It has been known for some time that the application of ultrasound can enhance the efficacy of thrombolytic medications such as recombinant tissue plasminogen activator (rt-PA). Potential clinical applications of this ultrasound enhanced thrombolysis (UET) include the treatment of myocardial infarction, acute ischemic stroke, deep venous thrombosis, and other thrombotic disorders. It may be possible to reduce the dose of rt-PA while maintaining lytic efficacy, however there is little data on the rt-PA concentration dependence of UET. In this work, the rt-PA concentration dependence of clot lysis resulting from 120 kHz UET exposure was measured in an in-vitro human clot model. Clots were exposed to rt-PA for 30 minutes, with (UET treated) or without 120 kHz ultrasound (rt-PA treated) at 37° C, and the clot width measured as a function of time. The rt-PA concentration ranged from 0 to 10 µg/ml. The initial lytic rate for the UET treated group was greater than that of the rt-PA group at almost all rt-PA concentrations, and exhibited a maximum over concentration values of 1 to 3 µg/ml.
Ultrasound enhanced thrombolysis; tissue plasminogen activator; acute ischemic stroke
A simple venous thrombosis model in rabbits was used for the quantitative evaluation of the thrombolytic effect of human extrinsic (tissue-type) plasminogen activator as compared with urokinase.
A thrombus was formed in an isolated segment of the jugular vein from a mixture of 125I-labeled fibrinogen, whole rabbit blood, and thrombin. In order to immobilize the thrombus during lysis, it was formed around a woolen thread introduced longitudinally in the lumen of the vein. Thrombotic extension of the clot was prevented by subcutaneous injection of heparin. The extent of thrombolysis was measured as the difference between the radioactivity introduced in the clot and that recovered in the vein segment at the end of the experiment. In control animals the extent of thrombolysis was 5.6±1.4% (n = 5) after 6 h, 14.5±1.7% (n = 10) after 30 h, 16.0±1.5% (n = 11) after 78 h, and 48.1±2.7% (n = 10) after 174 h (mean±SEM).
Extrinsic (tissue-type) plasminogen activator, highly purified from the culture fluid of a human melanoma cell line, was administered systemically or locally over a time period of 4 h and the percent thrombolysis measured 2 h after the end of the infusion. One- and two-chain extrinsic plasminogen activator had very similar thrombolytic potency. Systemic infusion resulted in a dose-dependent degree of thrombolysis. The activator-induced thrombolysis, after infusion of 100,000 IU (≅1 mg protein), was ∼75% for fresh clots, 35% for 1-d-old clots, 30% for 3-d-old clots, and 50% for 7-d-old clots. The thrombolytic activity of urokinase was more than five times lower than that of extrinsic plasminogen activator: Infusion of 500,000 IU resulted in ∼40% lysis of fresh clots and 25% of 1-3-d-old clots, while 7-d-old clots appeared to have become resistent to urokinase. Local infusion resulted in a 5-10 times higher thrombolytic effect of both extrinsic plasminogen activator and urokinase.
Thrombolysis with extrinsic plasminogen activator was not associated with systemic activation of the fibrinolytic system as evidenced by unaltered plasma levels of fibrinogen, plasminogen, and α2-antiplasmin. Systemic infusion of urokinase resulted in significant thrombolysis only at doses that were associated with disseminated plasminogen activation. Local infusion of urokinase required a 5-10-fold higher dose than extrinsic plasminogen activator to obtain a similar degree of thrombolysis, which also occurred in the absence of systemic activation of the fibrinolytic system.
It is concluded that the extent of thrombolysis by extrinsic plasminogen activator is mainly determined by the dose of activator and its delivery in the vicinity of the thrombus and much less by the age of the thrombus or the molecular form of the activator. Extrinsic plasminogen activator appears to be superior to urokinase because of its higher (5-10-fold) specific thrombolytic activity and the absence of systemic activation of the fibrinolytic system, which results in defibrinogenation and a bleeding tendency.
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.
Ultrasound enhanced thrombolysis; Duty cycle; Tissue plasminogen activator; Human whole blood clot
Fibrinolytics such as recombinant tissue plasminogen activator (rt-PA) are used to treat thrombotic disease such as acute myocardial infarction (AMI) and ischemic stroke. Interest in increasing efficacy and reducing side effects has led to the study of adjuncts such as GP IIb-IIIa inhibitors and ultrasound (US) enhanced thrombolysis. Currently, GP IIb-IIIa inhibitor and fibrinolytic treatment are often used in AMI, and are under investigation for stroke treatment. However, little is known of the efficacy of combined GP IIb-IIIa inhibitor, fibrinolytic and ultrasound treatment. We measure the lytic efficacy of rt-PA, eptifibatide (Epf) and 120 kHz ultrasound treatment in an in-vitro human clot model.
Materials and Methods
Blood was drawn from 15 subjects after IRB approval. Clots were made in 20 μL pipettes, and placed in a water tank for microscopic visualization during lytic treatment. Clots were exposed to control, rt-PA (rt-PA), eptifibatide (Epf), or rt-PA+eptifibatide (rt-PA+Epf), with or without ultrasound for 30 minutes at 37°C in human plasma. Clot lysis was measured over time, using a microscopic imaging technique. The fractional clot loss (FCL) and initial lytic rate (LR) were used to quantify lytic efficacy.
