Dissolution of peripheral arterial thrombi by ultrasound

BACKGROUND. We have previously shown that continuous-wave ultrasound can rapidly dissolve human thrombi in vitro, with 99% of all residual particles measuring less than 10 microns in diameter. To assess the effects of pulsed-wave ultrasound energy on whole blood clots, 1) in vitro studies were preformed to assess precisely the rates of clot disruption and to quantify particulate size, and 2) in vivo studies were performed to assess the efficacy and safety of catheter-delivered ultrasound for intra-arterial thrombus dissolution. METHODS AND RESULTS. In vitro, we studied 50 samples of human whole blood clots and using an 89-cm-long wire probe, applied pulse-wave energies from 8 to 23 W. The corresponding peak-to-peak tip displacement range was 63.5 - 102 microns. We studied arterial thrombosis in vivo in 21 canine superficial femoral arteries. To produce an acute thrombosis, 200 units of thrombin followed by 2 ml of 72-hour-old autologous clot were injected into a 5-7-cm segment of femoral artery and left to coagulate for 2 hours. Ultrasound energy was intermittently applied at a frequency of 20 kHz with a prototype ultrasound wire ensheathed in a catheter and directed to clots by fluoroscopy. In nine cases, angioscopic guidance was used to put the probe into direct contact with the intra-arterial thromboses. In vitro clot dissolution times were inversely related to the ultrasound power output (r = 0.95). All in vivo canine thromboses were disrupted in 4 minutes or less. All successful recanalizations were confirmed by angiography and in nine cases by angioscopy as well. Angioscopy demonstrated that probe activation caused rapid clot disruption. Histological studies of the vessels showed no evidence of thermal or cavitation injury, occlusive distal embolization, or perforation. CONCLUSIONS. Our findings in this experimental canine model suggest that ultrasound clot dissolution has the potential to be an effective and safe alternative to current treatment modalities for peripheral arterial thrombosis.     

The Effects of Streptokinase and Hydroxyethyl Starch on in vitro Clot Disruption by Ultrasound

Background: In vitro studies showed that low-frequency ultrasound (US) causes blood clot dissolution. This effect is augmented with thrombolytics, microbubbles and microparticles. However, in animal models of transcutaneous delivery, US alone is not effective, probably due to attenuation of US energy by overlying skin. When combined with thrombolytics or microbubbles, transcutaneous US is highly effective. Purpose: To assess the synergistic effect of low-intensity low-frequency US and saline, hydroxyethyl starch (HAES) (a non-gas filled microparticle containing solution), streptokinase (STK), and their combination on blood clot disruption. Methods: Human blood clots from 4 healthy donors, 2–4 hours old, were immersed for 0, 15, or 30 min in 37°C in 10 ml of the above-mentioned solutions, and then were randomized to 10 sec of 20 kHz US or no US. The % difference in weight was calculated. Results: Immersion for 30 min without US resulted in 13.8 ± 1.2% clot lysis in saline, and 22.0 ± 1.3%, 21.7 ± 2.1%, and 23.2 ± 1.9% in STK, HAES, and STK + HAES, respectively (p = 0.002). US augmented clot lysis in all groups and at all time points. With low-intensity US, HAES was not better than saline. However, the combination of HAES + STK with US resulted in larger clot disruption at 15 sec incubation time (46.7 ± 3.2%) than with saline (29.6 ± 2.1%), HAES (29.6 ± 2.5%), and STK (32.8 ± 3.6%) (p < 0.001). Conclusion: low-frequency, low-intensity US combined with HAES and STK resulted in greater clot disruption at short incubation times. This combination may assist in achieving faster reperfusion in in vivo models.     

