Human Clots Research Articles

Molecular Coronary MR Imaging of Human Thrombi using EP-2104R, a Fibrin-Targeted Contrast Agent: Experimental Study in a Swine Model

The aim of this study was to investigate the use of a fibrin-specific contrast agent (EP-2104R, EPIX Pharmaceuticals, Lexington, Massachusetts, USA) for targeted molecular magnetic resonance (MR) imaging of human clot material removed from patients in a model of coronary thrombosis in swine. Freshly ex vivo engineered clots from human blood and human in situ developed clots removed from patients were delivered into the coronary arteries of nine domestic swine. For MR imaging a navigator-gated, free-breathing, cardiac-triggered 3D inversion recovery black-blood gradient echo sequence was performed prior to clot delivery (baseline), after clot delivery but prior to contrast media administration, and two hours after systemic (i.v.) injection of 4 micromol/kg EP-2104R. MR images were analyzed by two investigators and the contrast-to-noise ratio and Gadolinium (Gd) concentration in the clots were assessed. On baseline images and prior to contrast media application no thrombi were visible. Post contrast administration all 10 coronary emboli (five ex vivo engineered clots and five human clots removed from patients) were selectively visualized as white spots with a mean contrast-to-noise ratio to the blood pool and the surrounding tissue of >12 and a mean Gd concentration of >100 microM. Molecular MR imaging using the fibrin-targeted contrast agent EP-2104R allows selective visualization of human clot material in a model of coronary thrombosis in swine.

Abstract 804: Noninvasive Integrated SPECT/CT Molecular Imaging of Activated Factor XIII Activity in Thrombosis

Background : Activated factor XIII (FXIIIa) is a blood transglutaminase that mediates fibrinolytic resistance and is a hallmark of acute thrombi. Noninvasive molecular imaging of FXIIIa may offer a novel approach to identify acute thrombi and to gauge fibrinolytic resistance in vivo. Here we developed and validated a FXIIIa thrombosis imaging strategy using noninvasive integrated SPECT/CT. Methods: A FXIIIa-targeted peptide agent (F13) was synthesized using NQEQVSPLTLLK chelated to DOTA and then labeled with 111 InCl 3 . A control agent (C13, 111 In-NAEQVSPLTLLK) was analogously synthesized. In vitro validation of the F13 agent was performed in human plasma clots. Next, the in vivo blood-half life of F13 was determined in mice (n=4). In vivo thrombosis studies (n=15 mice) were then performed using 10% ferric chloride jugular venous thrombi aged 1 hour or 16 hours. Mice were intravenously injected with 200 μCi of F13 or C13. After 4 hours, mice underwent integrated CT angiography (72 μm isotropic resolution) and SPECT imaging (32 minute acquisition). In situ thrombi were then resected for radioactivity and weight measurements. Results : Human plasma clots incubated with F13 showed 280–740% greater counts per minute (CPM) than controls (p<0.01). F13 binding was dose-dependent and >90% inhibited by pretreatment with iodoacetamide, an alkylating agent. The blood half-life of F13 was calculated to be 16 minutes. In one hour thrombi, in vivo SPECT/CT imaging revealed strong focal F13 SPECT signal in the co-registered ipsilateral venous thrombi but not the contralateral normal jugular vein. One hour thrombi in the F13 group had 15-fold greater radioactivity than the C13 group (4.6±3.6% vs. 0.3±0.2% injected dose per gram tissue, IDGT, p<0.01). Compared to 1 hour thrombi, 16 hour old thrombi had 4-fold less F13 radioactivity (1.1%±0.1% IDGT, p<0.05). Conclusions : Blood transglutaminase FXIIIa can be noninvasively detected using a FXIIIa-sensitive and specific imaging agent for integrated SPECT/CT. The current in vivo results further validate that activated factor XIII is a hallmark of acute thrombi and declines in activity over time. This clinically translatable imaging strategy could permit visualization of FXIIIa in patients with thrombotic syndromes.

