Blood Clot Lysis Research Articles

Seasonal and individual characteristics of the response of the fibrinolytic system of blood to physical exercise

The response of the fibrinolytic system of the blood of 33 untrained subjects (16 males and 17 females 18 years old) to a single 20-min bicycle-ergometric large-capacity load (N male = 2.66 W/kg, N female = 2.3 W/kg) was studied for 1 year. It is known that the blood fibrinolytic activity at rest has seasonal characteristics: it is relatively high in autumn and spring and low in winter and summer. On average, physical activity in winter and summer stimulates the blood clot lysis, whereas in autumn and spring this stimulation is not observed. The direction and intensity of the response of the fibrinolytic system to exercise have individual features: in autumn and spring they depend on its initial state (increase when the blood FA is low and decrease when it is high) and fluctuate widely, whereas in winter and summer this dependence is not observed. According to the direction of response to the load and its persistence, three types of responses can be distinguished: (a) hyperfibrinolytic, which is characterized by an increase in FA during exercise throughout the year, with a constantly low initial level of fibrinolytic activity at rest; (b) hypofibrinolytic, which is characterized by a stable suppression of fibrinolysis after exercise regardless of the season and the initial level of fibrinolytic activity, with a high fibrinolytic activity at rest in autumn and spring and a low fibrinolytic activity in winter and summer; and (c) unstable, in which the direction of the response of the fibrinolytic system to exercise varies. It is concluded that the development of thromboembolic complications after exercise can be expected with the highest and lowest probability in the subjects with the hypofibrinolytic and hyperfibrinolytic response, respectively.

A novel fibrin(ogen)olytic trypsin-like protease from Chinese oak silkworm (Antheraea pernyi): Purification and characterization

A novel fibrin(ogen)olytic protease from Antheraea pernyi (important economically insect), named cocoonase, was isolated by a combination of ion-exchange chromatography and gel filtration. Furthermore, the characterization of cocoonase was investigated using fibrin(ogen)olytic, thrombolysis, and hemorrhagic assays. The NH2-terminal sequence (IVGGY SVTID KAPYQ) was established by Edman degradation. Based on the N-terminal sequencing, cocoonase cDNA has been cloned by means of RT-PCR and 5′RACE. It is composed of 261 amino acid residues and possesses the structural features of trypsin-like serine protease. The purified cocoonase showed specific esterase activity on N-β-benzoyl-l-arginine ethyl (BAEE), and the kinetic constants, Km and Vmax were 2.577×10−3mol/L and 4.09×10−3μmol/L/s, respectively. Cocoonase showed strong activities on both fibrin and fibrinogen, preferentially hydrolyzed Aα and Bβ chains followed by γ-chains of fibrinogen. Cocoonase exhibited a thrombolysis activity both in vitro (blood-clot lysis activity assay) and in vivo (carrageenan-induced thrombosis model). These findings indicate that A. pernyi cocoonase ia a novel fibrin(ogen)olytic enzyme and may have a potential clinical application as an antithrombotic agent.

Assessment of Thrombolytic Activity of Five Bangladeshi Medicinal Plants: Potential Source for Thrombolytic Compounds

Aim: The aim of our project work was to assess the thrombolytic activity of five common Bangladeshi plant extract in different solvent. Five plants are Geodorum densiflorum (Shankhamul), Pistia stratiotes (Topa Pana), Smilax zeylanica (Kumarilata), Pandanus foetidus (Keya) & Tabernaemontana coronaria (Tagar). Plants were collected and air dried separately for three weeks. They were ground into a coarse powder. Cold extractions were performed for all plants by using different solvents. Place and Duration of Study: Department of Pharmacy, University of Chittagong and University of Science and Technology Chittagong, November, 2013. Methodology: Fresh blood was collected from healthy individuals ten volunteers (n=10). Blood was allowed to form clots in a pre-weighed sterile micro-centrifuge eppendorf tubes. After clot serum was removed and blood clot was weighed then blood clot was allowed to lysis by streptokinase. After lysis fluid was removed and the remaining of blood clot was again weighed along with the tube. Percentage of blood clot lysis was calculated on the basis of the weight difference. Weight difference of tubes obtained by weighing before and after clot lyses of blood. % clot lysis=(Weight after clot lysis/ Weight of clot before lysis)×100. This method was repeated for all extracts. Original Research Article Hossen et al.; IBRR, Article no. IBRR.2014.6.001 263 Result: Among the herbs studied Pandanus foetidus (C), Pandanus foetidus (PE), Smilax zeylanica (E) and Pistia stratiotes-Root (M) showed significant % of clot lysis 47.54% 41.49%, 43.35% and 35.85% respectively with reference to standard, streptokinase (70.24%). Conclusion: These extracts lyse the blood clots In-vitro, however, we need to know Invivo clot dissolving property. Further systemic research on these plants and may be a potential source of thrombolytic agent in future.

