Blood Clot Lysis Assay Research Articles

Phytochemical analysis and biological investigation of Cheilanthes tenuifolia (Burm.f.) Swartz.

Introduction: Cheilanthes tenuifolia is an evergreen ornamental small fern, belonging to the family Pteridaceae, that grows in warm and rocky regions worldwide. Many species of Cheilanthes genus are evidently endowed with important phytochemicals and bioactivities. This study aimed to perform a preliminary phytochemical analysis of Cheilanthes tenuifolia leaves alongside an evaluation of free radical scavenging, anti-inflammatory, antimicrobial, and clot lysis activities of extract fractions. Materials and methods: A preliminary phytochemical analysis was done after fractionation of ethanolic extract (ECT) with n-hexane (HCT) and chloroform (CCT). Then, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, egg albumin and RBC membrane stabilization tests, disc diffusion, and human blood clot lysis assays were performed. Results: Phytochemical investigations suggested that the plant is rich in alkaloids, glycosides, tannins, and flavonoids. All obtained fractions exhibited concentration-dependent radical scavenging, inhibition of egg protein denaturation and RBC membrane lysis capacities. Except for antifungal tests, ECT exhibited better DPPH radical scavenging, anti-inflammatory, antibacterial, and clot lysis capacities than HCT and CCT fractions. However, all fractions exhibited a mild anti-inflammatory activity. Conclusion: C. tenuifolia might be a good source of antioxidant, anti-microbial, and anti-atherothrombotic agents. Further studies are required to isolate and characterize the active principles liable for each bioactivity, along with possible molecular interactions.

Unveiling the Potent Fibrino(geno)lytic, Anticoagulant, and Antithrombotic Effects of Papain, a Cysteine Protease from Carica papaya Latex Using κ-Carrageenan Rat Tail Thrombosis Model

While fibrinolytic enzymes and thrombolytic agents offer assistance in treating cardiovascular diseases, the existing options are associated with a range of adverse effects. In our previous research, we successfully identified ficin, a naturally occurring cysteine protease that possesses unique fibrin and fibrinogenolytic enzymes, making it suitable for both preventing and treating cardiovascular disorders linked to thrombosis. Papain is a prominent cysteine protease derived from the latex of Carica papaya. The potential role of papain in preventing fibrino(geno)lytic, anticoagulant, and antithrombotic activities has not yet been investigated. Therefore, we examined how papain influences fibrinogen and the process of blood coagulation. Papain is highly stable at pH 4–11 and 37–60 °C via azocasein assay. In addition, SDS gel separation electrophoresis, zymography, and fibrin plate assays were used to determine fibrinogen and fibrinolysis activity. Papain has a molecular weight of around 37 kDa, and is highly effective in degrading fibrin, with a molecular weight of over 75 kDa. Furthermore, papain-based hemostatic performance was confirmed in blood coagulation tests, a blood clot lysis assay, and a κ-carrageenan rat tail thrombosis model, highlighting its strong efficacy in blood coagulation. Papain shows dose-dependent blood clot lysis activity, cleaves fibrinogen chains of Aα, Bβ, and γ-bands, and significantly extends prothrombin time (PT) and activated partial thromboplastin time (aPTT). Moreover, the mean length of the infarcted regions in the tails of Sprague–Dawley rats with κ-carrageenan was shorter in rats administered 10 U/kg of papain than in streptokinase-treated rats. Thus, papain, a cysteine protease, has distinct fibrin and fibrinogenolytic properties, suggesting its potential for preventing or treating cardiovascular issues and thrombosis-related diseases.

PRELIMINARY PHYTOCHEMICAL SCREENING AND BIOACTIVITY INVESTIGATIONS OF AVORRHEA CARAMBOLA L.

Averrhoa carambola L. (A. carambola) is particularly well-known for its distinctive star-shaped fruit (Star fruit), cultivated in Southeast Asia, the Indian subcontinent, and northern and southern America. Historically, it has been used for a long time to cure many ailments, including vomiting, fever, arthralgia, cough, hypertension, hangovers, chronic paroxysmal headache, hyperglycemia, and diabetic nephropathy. This study aimed to examine its preliminary phytochemical and some biological properties. For this, fresh leaves of A. carambola were extracted (hot and cold) with ethanol, which was then fractionated with n-hexane and chloroform. Following a preliminary investigation of phytochemicals, radical scavenging, membrane stabilizing, alpha-glucosidase inhibitory, and clot lysis effects were evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, egg albumin model, alpha-amylase assay, and human blood clot lysis assay, respectively using suitable reference standards. The results suggest that A. carambola contains alkaloids, glycosides, steroids, tannins, flavonoids, saponins, and reducing sugars. All the organic fractions demonstrated significant (p <0.05) radical scavenging, membrane stabilizing, alpha-glucosidase inhibitory, and clot lysis activities in a concentration-dependent approach. Except for DPPH radical scavenging, the ethanol fraction showed better activities than the n-hexane and chloroform fractions. In summary, A. carambola might be a potential source of antioxidant, anti-inflammatory, hypoglycemic, and atherothrombolytic agents. Further studies are required to isolate, characterize, and establish molecular mechanisms for each component’s biological effects.