Ultrasonic clot disruption: an in vitro study

We studied in vitro the efficacy of ultrasound in human blood clot disruption, as well as the effects of clot age, wire probe length, and streptokinase on the outcome. The study included sizing the resulting particulate debris. Clot age (1 to 7 days) had no effect on the time required for disruption. Three groups of 1-day-old clots (n = 10 for each) were exposed to the same ultrasonic power source via probes of different lengths. The time required for clot disruption varied approximately as the square of the length for probes of 31, 56, and 105 cm, but was less than 3 minutes even for the longest probe employed. Disrupted whole-blood clot as well as cell-free fibrin clot solutions were analyzed for particulates by the resistive-pulse technique (size range: 2.5 to 80 microns). Debris as large as 80 microns were seen after disruption of whole blood clots, while cell-free fibrin clots contributed little above 40 microns. In all size ranges, whole blood clots produced two orders of magnitude more particulates than cell-free fibrin clots. Addition of streptokinase (7500 U/mL) had little effect on the size distribution of debris, with 99% of all particulates being smaller than 10 microns. D-dimer analysis was performed on the dissolved cell-free fibrin clots with and without streptokinase. While the former had analytically higher D-dimer concentrations than the latter (from eight- to 16-fold), the levels in both cases would be below detectability if measured in vivo. Hence the present study supports the concept that ultrasound can be employed to disrupt human blood clots by mechanisms (mechanical and cavitational) other than fibrinolysis.     

Synergism of Aspirin and Heparin with a Low-Frequency Non-Invasive Ultrasound System for Augmentation of In-Vitro Clot Lysis

BACKGROUND: Aspirin, glycoprotein IIb/IIIa inhibitors and heparin are routinely used in acute coronary syndromes. Previously we showed that there is synergism between ultrasound and heparin and tirofiban in augmenting blood clot disruption. However, there is a little data on a possible synergism of low-frequency ultrasound with aspirin for in-vitro clot dissolution, and especially on the combination of aspirin with heparin and/or glycoprotein IIb/IIIa inhibitors. MATERIALS AND METHODS: Human blood clots (n = 320) were incubated for 10 or 20 minutes in saline containing aspirin alone or combined with heparin and/or tirofiban and/or eptifibatide. Clots were randomly treated with low-frequency ultrasound (27.3 kHz) or incubation only. The percent clot weight loss and the incremental effect of ultrasound were calculated. RESULTS: The most significant incremental effect of ultrasound on clot weight reduction was detected with aspirin alone (5.2 +/- 2.3% and 5.2 +/- 2.6% after 10' and 20', p = 0.04 and p = 0.06, respectively) and in combination with heparin (8.8 +/- 2.5% and 11.5 +/- 2.7% after 10' and 20', p = 0.01 and p = 0.0001, respectively). The greatest absolute magnitude of clot weight reduction was observed with ultrasound combined with aspirin and heparin (48.5 +/- 9.5% after 20'). The addition of tirofiban or eptifibatide to aspirin, heparin and ultrasound did not increase clot lysis. However, eptifibatide had significantly better synergism than tirofiban (p = 0.025 and p = 0.015, after 10 and 20 minutes, respectively). CONCLUSIONS: Aspirin alone or in combination with heparin results in significant augmentation of clot lysis and is synergistic with application of low-frequency ultrasound for 10 and 20 minutes only. These results may have important implications for a possible use of low-frequency ultrasound in treatment algorithms of acute coronary syndromes.     

An Investigation of the Physical Forces Leading to Thrombosis Disruption by Cavitation

Ultrasound therapy has proven to be an efficient and safe modality for the treatment of acute arterial occlusions, and the use of therapeutic ultrasound for the treatment of thrombosis and vascular diseases holds great promise in overcoming the limitations of other available therapies. Still, there exists little published work that covers the different phenomena that take place in a thorough and comprehensive way. In this paper, we endeavor to address the subject by reviewing work on the physical properties of ultrasound propagation in the blood arteries as it relates to the cavitation of microbubbles, and we compare the impact of the different forces at work for clot disruption. Our conclusion is that the most important effect of ultrasound in the treatment of thrombotic disorders is the liquid-jet impact forces that result from strong bubble collapses in the vicinity of solid boundaries.     

Direct action on the molecule is one of several mechanisms by which ultrasound enhances the fibrinolytic effects of reteplase.