In VitroWhole Blood Clot Lysis for Fibrinolytic Activity Study Using D-Dimer and Confocal Microscopy

This study aimed to evaluate in vitro whole blood (WB) clot lysis method for the assessment of fibrinolytic activity. Standardized unresected (uncut) retracted WB clot was incubated in pool platelet poor plasma (PPP) for varying incubation times and in streptokinase (SK) at different concentrations. The fibrinolytic activity was assessed by D-dimer (DD), confocal microscopy, and clot weight. DD was measured photometrically by immunoturbidimetric method. There was a significant difference in mean DD levels according to SK concentrations (P = 0.007). The mean DD ± SD according to the SK concentrations of 5, 30, 50, and 100 IU/mL was: 0.69 ± 0.12, 0.78 ± 0.14, 1.04 ± 0.14 and 2.40 ± 1.09 μg/mL. There were no significant changes of clot weight at different SK concentrations. Gradual loss and increased branching of fibrin in both PPP and SK were observed. Quantitation of DD and morphology of fibrin loss as observed by the imaging features are in keeping with fibrinolytic activity. Combination of DD levels and confocal microscopic features was successfully applied to evaluate the in vitro WB clot lysis method described here.

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.

Interactions between ultrasound stimulated microbubbles and fibrin clots

While it is well established that ultrasound stimulated microbubbles (USMBs) can potentiate blood clot lysis, the mechanisms are not well understood. Here we examine the interaction between USMBs and fibrin clots, which are comprised of fibrin networks that maintain the mechanical integrity of blood clots. High speed camera observations demonstrated that USMBs can penetrate fibrin clots. Two-photon microscopy revealed that penetrating bubbles can leave behind patent “tunnels” along their paths and that fluid can be transported into the clots. Finally, it is observed that primary radiation forces associated with USMBs can induce local deformation and macroscopic translation of clot boundaries.

LOCAL FIBRINOLYSIS IN SURGICAL TREATMENT OF NON-TRAUMATIC INTRACRANIAL HEMORRHAGES

More than 70% of hypertensive hemorrhages are located in deep brain structures. The removal of such hematomas using encephalotomy is accompanied by additional cerebral trauma and often results in unsatisfactory outcomes. The puncture aspiration with local fibrinolysis is one of the minimal invasive methods for treatment of intracerebral hematomas (ICH). The puncture and aspiration of liquid part of ICH (not more than 20% of ICH volume) is performed via small burr hole. Afterward the catheter is placed into ICH cavity and fibrinolytic is injected via this catheter in postoperative period for lysis of ICH solid part. The lysed blood is aspirated within 1-4 days. The last generations of fibrinolytics are very effective concerning intensity of blood clot lysis and practically have no systematic effect on blood coagulation system during their local usage. Morphological examinations showed that usage of fibrinolysis leads to formation of smaller cysts in the region of former hematoma as well as reparative processes in perihemorrhagical zone are expressed better comparing with treatment methods without usage of fibrinolytics. The morphological pattern is also confirmed by clinical signs of neurological deficit regress corresponding to damage focus. We operated 124 patients with parenchymal hemorrhages and 28 patients with intraventricular hemorrhages using described minimally invasive method at the base of Scientific Research Institute of Emergency Care n.a. N. V. Sklifosovsky. The applied method allowed decreasing lethality from 35% to 21% among patients with parenchymal ICH and from 98% to 48%--among patients with ventricular hemotamponade underwent usage of ventricular drainage combined with local fibrinolysis.