Exploring the Fibrin(ogen)olytic, Anticoagulant, and Antithrombotic Activities of Natural Cysteine Protease (Ficin) with the κ-Carrageenan-Induced Rat Tail Thrombosis Model.

Although fibrinolytic enzymes and thrombolytic agents help in cardiovascular disease treatment, those currently available have several side effects. This warrants the search for safer alternatives. Several natural cysteine protease preparations are used in traditional medicine to improve platelet aggregation and thrombosis-related diseases. Hence, this study aimed to investigate the effect of ficin, a natural cysteine protease, on fibrin(ogen) and blood coagulation. The optimal pH (pH 7) and temperature (37 °C) for proteolytic activity were determined using the azocasein method. Fibrinogen action and fibrinolytic activity were measured both electrophoretically and by the fibrin plate assay. The effect of ficin on blood coagulation was studied by conventional coagulation tests: prothrombin time (PT), activated partial thromboplastin time (aPTT), blood clot lysis assay, and the κ-carrageenan thrombosis model. The Aα, Bβ, and γ bands of fibrinogen are readily cleaved by ficin, and we also observed a significant increase in PT and aPTT. Further, the mean length of the infarcted regions in the tails of Sprague–Dawley rats was shorter in rats administered 10 U/mL of ficin than in control rats. These findings suggest that natural cysteine protease, ficin contains novel fibrin and fibrinogenolytic enzymes and can be used for preventing and/or treating thrombosis-associated cardiovascular disorders.

Optimization of high expression and purification of recombinant streptokinase and in vitro evaluation of its thrombolytic activity

Streptokinase (SK) is a potent plasminogen activator naturally produced by beta-hemolytic streptococcus bacteria and used as a thrombolytic drug. Optimize high yield production of recombinant streptokinase (rSK) in Escherichia coli and evaluate its thrombolytic activity . Synthetic gene encoding mature SK protein with optimization for rare codons and mRNA secondary structure was cloned into the expression vector pET-3a and transformed into Escherichia coli BL21 (DE3). Seed banks were established for high rSK expression clones. The native rSK protein expression was optimized using IPTG induction. The nonsoluble rSK inclusion bodies were purified, denatured in 6 M guanidinium chloride, and refolded using the rapid dilution method. The refolded rSK protein was purified using anion exchange chromatography and evaluated with ELISA. The activity of rSK was evaluated using the casein digestion method and in vitro blood clot lysis assay with reference drug Sedonase as standard. Seed banks with high stable expression of native rSk (MW 47 kDa) were established. High rSK expression was optimized using 1 mM IPTG at bacterial OD600 0.6. The refolded rSK was prepared and purified successfully with high productivity (494 mg purified rsk/L culture). Using ELISA, the purified rSK molecular identity and conservation of native SK epitopes were confirmed. The enzymatic activity of the purified rSK was 1.945x10 6 IU/mg with 62.94 ± 2.3% clot lysis efficiency. A high yield production of proper rSK protein with in vitro thrombolytic activity similar to commercial SK has been achieved, suggesting a more cost-effective industrial production of its biosimilar drug.

Targeted thrombolysis by using c-RGD-modified N,N,N-Trimethyl Chitosan nanoparticles loaded with lumbrokinase

Lumbrokinase (LK) has strong fibrinolytic and thrombolytic activities, but it has a short half-life, can be easily inactivated, and may cause hemorrhage as a side effect. This study develops a potential thrombolytic therapy by fabricating N,N,N-Trimethyl Chitosan (TMC) nanoparticles modified with the cyclic Arg-Gly-Asp-Phe-Lys peptide (c-RGD) and loaded with LK (i.e. c-RGD-LK-NPs). The binding of c-RGD to platelet membrane GPIIb/IIIa receptors is expected to enable targeted delivery of the c-RGD-conjugated TMC to the thrombus. The synthesized c-RGD-LK-NPs had a mean particle size of 232.0 nm, zeta potential of 19.8 mV, entrapment efficiency of 52.7% ± 2.5%, and loading efficiency of 17.4% ± 0.65%. Transmission electron microscopy showed that they were generally spherical. The c-RGD-LK-NPs gave a cumulative in vitro LK release of 80.6% over 8 h, and the activity of LK was close to 80%, indicating that the nanoparticles protected the activity of LK. In vitro blood clot lysis assays were carried out and in vivo thrombolysis effect was tested in Sprague-Dawley rats carotid artery thrombus model. In all cases, the c-RGD-LK-NPs showed superior performance compared with the free LK and the unmodified TMC nanoparticles loaded with LK. The c-RGD-LK-NPs reagent is expected to be potentially useful in treating thromboembolic diseases.