Enhanced fibrinolytic reperfusion therapy may improve the outcome in embolic stroke, where ultrasound exposure has been shown to be one option. We recently verified that the fibrinolytic properties of streptokinase were modulated following ultrasound exposure of the molecule. We have now explored this possibility following ultrasound exposure of the reteplase molecule. The effects on clot lysis of reteplase and ultrasound both separately and in combination were studied by evaluating cumulated release of haemoglobin from whole blood clots following 1 h of exposure. Specifically, we investigated how clot lysis was modulated following pulsed 1 MHz ultrasound pre-exposure of the reteplase solution at intensities ranging between 0.125 and 4 W/cm2 spatial-average temporal-average intensity (SATA) and the effects of reteplase following 1 h of pre-exposure of clots to ultrasound at high intensity (4 W/cm2SATA). Significant enhancement of clot lysis during concomitant reteplase and pulsed ultrasound exposure were observed in two intensity ranges: 0.125-0.25 and 2-4 W/cm2SATA. Pre-exposing reteplase solution to ultrasound significantly increased clot lysis only in the lower intensity range. At high ranges, pre-exposure of clots to ultrasound was followed by an increased fibrinolytic action of reteplase. Pre-exposing reteplase solution to low-intensity ultrasound induced changes in the reteplase molecule that enhanced its fibrinolytic effects. Although this effect disappeared at moderately higher ultrasound intensity, the pre-exposure of clots to ultrasound of higher intensity induced increased fibrinolytic effects of reteplase solution.     

Procoagulant State of Sleep Apnea Depends on Systemic Inflammation and Endothelial Damage

IntroductionGrowing evidence shows a hypercoagulable state in obstructive sleep apnea (OSA) that could be a risk factor for thromboembolic disease.ObjectivesWe aimed to elucidate mechanisms involved in the procoagulant profile observed in patients with OSA and to investigate the potential utility of global tests in its characterization.MethodsThirty-eight patients with severe OSA without previous history of thrombosis and nineteen healthy age- and sex-matched controls were included.Kinetic of clot formation was determined using rotational thromboelastometry.Haemostatic capacity of plasma and microparticles was determined by Calibrated Automated Thrombinography.Platelet surface receptors, activation markers and formation of platelet/leukocytes aggregates were analyzed by flow cytometry.ResultsThromboelastometry showed a procoagulant state in patients with OSA that did not seem to be related to a basal activation of platelets but by the increased existence of platelet/leukocyte aggregates.Patients with OSA presented many signs of endothelial damage such as increased plasma levels of E-selectin and cfDNA and enhanced thrombin generation due to the presence of microparticles rich in tissue-factor, which is related to OSA severity.ConclusionsOSA induces an enhancement in the dynamics of clot formation which appears to be caused by at least two pathological mechanisms. First, a greater formation of platelet-leukocyte aggregates; secondly, endothelial damage which provokes a greater procoagulant potential due to the increase in tissue factor-rich microparticles. Moreover, this study has identified thromboelastometry and thrombin generation assay as useful tools to evaluate the prothrombotic state in these patients.     

Rapid and accurate diagnosis of pulmonary emboli in a canine model using intravascular ultrasound imaging

We utilized intravascular ultrasound (IVUS) imaging in a canine pulmonary embolism model to visualize experimental emboli. The images obtained were compared with those obtained by single-plane pulmonary arteriography in each of six animals. The vessel lumen appeared patent by both methods prior to injection of autologous clot. After thrombi were injected, the vessels were again imaged using both techniques. Intravascular ultrasound was 100 percent sensitive in detecting emboli, and visualization was always rapid. There were no complications. It appears that IVUS imaging is a sensitive method for documenting the presence of clot in a canine pulmonary embolism model.     

Impact of microparticles derived from erythrocytes on fibrinolysis

It has long been known that negatively charged membranes of erythrocyte-derived microparticles display procoagulant activity. However, relatively little is known about the possible fibrinolytic activity of such microparticles. This issue becomes particularly important during red blood cell storage, which significantly increases the number of microparticles. Whole blood was collected from 30 healthy donors. Microparticles were isolated on days 7, 14, 21, and 28 of erythrocyte storage. The effect of microparticles on the fibrinolytic activity of the donor plasma was determined by coagulation and optical (chromogenic substrate) methods. We demonstrated that erythrocyte microparticles had a prominent fibrinolytic activity which cleaves not only fibrin but also chromogenic substrates. Microparticles present fibrinolytic activity mainly due to the presence of plasminogen on them. Microparticles derived from erythrocytes significantly enhance cleavage of the chromogenic substrate by the streptokinase-plasminogen complex, but to a lesser extent accelerate euglobulin clot lysis time. Erythrocyte-derived microparticles display prominent fibrinolytic activity, which significantly decreases during storage of red blood cells.     

