activity levels of serum thrombin-activated fibrinolysis inhibitor and plasmin strongly correlated with the degree of renal function impairment. We made similar observations in rats after subtotal nephrectomy and tested whether pharmacologic inhibition of thrombin-activated fibrinolysis inhibitor with UK-396082 could reduce renal fibrosis and improve renal function. Compared with untreated animals, UK-396082-treated animals had reduced glomerular and tubulointerstitial fibrosis after subtotal nephrectomy. Renal function, as measured by an increase in creatinine clearance, was maintained and the rate of increase in proteinuria was reduced in UK-396082-treated animals. Furthermore, cumulative survival improved from 16% to 80% with inhibition of thrombin-activated fibrinolysis inhibitor. Taken together, these data support the importance of the fibrinolytic axis in regulating renal fibrosis and point to a potentially important therapeutic role for suppression of thrombin-activated fibrinolysis inhibitor activity.

[Screening and identification of Bacillus pumilus producing double active protein of anticoagulation and thrombolysis].

OBJECTIVE: The aim of this study was to screen bacteria that can produce antithrombotic. METHODS: We screened the target bacteria on VY/4 plate and casein plate from more than 20 samples such as water, soil, rabbit manure, sheep manure and deadwood. We detected the antithrombotic activity by fibrin plate and fibrin tube. We identified the target bacteria by morphological characteristics, physical and chemical properties and 16S DNA sequence homology. RESULTS: We obtained 5 strains that can produce antithrombotic. We found that the extracellular protein of strain LDS33 shows both stronger fibrinolytic activity and stronger anticoagulation activity. According to the morphology, physiochemical properties, 16S DNA sequencing and phylogenetic tree, strain LDS33 is identified as Bacillus pumilus. CONCLUSION: Bacillus pumilus LDS33 can produce highly active anticoagulation and thrombolysis double active protein.

Impact of stearic acid and oleic acid on hemostatic factors in the context of controlled diets consumed by healthy men.

The effects of stearic acid (STA) on cardiovascular disease risk beyond lipid and lipoprotein risk factors, including hemostasis, are unclear, particularly when compared with unsaturated fatty acids. The aim of the present study is to compare the effects of STA with those of oleic acid (OL) on markers of hemostasis. In a randomized crossover study, 50 men consumed six controlled diets for 5 weeks each (39% energy from fat, 15% energy from protein, 46% energy from carbohydrate (CHO)). Fat (8% energy) was replaced across diets by: STA, OL, CHO (control), trans fatty acids (TFAs), TFA/STA and 12:0-16:0 saturated fatty acids. Factor VIIc, plasminogen activator inhibitor-1 (PAI-1) and plasmin alpha-2-antiplasmin complex concentrations were not different between OL and STA (P>0.05). Compared with control, OL increased factor VIIc and PAI-1 (P≤0.05), whereas there were no differences with STA (P>0.05). STA and OL similarly affect markers of hemostasis in healthy men, within the context of a highly controlled diet.

Effects of black cohosh on the plasminogen activator system in vascular smooth muscle cells.

OBJECTIVE: The rhizome of the Cimicifuga racemosa plant (commonly known as black cohosh) has been used for menopausal complaints. Studies regarding the cardiovascular effects of black cohosh are lacking. We investigated the effect of black cohosh on the plasminogen activator system in cultured vascular smooth muscle cells (VSMCs). METHODS: VSMCs were isolated from rat aortae. Expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) proteins were evaluated by Western blot analysis and enzyme-linked immunosorbent assay, respectively. The activities of PAI-1 and t-PA in the conditioned media were assessed by fibrin overlay zymography. A 40% 2-propanol extract of black cohosh was used. RESULTS: Black cohosh extract (BcEx) stimulated the protein expression of PAI-1, but it did not affect that of t-PA. Vitamin E, a potent antioxidant, inhibited the BcEx-induced increase in PAI-1 expression, while ICI 182,780, an estrogen receptor antagonist, had no effect. Fibrin overlay zymography revealed that BcEx increased the activity of PAI-1 in the conditioned media, while concurrently decreasing that of free t-PA by inducing a binding to PAI-1. CONCLUSIONS: BcEx induces PAI-1 protein expression in the VSMCs likely via an oxidant mechanism. It also stimulates the enzyme activity of PAI-1 and reduces that of free t-PA. These findings suggest that black cohosh might exert a negative influence on fibrinolysis.