Annexin V-Conjugated Mixed Micelles as a Potential Drug Delivery System for Targeted Thrombolysis

To alleviate the hemorrhagic side effect of thrombolysis therapy, a thrombus targeted drug delivery system based on the specific affinity of Annexin V to phosphatidylserine exposed on the membrane surface of activated platelet was developed. The amphiphilic and biodegradable biomaterial, polycaprolactone-block-poly(2-(dimethylamino)ethyl methacrylate)-block-poly(2-hydroxyethyl methacrylate) (PCL-b-PDMAEMA-b-PHEMA (PCDH)) triblock polymer, was synthesized via ring opening polymerization (ROP) and atom transfer radical polymerization (ATRP) to use as the nanocarriers of thrombolytic drug. In order to conjugate Annexin V to the polymer, PCDH was modified by succinic anhydride via ring-opening reaction to introduce the carboxyl group (PCDH-COOH). After preparation of PCDH/PCDH-COOH (9/1, m/m) mixed micelles, Annexin V was coupled with the micelles using carbodiimide chemistry. The blood clot lysis assay in vitro confirmed that lumbrokinase-loaded targeted micelles (LKTM) had stronger thrombolysis potency than free lumbrokinase (LK) and LK-loaded nontargeted micelles (LKM, P < 0.05). In vivo thrombolytic assay, multispectral, optoacoustic tomography (MSOT) was used to assess the target ability of LKTM. The results of MSOT images indicated the fluorescence intensity of the LKTM group located in the blood clot position were significantly stronger than the LKM group. A 5 mm of carotid artery containing blood clot was cut out 24 h later after administration to assess the degree of thrombolysis. The results of thrombolytic assay in vivo were consistent with the assay in vitro, which the differences between LK, LKM, and LKTM groups were both statistically significant. All the results of thrombolysis assays above proved that the capacity of thrombolysis in the LKTM group was optimal. It suggested that Annexin V-conjugated micelles will be a potential drug delivery system for targeted thrombolysis.

Thrombin-Activatable Fibrinolysis Inhibitor in Chronic Thromboembolic Pulmonary Hypertension.

The pathogenesis of chronic thromboembolic pulmonary hypertension (CTEPH) remains to be elucidated. Thrombin-activatable fibrinolysis inhibitor (TAFI) inhibits fibrinolysis. It remains to be elucidated whether TAFI is directly involved in the pathogenesis of CTEPH. We examined potential involvement of TAFI in the pathogenesis of CTEPH in humans. We enrolled 68 consecutive patients undergoing right heart catheterization in our hospital, including those with CTEPH (n=27), those with pulmonary arterial hypertension (n=22), and controls (non-pulmonary hypertension, n=19). Whole blood clot lysis assay showed that the extent of clot remaining after 4 hours was significantly higher in CTEPH compared with pulmonary arterial hypertension or controls (41.9 versus 26.5 and 24.6%, both P<0.01). Moreover, plasma levels of TAFI were significantly higher in CTEPH than in pulmonary arterial hypertension or controls (19.4±4.2 versus 16.1±4.5 or 16.3±3.3 μg/mL, both P<0.05), which remained unchanged even after hemodynamic improvement by percutaneous transluminal pulmonary angioplasty. Furthermore, the extent of clot remaining after 4 hours was significantly improved with CPI-2KR (an inhibitor of activated TAFI) or prostaglandin E1 (an inhibitor of activation of platelets). Importantly, plasma levels of TAFI were significantly correlated with the extent of clot remaining after 4 hours. In addition, the extent of clot remaining after 4 hours was improved with an activated TAFI inhibitor. These results indicate that plasma levels of TAFI are elevated in patients with CTEPH and are correlated with resistance to clot lysis in those patients.