Noninvasive transcutaneous low frequency ultrasound enhances thrombolysis in peripheral and coronary arteries

Previous studies have shown that external ultrasound with low frequencies and high intensities can enhance thrombolytic drug-induced clot dissolution during in vitro experiments. In this series of studies, we evaluated the efficacy of peripheral and coronary thrombolysis in vivo in animals by using noninvasive transcutaneous ultrasound combined with thrombolytic drugs (streptokinase and tPA) and/or microbubbles agents (dodecafluoropentane [DDFP] and perfluorocarbon-exposed sonicated dextrose albumin [PESDA]). Thrombotic occlusions were induced in 74 rabbit iliofemoral arteries and 24 canine left anterior descending (LAD) coronary arteries in this in vivo study. By using the combination of transcutaneous ultrasound and streptokinase, the angiographic patency rate in rabbit iliofemoral arteries was higher (56%-100%) than with ultrasound (6%; P < or = 0.0036) and streptokinase alone (6%; P < or = 0.0012). Also, with transcutaneous ultrasound and microbubbles, the angiographic patency rates were 76%-100% as compared with ultrasound alone (0%, P < or = 0.0003) or microbubbles alone (9%, P < or = 0.0001). In the canine study of acute myocardial infarction, thrombolysis in myocardial infarction (TIMI) grade flow at 90 minutes in the tPA alone group was 0.92 +/- 1.4 as compared with 2.42 +/- 1.9 in the tPA plus transthoracic ultrasound group (P = 0.006). There was much improved reperfusion with tPA plus ultrasound as compared with tPA alone. In vivo animal studies demonstrate that noninvasive transcutaneous ultrasound can greatly enhance the effect of clot dissolution with thrombolytic drugs and/or microbubbles, and has the potential for clinical application as an adjunctive method to improve arterial thrombolysis.     

Potentiating intra-arterial sonothrombolysis for acute ischemic stroke by the addition of the ultrasound contrast agents (Optison™ &; SonoVue<Superscript>®</Superscript>)

Transcranial ultrasound in combination with intravenously administered ultrasound contrast agents (UCA) in the presence or absence of recombinant tissue plasminogen activator (rt-PA) has been widely evaluated as a new modality for treatment of ischemic stroke. Despite the successful demonstration of accelerated clot lysis there are inherent limitations associated with this modality such as inconsistency in temporal window thickness and/or potential serious cardiopulmonary reactions to intravenous administration of UCA that prevent broad application to ischemic stroke populations. As a complementary modality, we evaluated potential lysis enhancement by intra-arterial ultrasound with concurrent intra-clot delivery of UCA and rt-PA. To this end, clots were formed with average pore diameter similar to clinically retracted clots by adjusting the thrombin concentration. Physical characteristic and retention of UCA after delivery through the catheter as a function of clinically relevant flow rates of 6, 12, 18 ml/h were determined using a microscopic method. The ability of the UCA employed in this study, Optison and SonoVue, to penetrate into the clot was verified using ultrasound B-mode imaging. Clot lysis as a function of rt-PA concentration, 0.009 through 0.5 mg/ml, in the presence and absence of UCA diluted to 1:10, 1:100, and 1:200 v/v at two Peak rarefaction acoustic pressures of 1.3 and 2.1 MPa were evaluated using a weighing method. The study results suggest the addition of only 0.02 ml of 1:100 diluted UCA to rt-PA of 0.009, 0.05, 0.3, and 0.5 mg/ml can enhance the lysis rate by 3.9, 2.6, 1.9 and 1.8 fold in the presence of peak rarefaction acoustic pressure of 1.3 MPa and by 5.1, 3.4, 2.6, 3.1 in the presence of peak rarefaction acoustic pressure of 2.1 MPa, respectively. In addition, Optison and SonoVue demonstrated comparable effectiveness in enhancing the clot lysis rate. Addition of UCA to intra-arterial sonothrombolysis could be considered as a viable treatment option for ischemic stroke patients.     