Molecular MR Imaging of Human Thrombi in a Swine Model of Pulmonary Embolism Using a Fibrin-Specific Contrast Agent

Molecular targeted MR imaging of human clots material in a model of pulmonary embolism using a fibrin-specific magnetic resonance imaging contrast agent (EP-2104R, EPIX Pharmaceuticals, Cambridge, MA). Fresh ex vivo engineered thrombi (human blood) and human clots removed from patients were delivered in 11 swine. Molecular MR imaging with a 3D gradient-echo [3D fast field echo (3DFFE)] sequence and a navigator-gated and cardiac-triggered 3D inversion-recovery black-blood gradient-echo sequence (IR) was performed before thrombus delivery, after thrombus delivery but before contrast media application, and 2 hours after i.v. administration of 4 micromol/kg EP-2104R. MR images were analyzed by 2 investigators and contrast-to-noise ratio (CNR) was assessed. Thrombi were removed for assessment of gadolinium (Gd) concentration. Only after contrast media application were pulmonary emboli [freshly engineered thrombi (n = 23) and human clot material removed from patients (n = 25)] visualized as white foci on MR images. CNR was 13 +/- 3 (ex vivo engineered clot) and 22 +/- 9 (patient clot material) for the fast field echo (FFE)-sequence and 29 +/- 9 (ex vivo engineered clot) and 43 +/- 18 (patient clot material) for the IR-sequence, respectively. A high Gd concentration in the clots was found (82 +/- 43 microM for the freshly engineered and 247 +/- 44 microM for the clots removed from patients, respectively). EP-2104R allows for molecular MR imaging of human clot material in the pulmonary vessels of a swine model.

Fibrin-Targeted Perfluorocarbon Nanoparticles for Targeted Thrombolysis

Reperfusion of the ischemic brain is the most effective therapy for acute stroke, restoring blood flow to threatened tissues. Thrombolytics, such as recombinant tissue plasminogen activator, administered within 3 h of symptom onset can improve neurologic outcome, although the potential for adverse hemorrhagic events limits its use to less than 3% of acute ischemic stroke patients. Targeting of clot-dissolving therapeutics has the potential to decrease the frequency of complications while simultaneously increasing treatment effectiveness, by concentrating the available drug at the desired site and permitting a lower systemic dose. We aimed to develop a fibrin-specific, liquid perfluorocarbon nanoparticle that is surface modified to deliver the plasminogen activator streptokinase. We also aimed to evaluate its effectiveness for targeted thrombolysis in vitro using quantitative acoustic microscopy. Human plasma clots were formed in vitro and targeted with streptokinase-loaded nanoparticles, control nanoparticles or a mixture of both. Depending on the treatment group, clots were then exposed to either phosphate-buffered saline (PBS), PBS with plasminogen or PBS with plasminogen and free streptokinase. Spatially registered ultrasound scans were performed at 15-min intervals for 1 h to quantify changes in clot morphology and backscatter. Nanoparticles bound to the clot significantly increased the acoustic contrast of the targeted clot surface, permitting volumetric estimates. Profile plots of detected clot surfaces demonstrated that streptokinase-loaded, fibrin-targeted perfluoro-octylbromide nanoparticles in the presence of plasminogen induced rapid fibrinolysis (<60 min) without concurrent microbubble production and cavitation. Streptokinase-loaded or fibrin-targeted control nanoparticles insonified in PBS did not induce clot lysis. Morphologic changes in the treated group were accompanied by temporal and spatial changes in backscatter. Ultrasound exposure had no effect on the digestion process. Effective concentrations of targeted streptokinase were orders of magnitude lower than equivalently efficacious levels of free drug. Moreover, increasing competitive inhibition of fibrin-bound streptokinase nanoparticles reduced clot lysis in a monotonic fashion. As little as 1% surface targeting of streptokinase nanoparticles produced significant decreases in clot volumes (approximately 30%) in 1 h. This new nanoparticle-based thrombolytic agent provides specific and rapid fibrinolysis in vitro and may have a clinical role in early reperfusion during acute ischemic stroke.