Pump blood processing, salvage and re-transfusion improves hemoglobin levels after coronary artery bypass grafting, but affects coagulative and fibrinolytic systems.

Cell saving systems are commonly used during cardiac operations to improve hemoglobin levels and to reduce blood product requirements. We analyzed the effects of residual pump blood salvage through a cell saver on coagulation and fibrinolysis activation and on postoperative hemoglobin levels. Thirty-four elective coronary artery bypass graft (CABG) patients were randomized. In 17 patients, residual cardiopulmonary bypass (CPB) circuit blood was transfused after the cell saving procedure (cell salvage group). In the other 17 patients, residual CPB circuit blood was discarded (control group). Activation of the coagulative, fibrinolytic and inflammatory systems was evaluated pre-operatively (Pre), 2 hours after the termination of CPB (T0) and 24 hours postoperatively (T1), measuring prothrombin fragment 1.2 (PF 1.2), plasmin-anti-plasmin (PAP), plasminogen activator inhibitor-1 (PAI-1) and interleukin-6 (IL-6). The cell salvage group of patients had a significant improvement in hemoglobin levels after processed blood infusion (2.7 ± 1.7 g/dL vs 1.2 ± 1.1 g/dL; p=0.003). PF1.2 levels were significantly higher after infusion (T0: 1175 ± 770 pmol/L vs 730 ± 237 pmol/L; p=0.037; T1: 331 ± 235 pmol/L vs 174 ± 134 pmol/L; p=0.026). Also, PAP levels were higher in the cell salvage group, although not significantly (T0: 253 ± 251 ng/mL vs 168 ± 96 ng/mL; p: NS; T1: 95 ± 60 ng/mL vs 53 ± 32 ng/mL; p: NS). No differences were found for PAI-1, IL-6, heparin levels or for red blood cell (RBC) transfusions. The cell salvage group of patients had increased chest tube drainage (749 ± 320 vs 592 ± 264; p: NS) and fresh frozen plasma transfusion rate (5 (29%) pts vs 0 pts; p<0.04). Pump blood salvage with a cell saving system improved postoperative hemoglobin levels, but induced a strong thrombin generation, fibrinolysis activation and lower fibrinolysis inhibition. These conditions could generate a consumption coagulopathy.

Sanguisorba minor extract suppresses plasmin-mediated mechanisms of cancer cell migration.

BACKGROUND: Sanguisorba minor, as well as several other edible herbs and vegetables, has been used extensively in traditional medicine. The observed beneficial effects can be attributed at least in part to the direct modulation of several enzymatic activities by its polyphenolic constituents. METHODS: The ethanol extract of Sanguisorba minor was characterized by reversed-phase liquid chromatography, and most relevant analytes were identified by multiple stage mass spectrometry. The whole extract and the most relevant isolated constituents were tested for their ability to modulate the activity of human plasmin both toward a synthetic substrate and in human breast cancer cell culture models. Kinetic and equilibrium parameters were obtained by a concerted spectrophotometric and biosensor-based approach. RESULTS: Quercetin-3-glucuronide was recognized as the compound mainly responsible for the in vitro plasmin inhibition by S. minor extract, with an inhibition constant in the high nanomolar range; in detail, our approach based on bioinformatic, enzymatic and binding analyses classified the inhibition as competitive. Most interestingly, cell-based assays showed that this flavonoid was effective in suppressing plasmin-induced loss of cancer cell adhesion. GENERAL SIGNIFICANCE: Our results show that the extract from Sanguisorba minor limits plasmin-mediated tumor cell motility in vitro, mostly due to quercetin-3-glucuronide. This glucuronated flavonoid is a promising template for rational designing of anticancer drugs to be used in the treatment of pathological states involving the unregulated activity of plasmin.

t-PA-specific modulation of a human blood-brain barrier model involves plasmin-mediated activation of the Rho kinase pathway in astrocytes.