Ultrasonic determination of clot deposition rates in a milk-based, in-vitro procedure for thrombogenicity assessment

BACKGROUND AND AIM OF THE STUDY: Thrombosis and thromboembolism remain the main problems associated with mechanical heart valves. We have devised a milk-based clotting technique to simulate in-vitro clinical incidence of thrombosis. Early results with the technique revealed good correlation between milk clot deposition and clinical thrombosis, but were limited in their ability to predict the course of clot deposition, as deposition could only be measured upon dismantling the apparatus. METHODS: Clot deposition was observed ultrasonically for both steady and pulsatile flows of a milk preparation through a cylindrical test chamber containing axisymmetric test bodies similar in shape to those used in earlier studies of thrombosis. Echo ultrasound images were recorded at regular time intervals, depicting the interface between clot deposits and flowing milk. From these images, the thickness of the clot deposit could be determined as a function of time. RESULTS: Milk clot deposition on the test bodies, while observed, could not be measured accurately due to faint reflections arising from significant differences between the angle of incidence and the angle of reflection of the ultrasound beam. However, measurement was possible at the wall of the test chamber where deposition rates revealed steady growth of clot, following an initial 'lag', with growth continuing until a maximum thickness is reached. In some experiments shedding of parts of the deposit was observed. In pulsatile flow, wall clot deposition rates and final clot thickness attained were significantly lower than in steady flow. CONCLUSION: Ultrasonic measurement of clot deposition rates is possible in our thrombogenicity assessment apparatus on surfaces perpendicular to the line of incidence of the ultrasound beam. With suitably designed viewing windows in an artificial heart, such measurements should enable the time course of clot deposition on artificial valves to be determined, with a view to identifying initial deposition sites and the dynamics of clot growth. Observations of the growth of clot on the test chamber wall in this study suggest that both the rate of deposition and nature of deposit formed are strongly influenced by fluid mechanical properties such as shear and mass transfer rates. In particular, our results appear to suggest a different structure of deposit, whose rate of deposition is relatively slow, under conditions of high shear.     

In vitro ultrasound augmented clot dissolution--what is the optimal timing of ultrasound application?

The mechanism of ultrasound augmentation of pharmacological thrombolysis is yet unknown. The goal of this study is to find the best timing regimen for in-vitro ultrasound augmented clot dissolution by streptokinase, heparin and their combination. Blood clots from 4 donors were cut into 200–400 mg sections and randomized to no treatment with ultrasound; pre-treatment with ultrasound (before immersion); early treatment with ultrasound; or late treatment with ultrasound. Clots were placed in tubes containing either saline; heparin; streptokinase or streptokinase +heparin. All groups showed significant weight reduction (p < 0.001). Using the one way ANOVA test, we showed that ultrasound application resulted in a significantly higher rate of clots dissolution (p < 0.05) than without ultrasound in all of the solutions tested. We found no statistically significant difference between the three ultrasound regimens tested. In conclusion, in our in-vitro model, no single ultrasound timing schedule was found to provide better clot dissolution than the other schedules. This finding may suggest an additive effect between the ultrasound and the different solutions rather than a synergistic effect.     

Effect of ultrasound on tissue-type plasminogen activator-induced thrombolysis.

BACKGROUND. The efficacy of fibrinolytic therapy is limited by the small surface area of the clot that is available for the binding of the thrombolytic agent, such as tissue-type plasminogen activator (t-PA). We hypothesized that exposure of the clot to ultrasound during thrombolytic treatment could enhance lysis through perturbation of the thrombus, which would expose additional fibrin binding sites for t-PA. METHODS AND RESULTS. Whole human blood clots containing radiolabeled fibrinogen were incubated in vitro for 200 minutes with Tris-albumin buffer containing t-PA at concentrations ranging from 3 to 3,000 IU/ml. In paired experiments, one of the clots also was exposed to intermittent ultrasound (1 MHz, 1.75 W/cm2) throughout the experiment. The ultrasound was delivered as a 2-second exposure followed by a 2-second rest interval. The overall difference in mean clot lysis between thrombi receiving ultrasound and those receiving no ultrasound was significant (p less than 0.001) at all concentrations of t-PA. For clots incubated with t-PA at a concentration of 300 IU/ml, ultrasound increased the percent lysis at 200 minutes from 42 +/- 5% (mean +/- SEM) to 64 +/- 10%. In six paired experiments in a rabbit jugular vein thrombosis model, rabbits received 1 mg t-PA alone or t-PA and intermittent ultrasound (1 MHz, 1.75 W/cm2) for 200 minutes. For rabbits receiving ultrasound and t-PA, lysis was 55 +/- 11% at 100 minutes compared with 30 +/- 12% for rabbits receiving only t-PA. Lysis was 6 +/- 10% for rabbits (n = 4) receiving ultrasound alone. No evidence for tissue damage was noted in rabbits exposed to intermittent ultrasound. CONCLUSIONS. Exposure of whole blood clots in vitro to intermittent ultrasound combined with t-PA caused a significant enhancement of thrombolysis compared with t-PA alone. Intermittent ultrasound also showed a trend toward enhancement of t-PA-induced clot lysis in an animal thrombosis model. These data suggest that noninvasive intermittent ultrasound may be a useful adjunct to thrombolytic therapy.     