Ultrasound-Induced Thermal Elevation in Clotted Blood and Cranial Bone

Ultrasound thermal effects have been hypothesized to contribute to ultrasound-assisted thrombolysis. To explore the thermal mechanism of ultrasound-enhanced thrombolysis with recombinant tissue plasminogen activator (rt-PA) for the treatment of ischemic stroke, a detailed investigation is needed of the heating produced in skull, brain and blood clots. A theoretical model is developed to provide an estimate for the worst-case scenario of the temperature increase in blood clots and on the surface of cranial bone exposed to 0.12- to 3.5-MHz ultrasound. Thermal elevation was also assessed experimentally in human temporal bone, human clots and porcine clots exposed to 0.12 to 3.5-MHz pulsed ultrasound in vitro with a peak-to-peak pressure of 0.25 MPa and 80% duty cycle. Blood clots exposed to 0.12-MHz pulsed ultrasound exhibited a small temperature increase (0.25° C) and bone exposed to 1.0-MHz pulsed ultrasound exhibited the highest temperature increase (1.0° C). These experimental results were compared with the predicted temperature elevations. (E-mail: nahirnvm@email.uc.edu)

Arrhenius temperature dependence of in vitro tissue plasminogen activator thrombolysis

Stroke is a devastating disease and a leading cause of death and disability. Currently, the only FDA approved therapy for acute ischemic stroke is the intravenous administration of the thrombolytic medication, recombinant tissue plasminogen activator (tPA). However, this treatment has many contraindications and can have dangerous side effects such as intra-cerebral hemorrhage. These treatment limitations have led to much interest in potential adjunctive therapies, such as therapeutic hypothermia (T ⩽ 35 °C) and ultrasound enhanced thrombolysis. Such interest may lead to combining these therapies with tPA to treat stroke, however little is known about the effects of temperature on the thrombolytic efficacy of tPA. In this work, we measure the temperature dependence of the fractional clot mass loss Δm(T) resulting from tPA exposure in an in vitro human clot model. We find that the temperature dependence is well described by an Arrhenius temperature dependence with an effective activation energy Eeff of 42.0 ± 0.9 kJ mole−1. Eeff approximates the activation energy of the plasminogen-to-plasmin reaction of 48.9 kJ mole−1. A model to explain this temperature dependence is proposed. These results will be useful in predicting the effects of temperature in future lytic therapies.

Duty Cycle Dependence of Ultrasound Enhanced Thrombolysis in a Human Clot Model

Combined ultrasound and tissue plasminogen activator (rt-PA) therapy, or ultrasound enhanced thrombolysis (UET), has been shown to improve recanalization in patients with acute ischemic stroke. We measured the effect of ultrasound duty cycle on the lytic efficacy of 120 kHz UET in an in vitro human clot model. The hypothesis was that an increase in duty cycle increases rt-PA lytic efficacy. Human whole blood clots were exposed to 120-kHz ultrasound and rt-PA for 30 min in human plasma. The duty cycle ranged from 0% to 80%, where 0% represents sham exposure. Clot lytic rate was measured by recording the clot width over time. The clot width after 30 min exposure to rt-PA and ultrasound decreases with increasing duty cycle. The initial lytic rate increased linearly with duty cycle. (E-mail: meuniejn@uc.edu)

Molecular Imaging of Human Thrombus With Novel Abciximab Immunobubbles and Ultrasound