Potential of Magnetic Resonance-Guided Focused Ultrasound for Intracranial Hemorrhage: Anin VitroFeasibility Study

Intracranial hemorrhage has a mortality rate of up to 40-60% due to the lack of effective treatment. Magnetic resonance-guided focused ultrasound may offer a breakthrough noninvasive technology, by allowing accurate delivery of focused ultrasound, under the guidance of real-time magnetic resonance imaging. The purpose of the current study was to optimize the acoustic parameters of magnetic resonance-guided focused ultrasound for effective clot liquefaction, in order to evaluate the feasibility of magnetic resonance-guided focused ultrasound for thrombolysis. Body (1·1 MHz) and brain (220 kHz) magnetic resonance-guided focused ultrasound systems (InSightec Ltd, Tirat Carmel, Israel) were used to treat tube-like (4 cc), round (10 cc), and bulk (300 cc) porcine blood clots in vitro, using burst sonications of one-second to five-seconds, a duty cycle of 5-50%, and peak acoustic powers between 600 and 1200 W. Liquefied volumes were measured as hyperintense regions on T2-weighted magnetic resonance images for body unit sonications (duration of one-second, duty cycle of 10%, and power of 500-1200 W). Liquefaction efficiency was calculated for brain unit sonications (duration of one-second, duty cycle of 10%, power of 600 W, and burst length between 0·1 ms and 100 ms). Liquified lesion volume increased as power was raised, without a thermal rise. For brain unit sonications, a power setting of 600 W and ultrashort sonications (burst length between 0·1 and 1·0 ms) resulted in liquefaction efficacy above 50%, while longer burst duration yielded lower efficacy. These results demonstrate the feasibility of obtaining reproducible, rapid, efficient, and accurate blood clot lysis using the magnetic resonance-guided focused ultrasound system. Further in vivo studies are needed to validate the feasibility of magnetic resonance-guided focused ultrasound as a treatment modality for intracranial hemorrhage.

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.

No Exacerbation of Perihematomal Edema with Intraventricular Tissue Plasminogen Activator in Patients with Spontaneous Intraventricular Hemorrhage

In severe spontaneous intraventricular hemorrhage (IVH), intraventricular (IVR) administration of tissue plasminogen activator (rtPA) clears blood from the ventricles more rapidly than with external ventricular drainage (EVD) alone. However, experimental studies suggest tPA may be neurotoxic in compromised brain tissue and may exacerbate perihematomal edema. We used computerized volumetrics to assess change in intracerebral hemorrhage (ICH), IVH, ventricular, and perihematomal edema (PHE) volumes at 2-4 (T1) and 5-9 (T2) days following diagnostic CT scans (T0) of 24 patients (12 tPA-treated; 12 controls) with IVH requiring EVD. Controls from a hospital registry were matched by IVH and ICH volume to tPA-treated patients who came from a multicenter trial involving 52 patients with IVH. There were no significant differences between matched pairs in admission ICH and IVH volumes. IVR tPA resulted in more rapid clearance of IVH as determined by T2-T0 decrease in median IVH volume (tPA: -18.7 cc, iqr 14.9; control:-6.9 cc, iqr 6.4; P = 0.002). Median ratios of PHE to ICH volume were not significantly different in control versus tPA-treated patients at T1 and T2 [control:tPA = 0.55:0.56 (T1); P = 0.84 and 0.81:0.71 (T2); P = 1.00]. Total ventricular volume was significantly larger in the control group at T2 (mean: 57.57 ± 10.32 vs. tPA: 24.80 ± 2.67 cc; P = 0.01). Bacterial ventriculitis was more frequent in the control group (5 vs. 1 episodes; P = 0.06) as was shunt dependence (4 vs. 0 cases; P = 0.03). For case matched large IVH with small ICH volume, IVR tPA enhances lysis of intraventricular blood clots and has no significant impact on PHE.

Experimental immunology Complex function of magnesium in blood clot formation and lysis

ENWEndNote BIBJabRef, Mendeley RISPapers, Reference Manager, RefWorks, Zotero AMA Jankun J, Skrzypczak-Jankun E, Lipinski B. Experimental immunologyComplex function of magnesium in blood clot formation and lysis. Central European Journal of Immunology. 2013;38(2):149-153. doi:10.5114/ceji.2013.35203. APA Jankun, J., Skrzypczak-Jankun, E., & Lipinski, B. (2013). Experimental immunologyComplex function of magnesium in blood clot formation and lysis. Central European Journal of Immunology, 38(2), 149-153. https://doi.org/10.5114/ceji.2013.35203 Chicago Jankun, Jerzy, Ewa Skrzypczak-Jankun, and Bogusław Lipinski. 2013. "Experimental immunologyComplex function of magnesium in blood clot formation and lysis". Central European Journal of Immunology 38 (2): 149-153. doi:10.5114/ceji.2013.35203. Harvard Jankun, J., Skrzypczak-Jankun, E., and Lipinski, B. (2013). Experimental immunologyComplex function of magnesium in blood clot formation and lysis. Central European Journal of Immunology, 38(2), pp.149-153. https://doi.org/10.5114/ceji.2013.35203 MLA Jankun, Jerzy et al. "Experimental immunologyComplex function of magnesium in blood clot formation and lysis." Central European Journal of Immunology, vol. 38, no. 2, 2013, pp. 149-153. doi:10.5114/ceji.2013.35203. Vancouver Jankun J, Skrzypczak-Jankun E, Lipinski B. Experimental immunologyComplex function of magnesium in blood clot formation and lysis. Central European Journal of Immunology. 2013;38(2):149-153. doi:10.5114/ceji.2013.35203.