Portal vein reflectors: duplex sonographic appearance.

Duplex sonography has established utility in the noninvasive evaluation of the portal venous system. Recently, the duplex sonographic features of suspected portal venous air have been described. We report on an experimental study in dogs undertaken to establish if the same sonographic features could be reproduced in a laboratory setting and to determine if small fragments of clot emboli could produce similar gray-scale and Doppler findings. Injections of microbubbles of air and clot fragments into the portal venous system were monitored using duplex ultrasound. The gray-scale and Doppler features of flow in the main portal vein were indistinguishable in both microbubble and clot injections. Superimposition of high-amplitude spikes on the normal portal venous waveform was seen in 19 of 24 (79%) microbubble and 13 of 23 (56%) clot fragment injections. We conclude that the appearance of rapidly moving, bright intraluminal echoes coupled with spike-like aberrations of the portal venous waveform can be associated with portal venous air bubbles or small blood clot emboli.     

Echographic aspects of blood clots

The ultrasonic imaging of blood clots depends on the degree of compactness of the red cells which it contains. When the clot is densely compact, the number of targets struck by the ultrasound beam is large, and major diffusion occurs, providing a visible image. When the clot is less dense, there are insufficient targets and it is not possible to pick-up a reflected signal able to create a visible image. The former situation arises in the case of a recently-formed clot, whereas the latter occurs in ageing clots.     

Cooling System Permits Effective Transcutaneous Ultrasound Clot Lysis In Vivo Without Skin Damage

Previous in vivo studies have shown that transcutaneous ultrasound enhances clot dissolution in the presence of either streptokinase or microbubbles. However, ultrasound-induced skin damage has been a major drawback. The objective was to evaluate the effect of a cooling system to prevent the skin damage that has heretofore been associated with transcutaneous low-frequency, high-intensity ultrasound clot dissolution. After thrombi were induced in both iliofemoral arteries in 15 rabbits, streptokinase (25,000 U/kg) was given intravenously and dodecafluoropentane was injected slowly (2 mL/15 min) through an infusion catheter into the abdominal aorta. One iliofemoral artery was randomized to receive ultrasound treatment, and the contralateral artery was treated as a control (receiving streptokinase and dodecafluoropentane alone). In six rabbits (group 1), the skin below the ultrasound transducer was protected by the use of a balloon cooling system, and in the other nine rabbits (group 2), ultrasound was used without a cooling system. Seven of nine (78%) arteries treated without the cooling system, and six of six (100%) arteries treated with the cooling system were angiographically recanalized after ultrasound + streptokinase + dodecafluoropentane treatment. Thermal damage was present in the skin and soft tissues of all nine rabbits treated without a cooling system. However, the skin and soft tissues were grossly and histologically normal in the six rabbits in which the transcutaneous ultrasound was used with the cooling system. Low-frequency, high-intensity ultrasound energy can be delivered transcutaneously for clot dissolution without concomitant tissue damage when coupled with the use of a cooling system to prevent thermal injury.     