Molecular imaging of therapeutic interventions with targeted agents that simultaneously carry drugs or genes for local delivery is appealing. We investigated the ability of a novel microbubble carrier (immunobubble) for abciximab, a glycoprotein IIb/IIIa receptor inhibitor, for ultrasonographic molecular imaging of human clots. Human thrombi were incubated with immunobubbles conjugated with abciximab. Control clots were incubated in either saline or with immunobubbles conjugated with nonspecific antibody. We evaluated immunobubble suspensions with variable concentrations of encapsulated gas and measured mean acoustic intensity of the incubated clots. In vivo molecular imaging of human thrombi with abciximab immunobubbles was evaluated in a rat model of carotid artery occlusion. Mean acoustic intensity was significantly higher for abciximab immunobubbles as compared with control immunobubbles under all conditions tested with maximum difference in intensity at a gas volume of 0.2 microL (P=0.0013 for mechanical index 0.05, P=0.0001 for mechanical index 0.7). Binding of abciximab immunobubbles to clots in vitro led to enhanced echogenicity dependent on bubble concentration. In vivo ultrasonic detectability of carotid thrombi was significantly higher for clots targeted with abciximab immunobubbles (P<0.05). Quantification of in vivo contrast enhancement displayed a highly significant increment for abciximab immunobubble-targeted clots compared with nonspecific immunobubble-targeted clots (P<0.0001) and to native clots (P<0.0001). This study demonstrates the feasibility of using a therapeutic agent for selective targeting in vascular imaging. Abciximab immunobubbles improve visualization of human clots both in vitro and in an in vivo model of acute arterial thrombotic occlusion.

Comparison of the in vitro performance of 6 and 7 French aspiration catheters.

A comparison of aspiration catheters that have been approved for real-world use was carried out in vitro. Myocardial damage occurs during therapeutic aspiration of thrombus. The relative efficiency of aspiration may be important in this regard. Using saline and human clot, nine aspiration catheters were compared Thrombuster III(R)N (6 Fr and 7 Fr), ZEEK (6 Fr), Rebirth (7 Fr), Eliminate (6 Fr and 7 Fr), Pronto (6 Fr), and Export(R) (6 Fr and 7 Fr). Tracking was assessed from the resistance required to pass the catheter through a vessel model. Pushability was determined from the difference between the load at the hand piece and tip of the catheter during advancement through the vessel model. The Thrombuster III(R)N (6 Fr and 7 Fr) showed significantly better aspiration performance, although the ranking order of the catheters was not the same for saline and clot. The Thrombuster III(R)N also showed the best tracking with low resistance and was the easiest catheter to advance, as evaluated based on pushability. In the present in vitro evaluation system, the Thrombuster III(R)N performed better than other catheters.

120 kHz pulsed ultrasound enhanced thrombolysis with tissue plasminogen activator-loaded echogenic liposomes

Echogenic liposomes (ELIP), phospholipid vesicles filled with gas and fluid, can be manufactured to incorporate the thrombolytic drug tissue plasminogen activator (tPA). Real‐time thrombolysis of blood clots exposed to tPA‐incorporating ELIP (t‐ELIP) was monitored using video microscopy with an inverted optical microscope. Human whole blood clots on silk sutures were exposed to tPA alone (3.15 micrograms/ml), t‐ELIP alone (3.15 micrograms/ml), t‐ELIP and 120 kHz ultrasound (0.18 MPa peak negative pressure, 1.667 kHz pulse repetition frequency, 50% duty cycle), or tPA and ultrasound, for 30 min. The extent of thrombolysis was determined by assessing clot width as a function of time, using a time‐lapse microscopic imaging technique. The average percent change in clot width at 30 min for clots treated with t‐ELIP alone exceeded tPA alone (22.8% vs. 15.6%, respectively). Thrombolytic efficacy was similar for either tPA or t‐ELIP exposed to 120 kHz ultrasound. Thus, the thrombolytic drug could be effectively released by exposure to 120 kHz ultrasound. [This work was supported by The Distinguished Chair for Clinical Research in Emergency Medicine Foundation Award, K02‐NS056253, NIH 1RO1 NS047603, and NIH 1R01 HL074002.]

3-Mercaptopropionic acids as efficacious inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa)

A novel series of cyclic potent, selective, small molecule, thiol-based inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa) and the crystal structures of TAFIa inhibitors bound to porcine pancreatic carboxypeptidase B are described. Three series of cyclic arginine and lysine mimetic inhibitors vary significantly in their selectivity against other human basic carboxypeptidases, carboxypeptidase N and carboxypeptidase B. (−) 2a displays TAFIa IC 50 = 3 nM and 600-fold selectivity against CPN. Inhibition of TAFIa with (rac) 2a resulted in dose dependent acceleration of human plasma clot lysis in vitro and was efficacious as an adjunct to tPA in an in vivo rabbit jugular vein thrombolysis model.