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.

196 EXPRESSION OF TISSUE-TYPE PLASMINOGEN ACTIVATOR IN OOCYTES AND CUMULUS CELLS FROM MOUSE, PIG, AND COW

Tissue-type plasminogen activator (tPA) is one of the components of the plasminogen-plasmin (PLG-PLA) system, better known as fibrinolytic system for its role in the blood clot lysis. It has been demonstrated recently that the activation of plasminogen into the protease plasmin during the sperm-oocyte interaction in the pig and cow decreases the percentages of penetration and increases monospermy (Mondéjar et al. 2012). However, in the mouse species, it was showed that PLG-PLA system enhances fertilization (Huarte et al. 1993). Expression of tPA has been described in rat oocytes (Bicsak et al. 1989) and cumulus cells (Ny et al. 1987; O’Connell et al. 1987), but no clear evidence about its expression in mouse, pig, and cow oocytes or cumulus cells is available. We hypothesised that differences in the effect of PLG-PLA system on fertilization results between the species mentioned above could be related to differences in tPA expression. The aim of this study was the detection of mRNA encoding tPA in oocytes and cumulus cells in mouse, pig, and cow by molecular analysis. Total RNA was obtained from oocytes and cumulus cells and cDNA was synthesised with an oligo-dT as primer. These cDNAs were used as template in RT-PCR amplifications using specific primers designed based on the GenBank sequence for Mus musculus, Sus scrofa, and Bos taurus tPA (NM_ 008872, NM_214054, NM_174146, respectively). The results of this study showed a different expression in the 3 studied species. In mouse, amplicon encoding tPA was detected in oocytes and cumulus cells. In cow and pig, tPA transcripts were obtained only in cumulus cells. The relation between the differences in the tPA expression pattern and the role of PLG-PLA system on fertilization remains to be investigated. This study was supported by MICINN (AGL2009-12512-C02-01-02).

Abstract 3443: No Exacerbation of Perihematomal Edema or Ventricular Volume with Intraventricular Tissue Plasminogen Activator in Patients with Spontaneous Intraventricular Hemorrhage

Introduction: In severe spontaneous intraventricular hemorrhage (IVH), intraventricular (IVR) administration of tissue plasminogen activator (tPA) results in more rapid clearing of blood from the ventricles than with external ventricular drainage (EVD) alone. However, experimental studies suggest tPA may be neurotoxic in compromised brain tissue and may exacerbate perihematomal edema. Methods: We used computerized volumetrics to assess change in intracerebral hemorrhage (ICH), IVH, ventricular and peri-ICH edema (PHE) volumes at 2-5 (T1) and 5-9 (T2) days following diagnostic CT scans (T0) of 20 patients (10 tPA treated; 10 controls) with IVH requiring EVD. Control patients from a hospital database treated with EVD alone were matched by IVH and ICH volume to tPA-treated patients. Treated patients came from two multicenter trials involving 100 patients with IVH, and spontaneous ICH volume <30cc and received a median of 4 doses (range 1-8) of tPA. Results: There were no significant differences between matched pairs in terms of age, admission Glasgow Coma Score, Graeb IVH score, PHE volume, total ventricular volume, or ICH volume. Median IVH volume was larger in the tPA-treated group (26.2 cc iqr10.2 vs. 23.1 cc iqr9.2; P=0.02). IVR tPA resulted in more rapid clearance of IVH as determined by T2-T0 decrease in Graeb score and median IVH volume (tPA: -18.7 (10.8) cc; control:-7.6 (3.2) cc; P=0.005). Ratios of PHE to ICH volume were non-significantly higher in control vs. tPA treated patients at T1 and T2 [control:tPA = 0.82:0.77 (T1);P=0.44 and 0.64:0.56 (T2);P=0.48]. Total ventricular volume was significantly larger in the control group at T2 (median: 41.7 (36.9) cc vs. tPA: 24.5 (11.9) cc; P=0.02) and cumulative median cerebrospinal fluid drainage was non-significantly higher (control: 1079 (463) cc vs. tPA: 639 (428) cc; P=0.08). Ventriculitis was more frequent in the control group (4 vs. 2 episodes; P=0.08) as was shunt dependence (4 vs. 0 cases; P=0.04). Conclusions: For case matched large IVH with small ICH volume, IVR tPA enhances lysis of intraventricular blood clots, accelerates resolution of post-hemorrhagic hydrocephalus and has no significant impact on perihematomal edema.