The ultrasound contrast imaging properties of lipid microbubbles loaded with urokinase in dog livers and their thrombolytic effects when combined with low-frequency ultrasound in vitro

A new microbubble loaded with urokinase (uPA-MB) was explored in a previous study. However, its zeta potential and ultrasound contrast imaging properties and its thrombolytic effects when combined with low-frequency ultrasound (LFUS) were unclear. The zeta potential and ultrasound contrast imaging property of 5 uPA-MBs loading with 50,000 IU uPA was respectively detected using a Malvern laser particle analyzer and a Logiq 9 digital premium ultrasound system. Its ultrasound contrast imaging property was performed on the livers of two healthy dogs to compare with SonoVue. And the clot mass loss rate, D-dimer concentration and surface morphology of the clot residues were measured to evaluate the thrombolytic effect after treatment with three doses of 5 uPA-MBs combined with LFUS in vitro. The zeta potential of 5 uPA-MBs (?27.0 ± 2.40 mV) was higher than that of normal microbubbles (?36.95 ± 1.77 mV). Contrast-enhanced imaging of the hepatic vessels using 5 uPA-MBs was similar to SonoVue, while the imaging duration of 5 uPA-MBs (10 min) was longer than SonoVue (6 min). The thrombolytic effect of three doses of uPA-MBs combined with LFUS was significantly better than that of the control group and showed dose dependence. The 5 uPA-MBs have a negative charge on their surface and good echogenicity as ultrasound contrast agents. The 5 uPA-MBs combined with LFUS can promote thrombolysis in a dose-dependent manner.     

The Presence of Contractile Proteins in Platelet Microparticles Isolated from Human and Animal Platelet-free Plasma

S ummary . Microparticles have been prepared from human and animal plateletfree plasma by dilution of the plasma to lower the specific gravity followed by high-speed ultracentrifugation. The protein, cholesterol, and phospholipid content (and cholesterol-phospholipid ratios) of these microparticles have been determined and compared with values obtained with whole platelet homogenates and various isolated platelet subcellular fractions. Immunological investigations on the microparticles and on the density gradient fractions of platelet homogenates suggest that the particles may have their origin either in the surface membrane or within the intracellular membrane structures of the blood platelets. The microparticles have an associated ATP-ase activity which, though lower in specific activity than isolated platelet 'contractile protein' (thrombosthenin), has similar characteristics in response to divalent cations, mersalyl and ouabain. Thrombosthenin antisera and antisera to the microparticles both inhibit clot retraction in vitro at high dilution and also inhibit the Mg²⁺ ATP-ase activity of platelet homogenates and platelet thrombosthenin. When tested against platelet subcellular fractions from sucrose density gradients both these antisera showed precipitating antibodies to the low density membrane fraction. This fraction consists almost entirely of small vesiculated and larger sheet membrane fragments. In ultrastructure, the microparticles resemble the fine granular material present in the interior of thin-walled sacs which may often be seen in electron-micrographs prepared from fresh platelets. These sacs are believed to be the result of either budding of the pseudopodia or herniation of the cytoplasmic contents through the platelet wall.     

Ultrasound enhancement of thrombolytic therapy: observations and mechanisms

Fibrinolytic therapy is a proven approach for achieving reperfusion of occluded coronary arteries during myocardial infarction, resulting in reduced mortality and preservation of ventricular function. The amount of myocardial muscle loss is proportional to the duration of ischemia. Bleeding complications are not infrequent. Adjuvant therapy by ultrasound might enhance the rate of fibrinolysis and reduce the concentrations of lytic agents required to achieve an equivalent degree of clot lysis. ties and high frequencies, parameters that potentially could be applied and tolerated in vivo, have been proven to significantly accelerate the rate of fibrinolysis in both in vitro and in vivo models, in pure fibrin as well as whole blood clots. Such enhancement is not drug-specific. These effects were achieved by nonthermal mechanism. Ultrasound exposure did not cause mechanical fragmentation of the clot, did not alter the size of plasmatic derivates and degradation products. Ultrasound caused increased flow rate through thrombi, probably by cavitation-induced changes in fibrin ultrastructure; disaggregation of uncrosslinked fibrin fibers into smaller fibers has been shown. This resulted in increased trans-Noninvasive ultrasound at low intensi- port of the lytic agent into the clot, alteration of binding affinity and increased maximum binding. Presence of echo-contrast agent induced further acceleration of thrombolysis by ultrasound. (Int J Cardiovasc Intervent 2000; 3: 81-89)