Enhancement of recombinant tissue-plasminogen activator (rt-PA) activity with 2-MHz transcranial Doppler ultrasound

A video microscopy technique was developed to monitor the lytic activity of recombinant tissue plasminogen activator (rt-PA) in combination with 2-MHz transcranial Doppler ultrasound (TCD). Human whole blood clots formed on silk sutures were treated with TCD pulses alone [pulse length=10 cycles, pulse repetition frequency (PRF)=10.5 kHz, Pneg=0.18−0.47 MPa], rt-PA alone (3 μg/ml), and rt-PA with TCD pulses in human fresh frozen plasma for 30 min. An inverted optical microscope was used to acquire images of the clot during treatment. The extent of clot lysis was determined by assessing clot width as a function of time. The average percent change in clot width at 30 min was comparable (∼25%) for clots treated with rt-PA and TCD pulses for all output pressures. However, the initial lytic rate (within the first 5 min) was directly related to the TCD pressure output. The initial lytic activity of rt-PA and 2-MHz TCD pulses (Pneg=0.18 MPa, PRF=10.5 kHz, duty cycle=13%) was similar to the initial lytic activity of rt-PA and 120-kHz ultrasound pulses (Pneg=0.18 MPa, PRF=1667 Hz, duty cycle=20%).

The potential of a new stable ultrasound contrast agent for site-specific targeting. An in vitro experiment

Microbubble-based ultrasound contrast agents can be used for specific site targeting, but demonstrate time-limited opacification. We have previously demonstrated the potential of gold-bound microtubules to provide a stable ultrasound contrast effect. Aim of the present study was to test the feasibility of gold-bound microtubules specifically to bind to human thrombi and to inflammatory activated human umbilical vein endothelial cells (HUVEC) in vitro. HUVEC were incubated with tumor necrosis factor, to induce expression of adhesion molecules. Human clots and HUVEC were incubated with biotinylated monoclonal antifibrin and anti-E-selectin antibodies, respectively. Probes were incubated with excess avidin followed by biotinylated gold-bound microtubules and by secondary Cy3-anti-β-tubulin antibody and processed for immune fluorescence microscopy. Clots were transferred in copolymer foils filled with buffer and were ultrasonographically imaged before and after their treatment with the antifibrin antibody and with biotinylated microtubules, using a broadband harmonic transducer, transmitting and receiving at a mean frequency of 1.7 MHz and 3.2 MHz. The feasibility of specific gold-bound microtubules conjugation to antibody treated clots and HUVEC was confirmed using immune fluorescence analysis. Contrast intensities of the clots significantly increased after their treatment with antifibrin antibody and incubation with gold-bound microtubules (39 ± 2 dB versus 26 ± 2 dB, p < 0.001) and remained high after 20 min of ultrasound exposure (37 ± 2 dB versus 39 ± 2 dB, p = NS). Thus, gold-bound microtubules can specifically bind to human thrombi and to endothelial cells, providing a significant contrast effect which remains stable in the ultrasound field. This may be a promising approach to target thrombi and inflammatory active atherosclerotic plaques. (E-mail: gkorosoglou@hotmail.com)

Resistance of porcine blood clots to lysis relates to poor activation of porcine plasminogen by tissue plasminogen activator