Abstract 173: A Novel Dual Function Molecule for the Treatment of Acute Ischemic Stroke

Rapid restoration of blood flow to ischemic brain is essential to prevent irreversible tissue injury and minimize neuronal function impairment after acute ischemic stroke. However, intracerebral hemorrhage, reperfusion injury and the oxidative stress provoked by returned blood supply remain the major concerns for the use of effective thrombolytic agent; such as, recombinant tissue plasminogen activator (rt-PA). DC009 is a novel small molecule designed to have both thrombolytic and free radical scavenging activities, which were characterized in various in vitro and in vivo models. The thrombolytic effect of DC009 was demonstrated in vitro using whole blood clot lysis assay and euglobulin clot lysis assay. In comparison to rt-PA (1mg/ml), DC009 (11.11 mg/ml) exerts comparable (p value = 0.40) in vitro thrombolytic activity to rt-PA. Further investigation showed that DC009 induced endogenous t-PA protein secretion from endothelial vesicles, and it enhanced endothelial t-PA mRNA expression through cAMP pathway. Ability of DC009 in ABTS radical cation scavenging (2, 2-azinobis-(3-ethylbenzthiazoline-6-sulfonate) was measured and found to be dose dependent with an EC50 of 49 μM. Using a focal cerebral ischemia induced by arterial thrombosis model in aged mice (12-15 months old), DC009 showed a steady thrombolytic action and superior safety profile compared to vessel recanalization induced by rt-PA. We further investigated DC009’s effect on normal hemostasis and maximal tolerable dose (MTD) level in rodents in order to assess its therapeutic potential. At their comparable pharmacological dose, the tail amputation bleeding assay in mice showed that DC009 (up to 30mg/kg) did not cause bleeding prolongation; vs. rt-PA (10mg/kg) prolonged the bleeding to &gt;10 folds of normal bleeding time. According to the MTD determined in preliminary toxicity dose range finding studies, greater 50 folds of safety margin was identified in both mouse and rat. In conclusion, DC009 appears to both thrombolytic and free radical scavenging with minimal risk of bleeding. As a safe and dual functional small molecule, it is a drug candidate of great potential for the treatment of ischemic stroke.

Anticoagulant activity of crude extract of Holotrichia diomphalia larvae

Ethnopharmacological relevanceHolotrichia diomphalia larvae are one classical folk medicinal material in East Asia which has clinically been used to promote blood circulation and dispel blood stasis for several hundred years. Aim of the studyThe anticoagulant activity of crude extract of H. diomphalia larvae (CEHDL) in vitro and in vivo was evaluated to explore its mechanism as antithrombotic medicine. Materials and methodsThe effects of CEHDL on plasma recalcification time, platelet aggregation, bleeding time, hydrolysis of fibrinogen and fibrin were measured with normal human plasma, plasma-rich platelet, transected mouse tails and bovine fibrinogen; the anti-thrombosis activities of CEHDL in vitro and in vivo were analyzed with clots lysis assay and carrageenan-induced mouse tail thrombosis model. ResultsCEHDL was found to contain large numbers of proteins and could inhibit blood coagulation and platelet aggregation in a dose-dependent manner. Furthermore, CEHDL preferentially cleaved α- and β-chains followed by γ-chains of fibrinogen. Besides, CEHDL could directly degrade fibrin rather than activate plasminogen. It has been noted that fibrinogenolytic activity of CEHDL could be unaffected by metal ions such as Mg2+, Ca2+, Zn2+, Fe2+, Fe3+, Cu2+ and buffers with pH 3-10. Moreover, protease inhibitors like TPSI, aprotinin, leupetin, PMSF, DTT and EDTA only slightly or not inhibited fibrinogenolytic activity of CEHDL. However, CEHDL could be completely inactivated at 75°C and 100°C. In addition, CEHDL exhibited anti-thrombosis activities in both blood clot lysis assay and carrageenan-induced thrombosis model. ConclusionCEHDL possessed potent anticoagulant activity and several fibrin(ogen)olytic agents from H. diomphalia larvae were responsible for its antithrombotic effect as medicine.

Abstract 13304: Thrombin-activated Fibrinolysis Inhibitor in Chronic Thromboembolic Pulmonary Hypertension