Results and Conclusions
LR values for (−US) treated clots were 0.8±0.1(control), 1.8±0.3 (Epf), 1.5±0.2 (rt-PA), and 1.3±0.4 (rt-PA+Epf) (% clot width/minute) respectively. In comparison, the (+US) group exhibited LR values of 1.6±0.2 (control), 4.3±0.4 (Epf), 6.3±0.4 (rt-PA), and 4.6±0.6 (rt-PA+Epf). For (−US) treated clots, FCL was 6.0±0.8 (control), 9.2±2.5 (Epf), 15.6±1.7 (rt-PA), and 28.0±2.2% (rt-PA+Epf) respectively. FCL for (+US) clots was 13.5±2.4 (control), 20.7±6.4 (Epf), 44.4±3.6 (rt-PA) and 30.3±3.6% (rt-PA+Epf) respectively. Although the addition of eptifibatide enhances the in-vitro lytic efficacy of rt-PA in the absence of ultrasound, the efficacy of ultrasound and rt-PA is greater than that of combined ultrasound, rt-PA and eptifibatide exposure.
ultrasound; recombinant tissue plasminogen activator; eptifibatide; thrombosis
Thrombotic disease continues to account for significant morbidity and mortality. Ultrasound energy has been investigated as a potential primary and adjunctive treatment for thrombotic disease. We have previously shown that pulsed-high intensity focused ultrasound (HIFU) enhances thrombolysis induced by tissue plasminogen activator (tPA) in vitro, including describing the non-destructive mechanism by which tPA availability and consequent activity is increased. In this study we aimed to determined if the same effects could be achieved in vivo.
Materials and Methods
In this study, pulsed-HIFU exposures combined with tPA boluses was compared to treatment with tPA alone, HIFU alone and control in a novel in vivo clot model. Clots were formed in the rabbit marginal ear vein and verified using venography and infrared imaging. The efficacy of thrombolytic treatment was monitored via high resolution ultrasonography for five hours post treatment. The cross-sectional area of clots at 4 points along the vein was measured and normalized to the pre-treatment size.
At five hours the complete recanalization of clots treated with pulsed-HIFU and tPA was significantly different from the partial recanalization seen with tPA treatment alone. tPA treatment alone showed a significant decrease in clot versus control, where HIFU was not significantly different than control. Histological analysis of the vessel walls in the treated veins showed no apparent irreversible damage to endothelial cells or extravascular tissue.
This study demonstrates that tPA mediated thrombolysis can be significantly enhanced when combined with non-invasive pulsed-HIFU exposures.
High intensity focused ultrasound (HIFU); thrombolysis; tissue plasminogen activator (tPA); clot model; rabbit ear vein
Ultrasound (US) has been used to enhance thrombolytic therapy in the treatment of stroke. Considerable attenuation of US intensity is however noted if US is applied over the temporal bone. The aim of this study was therefore to explore possible changes in the effect of thrombolytic drugs during low-intensity, high-frequency continuous-wave ultrasound (CW-US) exposure.
Clots were made from fresh venous blood drawn from healthy volunteers. Each clot was made from 1.4 ml blood and left to coagulate for 1 hour in a plastic test-tube. The thrombolytic drugs used were, 3600 IU streptokinase (SK) or 0.25 U reteplase (r-PA), which were mixed in 160 ml 0.9% NaCl solution. Continuous-wave US exposure was applied at a frequency of 1 MHz and intensities ranging from 0.0125 to 1.2 W/cm2. For each thrombolytic drug (n = 2, SK and r-PA) and each intensity (n = 9) interventional clots (US-exposed, n = 6) were submerged in thrombolytic solution and exposed to CW-US while control clots (also submerged in thrombolytic solution, n = 6) were left unexposed to US.
To evaluate the effect on clot lysis, the haemoglobin (Hb) released from each clot was measured every 20 min for 1 hour (20, 40 and 60 min). The Hb content (mg) released was estimated by spectrophotometry at 540 nm. The difference in effect on clot lysis was expressed as the difference in the amount of Hb released between pairs of US-exposed clots and control clots. Statistical analysis was performed using Wilcoxon's signed rank test.
Continuous-wave ultrasound significantly decreased the effects of SK at intensities of 0.9 and 1.2 W/cm2 at all times (P < 0.05). Continuous-wave ultrasound significantly increased the effects of r-PA on clot lysis following 20 min exposure at 0.9 W/cm2 and at 1.2 W/cm2, following 40 min exposure at 0.3, 0.6, 0.9 and at 1.2 W/cm2, and following 60 min of exposure at 0.05 0.3, 0.6, 0.9 and at 1.2 W/cm2 (all P < 0.05).
Increasing intensities of CW-US exposure resulted in increased clot lysis of r-PA-treated blood clots, but decreased clot lysis of SK-treated clots.