Tissue-type plasminogen activator (t-PA) can modulate permeability of the neurovascular unit and exacerbate injury in ischemic stroke. We examined the effects of t-PA using in vitro models of the blood-brain barrier. t-PA caused a concentration-dependent increase in permeability. This effect was dependent on plasmin formation and potentiated in the presence of plasminogen. An inactive t-PA variant inhibited the t-PA-mediated increase in permeability, whereas blockade of low-density lipoprotein receptors or exposed lysine residues resulted in similar inhibition, implying a role for both a t-PA receptor, most likely a low-density lipoprotein receptor, and a plasminogen receptor. This effect was selective to t-PA and its close derivative tenecteplase. The truncated t-PA variant reteplase had a minor effect on permeability, whereas urokinase and desmoteplase were ineffective. t-PA also induced marked shape changes in both brain endothelial cells and astrocytes. Changes in astrocyte morphology coincided with increased F-actin staining intensity, larger focal adhesion size, and elevated levels of phosphorylated myosin. Inhibition of Rho kinase blocked these changes and reduced t-PA/plasminogen-mediated increase in permeability. Hence plasmin, generated on the cell surface selectively by t-PA, modulates the astrocytic cytoskeleton, leading to an increase in blood-brain barrier permeability. Blockade of the Rho/Rho kinase pathway may have beneficial consequences during thrombolytic therapy.

TSG-6 protein, a negative regulator of inflammatory arthritis, forms a ternary complex with murine mast cell tryptases and heparin.

TSG-6 (TNF-α-stimulated gene/protein 6), a hyaluronan (HA)-binding protein, has been implicated in the negative regulation of inflammatory tissue destruction. However, little is known about the tissue/cell-specific expression of TSG-6 in inflammatory processes, due to the lack of appropriate reagents for the detection of this protein in vivo. Here, we report on the development of a highly sensitive detection system and its use in cartilage proteoglycan (aggrecan)-induced arthritis, an autoimmune murine model of rheumatoid arthritis. We found significant correlation between serum concentrations of TSG-6 and arthritis severity throughout the disease process, making TSG-6 a better biomarker of inflammation than any of the other arthritis-related cytokines measured in this study. TSG-6 was present in arthritic joint tissue extracts together with the heavy chains of inter-α-inhibitor (IαI). Whereas TSG-6 was broadly detectable in arthritic synovial tissue, the highest level of TSG-6 was co-localized with tryptases in the heparin-containing secretory granules of mast cells. In vitro, TSG-6 formed complexes with the tryptases murine mast cell protease-6 and -7 via either heparin or HA. In vivo TSG-6-tryptase association could also be detected in arthritic joint extracts by co-immunoprecipitation. TSG-6 has been reported to suppress inflammatory tissue destruction by enhancing the serine protease-inhibitory activity of IαI against plasmin. TSG-6 achieves this by transferring heavy chains from IαI to HA, thus liberating the active bikunin subunit of IαI. Because bikunin is also present in mast cell granules, we propose that TSG-6 can promote inhibition of tryptase activity via a mechanism similar to inhibition of plasmin.

Influence of Aqueous Cream BP on corneocyte size, maturity, skin protease activity, protein content and transepidermal water loss.

BACKGROUND: Aqueous Cream BP is frequently prescribed for patients with eczema and is known to induce sensitivity in certain patients and also to decrease the thickness of the stratum corneum (SC). We have previously reported methodology to quantify corneocyte maturity and size, protease activity and protein content within different levels of the SC. OBJECTIVES: The aim of the present study was to investigate changes in corneocyte size, corneocyte maturity, selected protease activities, protein content and transepidermal water loss (TEWL) in normal skin after a 28-day application of Aqueous Cream BP. METHODS: The left and right mid volar forearms of six healthy female volunteers were selected as the study sites. Aqueous Cream BP was applied twice daily to treated sites for 28 days. At the end of this period, the site was tape-stripped and corneocyte maturity, corneocyte size and protease activity of the desquamatory kallikrein proteases, KLK5 and KLK7, and the inflammatory proteases tryptase and plasmin were measured. Protein content and TEWL measurements were also recorded. RESULTS: Corneocyte maturity and size decreased with increasing number of tape strips, and were significantly lower in treated sites compared with untreated sites. Protease activity and TEWL values were higher (P < 0·05) for the treated sites compared with untreated sites. The amount of protein removed from deeper layers of treated sites was significantly lower than from untreated sites. CONCLUSIONS: We report rapid minimally invasive measures of the effects of Aqueous Cream BP at the cellular and molecular level of the skin. Treatment with this formulation is associated with increased desquamatory and inflammatory protease activity. Changes in corneocyte maturity and size are also indicative of accelerated skin turnover induced by chronic application of this emollient. These findings question firmly the routine prescription of this preparation as a moisturizer in patients with atopic dermatitis.