Introduction: The pathogenesis of chronic thromboembolic pulmonary hypertension (CTEPH) remains to be elucidated. In the present study, we examined whether fibrinolysis capacity is impaired in CTEPH patients with a special reference to thrombin-activated fibrinolysis inhibitor (TAFI) and if so, whether TAFI is also directly involved in the pathogenesis of CTEPH in mice and humans. Methods and Results: We enrolled 68 consecutive patients undergoing right heart catheterization in our hospital, including those with CTEPH (n=27), those with pulmonary arterial hypertension (PAH, n=22) and controls (non-PH, n=19). Whole blood clot-lysis assay with monteplase (100 ng/ml) showed that thrombus reduction rate was significantly reduced in CTEPH compared with PAH or controls (58.1 vs. 73.5 and 75.1%, both P&lt;0.01). Plasma clot-lysis assay showed that the clot lysis time tended to be prolonged in CTEPH compared with PAH or controls (894 vs. 652 and 720 sec, both P=0.07). Moreover, serum levels of TAFI were significantly higher in CTEPH than in PAH or controls (46.1 vs. 39.1 and 38.7 μg/ml, both P&lt;0.05), which remained unchanged even after hemodynamic improvement by percutaneous transluminal pulmonary angioplasty (49.6 vs. 50.0 μg/ml, P=0.92). The levels of TAFI released from the platelets upon stimulation by thrombin (0.05 U/ml) were significantly higher in CTEPH than in PAH or non-PH (2.7 vs. 1.7 and 1.0 folds, both P&lt;0.05). Additionally, thrombus reduction rate was significantly improved with a carboxypeptidase R (an inhibitor of activated TAFI) (62.0 vs. 73.4%, P&lt;0.01) or prostaglandin E1 (an inhibitor of activation of platelets) (62.2 vs. 70.4%, P&lt;0.01). Importantly, plasma levels of TAFI were correlated with thrombus reduction rate and improved thrombus reduction rate by an activated TAFI-inhibitor (both P&lt;0.05). Finally, we newly developed TAFI-overexpressing mice (TAFI-Tg) and found that intravenous injection of thrombin (100 units/kg) caused accelerated thrombotic obstruction of the pulmonary artery in TAFI-Tg mice compared with littermate controls (5.4±1.2 vs. 1.8±0.6 vessels/lung fields) (n=9, P&lt;0.05). Conclusions: These results provide the first evidence that TAFI plays a crucial role in the pathogenesis of CTEPH.

Delivery of tissue plasminogen activator and streptokinase magnetic nanoparticles to target vascular diseases

Thrombolytic therapy for acute myocardial infarction standardly makes use of the medications streptokinase (SK) and tissue plasminogen activator (tPA). In this study, the potential of silica-coated magnetic nanoparticles (SiO2-MNPs) as nanocarriers clinical thrombolytic therapy was investigated. SiO2-MNPs for use in targeted therapeutic delivery of tPA and SK were prepared using a combined technique incorporating controlled precipitation and hydrothermal methods. Response surface methodology (RSM) was employed to evaluate the efficiency of the SiO2-MNPs. The production of SK secreted from Streptococcus equi was enhanced using random mutagenesis. The tPA and SK A were encapsulated by means of a silanizing agent with a surface rich in 3-aminopropyltrimethoxysilane layered around the SiO2-MNPs. Blood clot lysis assays and fibrin-containing agarose plates were used to carry out in vitro thrombolysis testing. The optimum conditions for producing MNPs were found to be at pH=13 and at a temperature of 75°C for 45min. Culture conditions of 2.75% NaCl concentration at initial pH=7.5 for 90s under UV resulted in maximum SK activity. The tPA/SK-conjugated SiO2-MNPs (SiO2-MNP-tPA-SK) increased operating stability in whole blood and storage stability in a buffer by 92%. More effective thrombolysis using magnetic targeting was indicated by a 38% reduction in blood clot lysis time achieved with SiO2-MNP-tPA-SK compared to administering the SiO2-MNPs without guidance. The silica-coated magnetic nanocarriers developed in this study show potential for improved clinical thrombolytic therapy.

Fibrinolytic enzyme production by newly isolated Bacillus cereus SRM-001 with enhanced in-vitro blood clot lysis potential.

The discovery of plasmin-like microbial fibrinolytic enzymes having high specificity and negligible side effects is crucial for thrombolytic therapy. Herein, we report one such extra-cellular fibrinolytic enzyme producing Bacillus cereus SRM-001 isolated from the blood-laden soil of a chicken dump yard. The potency of the enzyme was established with fibrin plate assay and in-vitro blood clot lysis assay. The shake-flask operating parameters and media composition were optimized for maximizing the productivity of the enzyme. The operating parameters, pH 7, 37°C, 1% inoculum volume and 24 h inoculum age, were found to be the optimum. The levels of media components, corn flour (0.3% w/v), soyabean powder (1.9% w/v) and MnSO4 (11.5 mM) were optimized by statistical analysis using Box-Behnken design derived RSM. This resulted in an almost 1.8 fold increase in fibrinolytic enzyme productivity. The 3D response surface plots showed soyabean powder and MnSO4 to be the key ingredients for enhancing the enzyme productivity, whereas corn flour had a marginal effect. The in-vitro blood clot lysis assay conducted at near physiological pH 7 at 37°C showed the enzyme to be a potential therapeutic thrombolytic agent.