In-vitro experimentation was performed on porcine and human blood to determine their comparative responsiveness to a novel fibrinolytic inhibitor and thereby assess whether the pig is a suitable animal model for subsequent in-vivo testing of this inhibitor. Thromboelastography showed the clots formed from porcine whole blood to be highly resistant to tissue plasminogen activator (t-PA)-catalyzed lysis, and this communication offers the resistance of porcine plasminogen to activation by t-PA as an explanation. Porcine blood containing 100 and 1500 IU/ml added t-PA lysed very slowly, having LY30 values of 1.9 +/- 1.4 and 2.9 +/- 1.9%, respectively. In contrast, the LY30 values for the human clots containing 100 and 1500 IU/ml t-PA were 77.1 +/- 6.3 and 93.3 +/- 1.3%, respectively. Moreover, purified porcine plasminogen was activated very slowly by added t-PA in the presence of both human and porcine fibrin. Activation of plasminogen by the endogenous activators, as measured by the euglobulin clot lysis time, was greatly prolonged for the pig (22 +/- 3 h) compared with the human (3.5 +/- 1.5 h). These results suggest caution in using the pig as an experimental model when studying the effects of various agents on fibrinolysis.

Acousto-mechanical and thermal properties of clotted blood

The efficacy of ultrasound-assisted thrombolysis as an adjunct treatment of ischemic stroke is being widely investigated. To determine the role of ultrasound hyperthermia in the process of blood clot disruption, the acousto-mechanical and thermal properties of clotted blood were measured in vitro, namely, density, speed of sound, frequency-dependent attenuation, specific heat, and thermal conductivity. The amplitude coefficient of attenuation of the clots was determined for 120 kHz, 1.0 MHz, and 3.5 MHz ultrasound at room temperature (20 +/- 2 degrees C). The attenuation coefficient ranged from 0.10 to 0.30 Np/cm in porcine clots and from 0.09 to 0.23 Np/cm in human clots. The experimentally determined values of specific heat and thermal conductivity for porcine clotted blood are (3.2 +/- 0.5) x 10(3) J/kg x K and 0.55 +/- 0.13 W/m x K, respectively, and for human clotted blood are (3.5 +/- 0.8) x 10(3) J/kg x K and 0.59 +/- 0.11 W/m x K, respectively. Measurements of the acousto-mechanical and thermal properties of clotted blood can be helpful in theoretical modeling of ultrasound hyperthermia in ultrasound-assisted thrombolysis and other high-intensity focused ultrasound applications.

Ultrasound hyperthermia in clotted blood and ex vivo cranial bone

Ultrasound thermal effects have been hypothesized to contribute moderately to ultrasound-assisted thrombolysis. Transcranial ultrasound adjuvant to thrombolytic treatment of ischemic stroke requires a detailed investigation of the heating produced by ultrasound exposure to the skull bone, brain, and blood clots. Thermocouple measurements of ex vivo cranial bone and blood clot heating were compared to theoretical predictions of ultrasound hyperthermia, which assumed adiabatic conditions and provided an estimate for the worst-case scenario. The in vitro experiments were conducted at two temperatures, 20&amp;lt;th&amp;gt;°C and 37&amp;lt;th&amp;gt;°C. Ex vivo human skull, and porcine or human blood clots were exposed to 120 kHz, 1 MHz, or 3.5 MHz pulsed ultrasound with an 80% duty cycle and an acoustic spatial peak intensity of 0.5 W/cm2. The thermal elevation in human cranial temporal bone was below 1&amp;lt;th&amp;gt;°C for all three frequencies. The ultrasound hyperthermia in porcine and human clots increased with an increase in center frequency, but did not exceed (0.33±0.06)&amp;lt;th&amp;gt;°C. These results provide the information necessary to quantify the contribution of a thermal mechanism for ultrasound-enhanced thrombolysis.