Chemical and biological studies of Kalanchoe pinnata (Lam.) growing in Bangladesh

ObjectiveTo isolate compounds from K. pinnata and elucidate their structures and to explore preliminary antioxidant, antimicrobial, cytotoxic and thombolytic activities of extractives of the plant. MethodsThe methanol extract of whole plant of K. pinnata has been subjected to different chromatographic separation and purification processes to isolate the secondary metabolites. The structures of the isolated compounds have been elucidated by extensive NMR studies. The free radical scavenging activity of the crude extract and its different Kupchan fractions were determined on stable radical DPPH. In vitro antimicrobial activity was determined by the disk diffusion method. Cytotoxicity screening has been performed against Artemia salina. Total phenolics content, membrane stabilizing activity and thombolytic activities were assessed by following established protocol. ResultsThe isolated compounds were identified as glut-5(6)-en-3-one, taraxerone, 3β-friedelanol, β-amyrin-3-acetate, 3,5,7,3′,5′-pentahydroxyflavone and β-sitosterol. The chloroform soluble fraction showed potent antioxidant activity of (IC50= 80.0 μg/mL) and significant cytotoxicity, while the crude extract demonstrated noticeable total polyphenol content (149.24 mg of GAE/gm of extractive), moderate membrane stabilizing activity and inhibition of clot lysis of blood. ConclusionsThe obtained results rationalize the folkloric use of the plant and can be further investigated to isolate the active compounds responsible for the biological activities.

Targeted delivery of tissue plasminogen activator by binding to silica-coated magnetic nanoparticle

Background and methodsSilica-coated magnetic nanoparticle (SiO2-MNP) prepared by the sol-gel method was studied as a nanocarrier for targeted delivery of tissue plasminogen activator (tPA). The nanocarrier consists of a superparamagnetic iron oxide core and an SiO2 shell and is characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, superconducting quantum interference device, and thermogravimetric analysis. An amine-terminated surface silanizing agent (3-aminopropyltrimethoxysilane) was used to functionalize the SiO2 surface, which provides abundant –NH2 functional groups for conjugating with tPA.ResultsThe optimum drug loading is reached when 0.5 mg/mL tPA is conjugated with 5 mg SiO2-MNP where 94% tPA is attached to the carrier with 86% retention of amidolytic activity and full retention of fibrinolytic activity. In vitro biocompatibility determined by lactate dehydrogenase release and cell proliferation indicated that SiO2-MNP does not elicit cytotoxicity. Hematological analysis of blood samples withdrawn from mice after venous administration indicates that tPA-conjugated SiO2-MNP (SiO2-MNP-tPA) did not alter blood component concentrations. After conjugating to SiO2-MNP, tPA showed enhanced storage stability in buffer and operation stability in whole blood up to 9.5 and 2.8-fold, respectively. Effective thrombolysis with SiO2-MNP-tPA under magnetic guidance is demonstrated in an ex vivo thrombolysis model where 34% and 40% reductions in blood clot lysis time were observed compared with runs without magnetic targeting and with free tPA, respectively, using the same drug dosage. Enhanced penetration of SiO2-MNP-tPA into blood clots under magnetic guidance was confirmed from microcomputed tomography analysis.ConclusionBiocompatible SiO2-MNP developed in this study will be useful as a magnetic targeting drug carrier to improve clinical thrombolytic therapy.