Hemostatic, milk clotting and blood stain removal potential of cysteine proteases from Calotropis gigantea (L.) R. Br. Latex.

Introduction:Plant latex is a natural source of biologically active compounds and several hydrolytic enzymes responsible for their diverse health benefits. Recent past has witnessed substantial progress in understanding their supplementary industrial and pharmaceutical utility. Calotropis gigantea is one of the important latex producing plants belonging to asclepediaceae family with wide ethnopharmacological applications and is rich in proteolytic enzymes. Present study investigates hemostatic, milk clotting and blood stain removal potential of C. gigantea latex proteases.Materials and Methods:The protease activity of crude enzyme (CE), obtained by centrifugation followed by ammonium sulphate precipitation and dialysis, was assayed using casein as the substrate. Effect of pH, temperature and specific inhibitors on protease activity was determined. Native PAGE and in gel protease activity of CE was performed. Hemostatic (Fibrinogen polymerization, fibrinogen agarose plate and blood clot lysis assays), milk clotting and blood stain removal efficacies of CE were determined.Results:CE exhibited high caseinolytic activity. Enzyme activity was optimum at 37-50ºC and pH 8.0. Fibrinogen polymerization assay showed concentration dependent increase in turbidity indicating thrombin like activity which was further confirmed by fibrinogen agarose plate assays. Clot lysis assay indicated 92.41% thrombolysis by CE in 90 min. CE also revealed significantly high ratio of milk clotting to protease activity (Milk Clotting Index, MCI = 827.59 ± 1.52). Complete destaining of blood stained fabric was observed when incubated with 1% detergent incorporated with 0.1mg/ml CE. The study highlights and validates the compound application potential of latex cysteine proteases from C. gigantea.

Comparative proteomic analysis of the sun- and freeze-dried earthworm Eisenia fetida with differentially thrombolytic activities

The dried earthworm is a traditional thrombolytic medicine in East Asia. Its thrombolytic mechanism has been extensively studied. However, the effects of drying process on thrombolysis were rarely investigated. Herein, we compared the thrombolytic activity of earthworm Eisenia fetida processed by sun-drying to that by freeze-drying. Fibrin plate and blood clot lysis assays showed that freeze-dried earthworms gave dramatically higher fibrinolytic and thrombolytic activities than the sun-dried earthworms. To address the thrombolytic difference, comparative proteomic analysis was carried out using fibrin zymography and two-dimensional gel electrophoresis (2-DE). The freeze- and sun-dried earthworms generated remarkably different 2-DE protein spot patterns. A total of 126 differential protein spots were detected, 83 of them were identified by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry and database searching with 13 quantitative changes and 70 qualitative changes. Five of these differential proteins were identified as fibrinolytic proteases (lumbrokinases), responsible for dissolving fibrin, the main protein component of thrombus. The total abundance of these fibrinolytic proteases in the freeze-dried earthworms was significantly higher, consistent with the results of fibrin zymography. Therefore, the higher concentration of fibrinolytic enzymes along with their broad substrate specificity explained the stronger fibrinolytic and thrombolytic activities of the freeze-dried earthworms. This study suggests that freeze-drying represents an improved processing method for earthworm as the thrombolytic therapy in the future. Thrombosis has become one of the biggest concerns all over the world. The dried earthworms have been intensively used as thrombolytic agents. Its thrombotic mechanism has been studied by the modern pharmacological researches. However, the drying procedure of the earthworm and its effects on the thrombolysis were rarely investigated. The present study compared the thrombolytic effects of the freeze-dried and the normal dried earthworm E. fetida. To better understand the underlying mechanisms for differential thrombolytic effects, the fibrin zymography and the two-dimensional gel electrophoresis (2-DE) were employed to identify sets of differential proteins. Therefore, this study provides not only the comparative proteomic analysis but also molecular mechanism underlying the differential thrombolytic effects.