Fibrinolytic efficacy of Amediplase, Tenecteplase and scu-PA in different external plasma clot lysis models

In this study, the in-vitro fibrinolytic efficacy of Tenecteplase, Amediplase and scu-PA was investigated in different external lysis models by measuring the lysis of human plasma clots after the addition of the plasminogen activators (PAs) to the surrounding plasma. The effect of TAFI was examined for each PA by neutralising TAFIa with potato carboxypeptidase inhibitor (PCI). The lytic efficacy of Amediplase was lower than that of Tenecteplase at low PA concentrations but slightly higher at therapeutic concentrations. The activity of scu-PA was clearly lower than that of either Tenecteplase or Amediplase. The TAFI system inhibited external clot lysis mediated by all the PAs when thrombomodulin was present in the model. In the therapeutic range (5-10 mug/ml) however, the TAFIa effect was negligible for both Amediplase and Tenecteplase. At lower PA concentrations the effect of TAFI on Amediplase was slightly stronger than that on Tenecteplase. Under static conditions the lysis rates were lower than with stirring. The role of TAFI was similar under both conditions. In conclusion, at therapeutic concentrations Amediplase was slightly more active than Tenecteplase and scu-PA under all conditions used. Therefore, Amediplase might possibly be a more potent thrombolytic agent at these concentrations and increase the efficacy of thrombolysis. The potential of TAFI for inhibiting thrombolytic therapy is probably low. However in conditions where the local PA concentrations are sub-optimal TAFI might affect the lysis rate.

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BioTechniquesVol. 39, No. 5 WebWatchOpen AccessWebWatchKevin AhernKevin AhernSearch for more papers by this authorPublished Online:30 May 2018https://doi.org/10.2144/05395WW01AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Seq and FindWant more out of your BLAST searches? Well, of course you do! In the latest wrinkle on online sequence analysis, Sequerome ups the ante for thousands of online BLAST sites on the web by offering more than just simple retrieval of homologs to entered query sequences. How about restriction enzyme maps or ORF identification for DNAs or secondary structure predictions for protein sequences? CpG islands anyone? Like a free online version of commercial DNA/protein sequence analysis packages, Sequerome is sure to be popular with everyone doing sequence analysis on a tight budget.sequerome.georgetown.edu/blast/index.jspBacteriacastingLive from New York (Ithaca, NY, to be specific), it's Microbeworld, produced cooperatively by Finger Lakes Productions and the American Society for Microbiology. Organized around a set of daily RealPlayer-formatted au-diocasts about microbes, Microbeworld is one of the most unusual sources of information about bacteria to be found on the web. Daily reports on Microbeworld span a wide gamut of interests, from rabies immunization of raccoons for slowing the spread of the disease to how Streptococcus bacteria dissolve human blood clots and facilitate their spread in the body. Spots are typically about 90 seconds long, providing a bacterial “thought for the day” that is long enough to inform, but short enough to keep even the MTV generation from getting bored.www.microbeworld.org/home.htmDriving Bugs NutsImagine you're a lonely Dipteran looking for a hot date on a summer night. Wouldn't it be great to find the perfect “cologne” to attract the mate of your dreams? More likely, you're a scientist looking to learn more about insect attractants. No matter how you look at insects, you're likely to be delighted with the discoveries to be had at Pherobase, an online database of pheromones and semiochemicals (chemicals affecting insect behavior) for over 7000 species. Users will love the site's easy access. Browse data according to many fields of interest—by taxonomic classification, by compound name, by synthesis, by mechanism of action, or even by reference—and then swim in the sea of results that are retrieved.www.pherobase.comStem Cell 101Is there a high-tech topic in the news that has generated more interest, curiosity, and controversy than stem cells? Probably not, so the time is ripe for a site to educate the curious and help the cognoscenti to navigate important data. Meeting the needs of this diverse group is the National Institutes of Health (NIH) Stem Cell Information Page that employs a four-part organizational scheme. Scientists can find what they're after in Research Topics. Federal Policy can be viewed in the section of the same name. News/Announcements (recent headlines) and Info Center (general information on the topic) round out the offerings. Serving very diverse audiences is a significant challenge, but NIH's Stem Cell Information Page is the rare effort that succeeds where many fail.stemcells.nih.gov/index.aspBeneath the WavesPeer deep into a drop of seawater. It's full of bacteria, as you might imagine, but you'll probably be surprised to learn that most of the life forms therein aren't readily propagated in the laboratory, and in many cases, it's not really clear why. Considering that over 70% of the earth's surface is covered by seawater, that's a LOT of earthly life that we hardly know anything about. How to study? Enter the Gordon and Betty Moore Foundation, which seeks to “improve the quality of life for future generations” by funding initiatives to increase our knowledge of species in marine habitats. Amazingly enough, some of the efforts use genomic DNA sequencing as a means to characterize an organism before anything else about it is known. How's that for technology turning biology on its ear?www.moore.org/microgenome/worldmap.aspFiguresReferencesRelatedDetails Vol. 39, No. 5 Follow us on social media for the latest updates Metrics History Published online 30 May 2018 Published in print November 2005 Information© 2005 Author(s)PDF download