Fibrinolytic enzyme from newly isolated marine bacterium Bacillus subtilis ICTF-1: Media optimization, purification and characterization

Fibrinolytic enzymes are important in treatment of cardiovascular diseases. The present work reports isolation, screening and identification of marine cultures for production of fibrinolytic enzymes. A potent fibrinolytic enzyme-producing bacterium was isolated from marine niches and identified as Bacillus subtilis ICTF-1 on the basis of the 16S rRNA gene sequencing and biochemical properties. Further, media optimization using L(18)-orthogonal array method resulted in enhanced production of fibrinolytic enzyme (8814 U/mL) which was 2.6 fold higher than in unoptimized medium (3420 U/mL). In vitro assays revealed that the enzyme could catalyze blood clot lysis effectively, indicating that this enzyme could be a useful thrombolytic agent. A fibrinolytic enzyme was purified from the culture supernatant to homogeneity by three step procedures with a 34.42-fold increase in specific activity and 7.5% recovery. This purified fibrinolytic enzyme had molecular mass of 28 kDa, optimal temperature and pH at 50 °C and 9, respectively. It was stable at pH 5.0-11.0 and temperature of 25-37 °C. The enzyme activity was activated by Ca(2+) and obviously inhibited by Zn(2+), Fe(3)(+), Hg(2+) and PMSF. The purified fibrinolytic enzyme showed high stability towards various surfactants and was relatively stable towards oxidizing agent. Considering these properties purified fibrinolytic enzyme also finds potential application in laundry detergents in addition to thrombolytic agent. The gene encoding fibrinolytic enzyme was isolated and its DNA sequence was determined. Compared the full DNA sequence with those in NCBI, it was considered to be a subtilisin like serine-protease.

Platelets retain high levels of active plasminogen activator inhibitor 1.

The vascular fibrinolytic system is crucial for spontaneous lysis of blood clots. Plasminogen activator inhibitor 1 (PAI-1), the principal inhibitor of the key fibrinolytic enzyme tissue-type plasminogen activator (tPA), is present in platelets at high concentrations. However, the majority of PAI-1 stored in platelets has been considered to be inactive. Our recent finding (Brogren H, et al. Blood 2004) that PAI-1 de novo synthesized in platelets remained active for over 24 h, suggested that PAI-1 stored in the α-granules might be active to a larger extent than previously reported. To re-evaluate this issue, we performed experiments where the fraction of active PAI-1 was estimated by analyzing the tPA-PAI-1 complex formation. In these experiments platelets were lysed with Triton X-100 in the presence of serial dilutions of tPA and subsequently the tPA-PAI-1 complex was evaluated by Western blot. Also, using a non-immunologic assay, tPA was labeled with 125I, and 125I-tPA and 125I-tPA-PAI-1 was quantified by scintigraphy. Interestingly, both methods demonstrated that the majority (>50%) of platelet PAI-1 is active. Further analyses suggested that pre-analytical procedures used in previous studies (sonication or freezing/thawing) may have substantially reduced the activity of platelet PAI-1, which has lead to an underestimation of the proportion of active PAI-1. Our in vitro results are more compatible with the role of PAI-1 in clot stabilization as demonstrated in physiological and pathophysiological studies.

Remarkable extension of PAI-1 half-life surprisingly brings no changes to its structure

Plasminogen activator inhibitor type 1 (PAI-1) is a serpin protein, a natural inhibitor of urokinase (uPA) and tissue plasminogen activators (tPA). By inhibiting uPA it can block growth of the cancer tumors by suppressing angiogenesis, while when acting on tPA in the blood it can avert conversion of plasminogen to plasmin preventing lysis of the clot. Furthermore, blocking PAI-1 activity can protect against thrombosis. Thus PAI-1 makes great impact on human homeostasis and is desirable for clinical application. Wild-type PAI-1 (wt-PAI-1) has a short span of activity with a t1/2 of ~2 h, being spontaneously converted into a latent form. An enormous effort has been made to create a more stable molecule with >600 PAI-1 variants constructed to study its structure-function relationship. In the present study, we evaluate the structure of the active recombinant VLHL-PAI-1 (very long half life, active >700 h) which is glycosylated similarly to wt-PAI-1 at N232 and N288, with the extended reactive center loop, intact engineered -S-S-bridge (Q174C, G323C) that precludes latency without affecting structure, and can be controlled by a reducing agent to terminate activity at will. We have already proven its usefulness to control cancer in human cancer cells, as well as preventing clot lysis in human whole blood and plasma and in a mouse model. Our results demonstrate the potential therapeutic applications (topical or systemic) of this protein in the treatment of cancer, for the trauma patients to ward off an excessive blood loss, or for people with the PAI-1 deficiency, especially during surgery.