Thrombus-Targeted Nanocarrier Attenuates Bleeding Complications Associated with Conventional Thrombolytic Therapy

To test the hypothesis that thrombus-specific tissue plasminogen activator (tPA)-loaded nanocarriers enhance thrombolytic efficacy and attenuate hemorrhagic complications. A series of pegylated and non-pegylated tPA-loaded liposomes were prepared and their surfaces were decorated with the peptide sequence (CQQHHLGGAKQAGDV) of fibrinogen gamma-chain that binds with GPIIb/IIIa expressed on activated platelets. All formulations were characterized for physical properties, stability and in vitro release profile. The thrombolytic activities of tPA-loaded liposomes were tested by visual end-point detection, fibrin agar-plate and human blood clot-lysis assays. The thrombus-specificity of the peptide-modified-liposomes was evaluated by studying the binding of fluorescent peptide-liposomes with activated platelets. The pharmacokinetic profile and thrombolytic efficacy were evaluated in healthy rats and an inferior vena-cava rat model of thrombosis, respectively. Both pegylated and non-pegylated peptide-modified-liposomes showed favorable physical characteristics and colloidal stability. Formulations exhibited an initial burst release (40-50% in 30min) followed by a continuous release of tPA (80-90% in 24h) in vitro. Encapsulated tPA retained >90% fibrinolytic activity as compared to that of native tPA. Peptide-grafted-liposomes containing tPA demonstrated an affinity to bind with activated platelets. The half-life of tPA was extended from 7 to 103 and 141min for non-pegylated and pegylated liposomes, respectively. Compared to native tPA, liposomal-tPA caused a 35% increase in clot-lysis, but produced a 4.3-fold less depletion of circulating fibrinogen. tPA-loaded homing-peptide-grafted-liposomes demonstrate enhanced thrombolytic activity with reduced hemorrhagic risk.

Cloning, Expression and Characterization of a Gene from Earthworm Eisenia fetida Encoding a Blood-Clot Dissolving Protein

A lumbrokinase gene encoding a blood-clot dissolving protein was cloned from earthworm (Eisenia fetida) by RT-PCR amplification. The gene designated as CST1 (GenBank No. AY840996) was sequence analyzed. The cDNA consists of 888 bp with an open reading frame of 729 bp, which encodes 242 amino acid residues. Multiple sequence alignments revealed that CST1 shares similarities and conserved amino acids with other reported lumbrokinases. The amino acid sequence of CST1 exhibits structural features similar to those found in other serine proteases, including human tissue-type (tPA), urokinase (uPA), and vampire bat (DSPAα1) plasminogen activators. CST1 has a conserved catalytic triad, found in the active sites of protease enzymes, which are important residues involved in polypeptide catalysis. CST1 was expressed as inclusion bodies in Escherichia coli BL21(DE3). The molecular mass of recombinant CST1 (rCST) was 25 kDa as estimated by SDS–PAGE, and further confirmed by Western Blot analysis. His-tagged rCST1 was purified and renatured using nickel-chelating resin with a recovery rate of 50% and a purity of 95%. The purified, renatured rCST1 showed fibrinolytic activity evaluated by both a fibrin plate and a blood clot lysis assay. rCST1 degraded fibrin on the fibrin plate. A significant percentage (65.7%) of blood clot lysis was observed when blood clot was treated with 80 mg/mL of rCST1 in vitro. The antithrombotic activity of rCST1 was 912 units/mg calculated by comparison with the activity of a lumbrokinase standard. These findings indicate that rCST1 has potential as a potent blood-clot treatment. Therefore, the expression and purification of a single lumbrokinase represents an important improvement in the use of lumbrokinases.

Evidence for an Enhanced Fibrinolytic Capacity in Liver Cirrhosis Measured with a New Global Fibrinolysis Test in Whole Blood.

It is known that liver cirrhosis is associated with hyperfibrinolysis, which might contribute to bleeding problems of patients with this disease. However, hyperfibrinolysis has been assessed by using tests (such as euglobulin, plasma or diluted whole blood clot lysis assays), which do not reflect all changes that might occur in the fibrinolytic parameters in blood. A recent study even questioned the presence of a hyperfibrinolytic state in nonbleeding cirrhotic patients, since no evidence of increased fibrinolysis was observed with a new plasma-based clot lysis assay (Gastroenterology 2001; 121:131). Therefore we decided to re-investigate the fibrinolytic state of cirrhotic patients with a recently developed test for global fibrinolytic capacity (GFC) using undiluted whole blood. Non-anticoagulated blood was collected from 30 healthy controls and 75 patients with cirrhosis of varying severity (34 Child-Pugh A, 28 Child-Pugh B and 13 Child-Pugh C). The GFC was determined immediately after collection by clotting the blood, incubating the clots during 3 hours at 37 degrees and then measuring the generated fibrin degradation products. Various haemostatic parameters were determined in plasma samples by using functional activity assays. The median GFC (25th–75th percentile) increased from 1.7 (0.8–3.5) mg/l in the controls to 4.0 (1.6–13.0) mg/l in Child-Pugh A (n.s.), to 11.1 (2.3–31.9) mg/l in Child-Pugh B (P<0.001) and to 22.5 (3.1–89.6) mg/l in Child-Pugh C (P<0.01). Plasma samples of the cirrhotic patients showed significantly decreased levels of antithrombin, plasminogen, alpha-2-antiplasmin and thrombin-activatable fibrinolysis inhibitor (TAFI), significantly increased levels of tissue-type plasminogen activator (tPA) and no significant change in plasminogen activator inhibitor-1 (PAI-1). Spearman correlation tests in the whole group (controls plus patients) showed significant correlations between GFC and antithrombin (r=−0.44), plasminogen (r=−0.38), alpha-2-antiplasmin (r=−0.45), TAFI (r=−0.40), tPA (r=0.77) and PAI-1 (r=−0.51) (all p-values <0.0001). In conclusion, the new GFC test, which includes all components present in blood, indicates that the fibrinolytic capacity is strongly increased in patients with severe cirrhosis. The anti-fibrinolytic effects of decreased plasminogen levels in cirrhosis are apparently overruled by the pro-fibrinolytic effects of decreased alpha-2-antiplasmin and TAFI levels and particularly of increased tPA activity levels.