IN FOCUS: A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity – a staphylokinase variant. I. In vitro study

We attempted to construct a new recombinant protein characterized by fibrin-specific properties of plasminogen activation combined with antithrombin and antiplatelet activities. To the C-terminal part of recombinant staphylokinase (r-SAK), which is a promising profibrinolytic agent, we assembled: (i) the Kringle 2 domain (K2) of tissue-type plasminogen activator (t-PA), containing a fibrin-specific binding site, (ii) the RGD sequence (Arg-Gly-Asp) for the prevention of platelet aggregation and (iii) the antithrombotic agent - hirudin. The cDNA for hybrid protein SAK-RGD-K2-Hir was cloned into pESP-3 yeast protein expression vector. The introduction of K2 t-PA, RGD sequence and hirudin into r-SAK molecule did not alter the SAK activity. The plasminogen activation rate (determined by K(M) and K(cat)) of SAK-RGD-K2-Hir was not significantly different from that of r-SAK. Affinity and binding strength of the recombinant protein to fibrin immobilized on the biosensor were higher than to r-SAK. We observed a higher clot lysis potency of SAK-RGD-K2-Hir as evidenced by a faster and more profound lysis of 125I-labeled human fibrin clots. The potency of thrombin inhibition by the hirudin part of the recombinant fusion protein SAK-RGD-K2-Hir was the same as that of r-Hir alone. In conclusion, the results of the in vitro study suggest that the SAK-RGD-K2-Hir construct can be a more potent and faster-acting thrombolytic agent with antithrombin and antiplatelet properties compared with standard r-SAK.

Ultrasound-Induced Blood Clot Dissolution without a Thrombolytic Drug Is More Effective with Lower Frequencies

Background and Purpose: Therapeutic ultrasound as stand-alone therapy or in combination with rt-PA has proven to be an effective measure for recanalisation of acute vessel occlusion in different in vitro and in vivo studies. Uncertainty still exists concerning the optimal frequency and intensity with regard to the thrombolytic efficacy of ultrasound. The purpose of this study was a direct comparison of different ultrasound frequencies, when otherwise using identical measurement settings and parameters. Methods: Ultrasound-induced dissolution of fresh human blood clots was studied in a flow system using low-frequency continuous wave ultrasound of 20, 40 and 60 kHz. After calibration of each ultrasound probe, blood clots were exposed to local time average intensities of either 0.12 or 0.2 W/cm². Exposure time of the clots to ultrasound was 10 min, the number of treated clots in each experimental group was 12. Results: As tested with 0.2 W/cm², we found the most pronounced thrombolytic effect with the 20-kHz probe (weight loss of blood clots: 52.4%) and the 40-kHz probe (49.4%), as compared to the 60-kHz probe (21.4%) and the control group (18.5%). The difference between the 20- and 60-kHz probes was statistically significant (p < 0.001). The treatment effect was clearly intensity dependent with a less pronounced, but still significant treatment effect at 0.12 W/cm² (24.5% at 20 kHz; p < 0.001 compared to 0.2 W/cm²; p = 0.045 compared to controls). Conclusions: These data show that therapeutic efficacy of ultrasound, in absence of a thrombolytic drug, is frequency and intensity dependent with best results at low frequencies. With continuous wave transmission, the benefit may be limited to the very low frequency range. The results are a basis for further evaluation in animal models.