Profibrinolytic effect of Enzamin, an extract of metabolic products from Bacillus subtilis AK and Lactobacillus.

Fibrinolytic system impairment contributes to the development of thrombotic disease such as cardiovascular disease and stroke. Therefore, an agent that increases fibrinolytic activity may be useful for the prevention of these diseases. In this study, to explore novel profibrinolytic agents, we examined the profibrinolytic effect of Enzamin, an extract of metabolic products from Bacillus subtilis AK and Lactobacillus in vitro and in vivo. Enzamin directly enhanced plasmin activity generated by tissue-type plasminogen activator (t-PA) by twofold but not by urokinase-type plasminogen activator (u-PA) in vitro, which was measured employing both the chromogenic substrate H-D: -Val-Leu-Lys-pNA (S-2251) and fibrin plate. Enzamin also increased plasmin activity generated by t-PA in the cell lysate and culture medium of endothelial cells, measured by fibrin zymography. Furthermore, the oral administration of a 1% concentration of Enzamin increased plasmin activity generated by t-PA by 1.7-fold but not by u-PA in the euglobulin fraction of mouse plasma. In conclusion, Enzamin has a unique ability to enhance the fibrinolytic activity through an increase in endogenous plasmin activity generated by t-PA released from endothelial cells, and may be a beneficial supplement for the prevention of thrombotic episodes.

[Cloning and expression of fibrinolytic enzyme cDNA sequence from Eupolyphaga sinensis].

OBJECTIVE: To obtain the cDNA sequence encoding fibrinolytic enzyme from Eupolyphaga sinensis and express it in prokaryotic and eukaryotic expression system. METHOD: The primers were designed according to the cDNA of other animals'fibrinolytic enzyme. The cDNA sequence was cloned by RT-PCR and 3 RACE. RESULT: Sequence 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. CONCLUSION: The 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.

Downregulation of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 by grape seed proanthocyanidin extract.

Urokinase plasminogen activator (uPA) system, comprising of uPA, its receptor uPAR and inhibitor, type 1 plasminogen activator inhibitor (PAI-1), plays a vital role in various biological processes involving extracellular proteolysis, fibrinolysis, cell migration and proliferation. The timely occurence of these processes are essential for normal wound healing. This study examines the regulation of uPA and PAI-1 by a natural polyphenol-rich compound, grape seed extract (GSE). GSE is reported to have beneficial effects in promoting wound healing. Fibroblast cells exposed to different doses of GSE for 18hours were processed for further studies such as ELISA, RT-PCR, western blotting, fibrinolytic assay, cell surface plasmin activity assay and in vitro wound healing assay. GSE treatment caused a significant downregulation of uPA and PAI-1 expression, both at the RNA and protein levels. ELISA also revealed a dose-dependent decrease in uPA and PAI-1 activities. Functional significance of the downregulation was evident in decreased fibrinolytic activity, concomittant with decreased cell-surface plasmin activity. In vitro wound healing studies showed that GSE also retarded the migration of cells towards the wounded region.

[Purification and activity calculated of fibrinolyric factors from Eupolyphaga sinensis].

Three kinds ( EFF-1, EFF-2 and EFF-3) of fibrinolytic factor were separated by ammonium sulphate precipitation, DEAE-cellulose and preparative PAGE electrophoresis from female Eupolyphaga sinensis Walker. Their molecular weights were proved to be 41kd, 32.9 kd and 30.6 kd respectively with SDS-PAGE electophoresis. Their Activities as plasminogen activator were 171.3 U/mg, 234.0 U/mg and 148.5 U/mg. In addition, EFF-2 and EFF-3 were not only fibrinolytic activities but also have plasminogen activator on fibrinous plate lacked of plasminogen . There had been no such components of plasminogen activator and fiberinolytic enzyme from Eupolyphaga sinensis reported yet.