A Reconstituted Dilute Blood Clot Lysis Assay for the Medium Throughput Screening of Thrombolytic Compounds

Antithrombotic and clotting factors have long been targets for drug discovery, necessitating the development of blood assays to determine the efficacy of lead compounds prior to animal testing. We have developed a reconstituted blood clot lysis assay which eliminates the need for on-site donors. The assay uses whole blood stored at 4°C obtained from a local blood bank, diluted 1:10 in phosphate buffer. This blood was supplemented with125I-labeled fibrinogen and the release of radioactive fibrinopeptides from formed clots was measured. The whole blood used in this assay, which had been stored at 4°C for several days, no longer formed solid or retracting clots. Thus, platelets 5–7 daysex vivo(165 × 106platelets) were added to the whole blood in the presence of thrombin (0.80 IU/ml) to form clots. Solid clots formed within 2 min of thrombin addition and began retracting shortly thereafter. In the absence of any thrombolytic agent, clots fully retracted within 2.5 h and remained stable. Thrombin-stimulated clot formation was completely inhibited by heparin. Clots could be lysed in a dose-dependent fashion in the presence of tissue-type plasminogen activator. Clot lysis could be completely inhibited in a dose-dependent fashion with plasminogen activator inhibitor type 1. To demonstrate the utility of this assay as a screen for thrombolytic agents, a 14-amino-acid PAI-1-inhibitory peptide relieved the PAI-1 effect on tPA in a dose-dependent fashion. These data describe an assay for the screening of potential pro-fibrinolytic agents that target PAI-1 inhibition in a human plasma-based system that is versatile, cost-effective, and physiologically relevant and does not rely on the availability of on-site blood donors.

Cross-linking of α2-antiplasmin to fibrin is a key factor in regulating blood clot lysis

The basal lysis rates ofex-vivoprepared blood clots from rats, mice, hamsters, dogs, and humans and levels of α2-antiplasmin (α2-AP) cross-linked to fibrin have been studied in the presence and absence of factor XIII (FXIII) inhibitors using a blood clot lysis assay and an α2-AP binding assay. Clots prepared from rat or human blood lysed spontaneously within 3-5h. Clots from hamster blood lysed completely within 30 min but clots prepared from murine or canine blood lysed only after addition of 1.0 IU human t-PA. To study the effect of activated FXIII (FXIII) inhibition on blood clot lysis the FXIII inhibitors L722151 and mono-dansylcadaverine (dansyl) were used. In the presence of the FXIII inhibitors human, murine, and canine blood clots showed increased lysis rates. The lysis rate of rat blood clots was not affected. Effects on hamster blood clots could not be detected because of the high basal lysis rate. In clots prepared from human, murine, or canine plasma about 20% of the plasma α2-AP concentration was cross-linked to fibrin. FXIII inhibitors inhibited crosslinking by more than 80%. No significant cross-linking of α2-AP could be detected in rat and hamster plasma clots. When 0.1 volume of human plasma was added to 0.9 volume of rat plasma the level of α2-AP cross-linking was equal to that in human plasma indicating that rat α2-AP can be cross-linked to human fibrin. The same effect was obtained with human FXIII deficient plasma but not with human fibrinogen deficient plasma. These experiments indicate that rat fibrinogen is not a substrate for the cross-linking of rat α2-AP catalysed by rat FXIII, Addition of human plasma to hamster plasma did not result in an increased level of α2-AP cross-linking which indicates that other factors are involved.