We studied the disaggregation of human platelets by tissue-type plasminogen activator (t-PA). When added to a suspension of human platelets induced to aggregate in plasma with adenosine 5'-diphosphate, t-PA promoted disaggregation of platelets over several minutes. Addition of fresh plasma or purified human fibrinogen to disaggregated platelets facilitated (reversible) aggregation and subsequent disaggregation. Aspirin treatment of platelets markedly potentiated the ability of t-PA to induce disaggregation. Disaggregation was inhibited by alpha-2-antiplasmin. Comparative analysis of the rate of proteolysis of platelet-bound fibrinogen with that of ambient plasma fibrinogen suggested that fibrinogenolysis of cohesive fibrinogen occurred more rapidly than fibrinogenolysis of ambient fibrinogen. These data demonstrate that t-PA facilitates platelet disaggregation in plasma through kinetically selective proteolysis of cohesive fibrinogen by plasmin, and suggest that thrombolytic mechanisms may serve both to remove platelets from platelet-fibrin thrombi and to disperse circulating platelet aggregates.
Pulsed ultrasound, when used as an adjuvant to recombinant tissue plasminogen activator (rt-PA), has been shown to enhance thrombolysis in the laboratory as well as in clinical trials for the treatment of ischemic stroke. The exact mechanism of this enhancement has not yet been elucidated. In this work, stable and inertial cavitation (SC and IC) are investigated as possible mechanisms for this enhancement. A passive cavitation detection scheme was utilized to measure cavitation thresholds at 120 kHz (80% duty cycle, 1667 Hz pulse repetition frequency) for four host fluid and sample combinations: plasma, plasma with rt-PA, plasma with clot and plasma with clot and rt-PA. Following cavitation threshold determination, clots were exposed to pulsed ultrasound for 30 min in vitro using three separate ultrasound treatment regimes: (1) no cavitation (0.15 MPa), (2) SC alone (0.24 MPa) or (3) SC + IC combined (0.36 MPa) in the presence of rt-PA. Percent clot mass loss after each treatment was used to determine thrombolysis efficacy. The highest percent mass loss was observed in the stable cavitation regime (26%), followed by the combined stable and inertial cavitation regime (20.7%). Interestingly, the percent mass loss in clots exposed to ultrasound without cavitation (13.7%) was not statistically significantly different from rt-PA alone (13%) [p > 0.05]. Significant enhancement of thrombolysis correlates with presence of cavitation and stable cavitation appears to play a more important role in the enhancement of thrombolysis.
Cavitation; Ultrasound-assisted thrombolysis; Cavitation detection
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.
Ultrasound; Hyperthermia; Thrombolysis
Ultrasound has been previously shown to act synergistically with a thrombolytic agent, such as recombinant tissue plasminogen activator (rt-PA) to accelerate thrombolysis. In this in vitro study, a commercial contrast agent, Definity®, was used to promote and sustain the nucleation of cavitation during pulsed ultrasound exposure at 120 kHz. Ultraharmonic signals, broadband emissions, and harmonics of the fundamental were measured acoustically by using a focused hydrophone as a passive cavitation detector and utilized to quantify the level of cavitation activity. Human whole blood clots suspended in human plasma were exposed to a combination of rt-PA, Definity®, and ultrasound at a range of ultrasound peak-to-peak pressure amplitudes, which were selected to expose clots to various degrees of cavitation activity. Thrombolytic efficacy was determined by measuring clot mass loss before and after the treatment and correlated with the degree of cavitation activity. The penetration depth of rt-PA and plasminogen was also evaluated in the presence of cavitating microbubbles using a dual antibody fluorescence imaging technique. The largest mass loss (26.2%) was observed for clots treated with 120 kHz ultrasound (0.32 MPa peak-to-peak pressure amplitude), rt-PA and stable cavitation nucleated by Definity®. A significant correlation was observed between mass loss and ultraharmonic signals (r=0.8549, p<0.0001, n=24). The largest mean penetration depth of rt-PA (222 µm) and plasminogen (241 µm) was observed in the presence of stable cavitation activity. Stable cavitation activity plays an important role in enhancement of thrombolysis and can be monitored to evaluate the efficacy of thrombolytic treatment.
Ultrasound-assisted thrombolysis; stroke therapy; ultraharmonics; stable cavitation; therapeutic ultrasound
Thrombolytics such as recombinant tissue plasminogen activator (rt-PA) have advanced the treatment of ischemic stroke, myocardial infarction, deep vein thrombosis and pulmonary embolism.
To improve the efficacy of this thrombolytic therapy, the synergistic effect of rt-PA and 120 kHz or 1.0 MHz ultrasound was assessed in vitro using a porcine clot model.
Materials and methods
Fully retracted whole blood clots prepared from fresh porcine blood were employed to compare rt-PA thrombolytic treatment with and without exposure to 120-kHz or 1-MHz ultrasound. For sham studies (without ultrasound), clot mass loss was measured as a function of rt-PA concentration from 0.003 to 0.107 mg/ml. For combined ultrasound and rt-PA treatments, peak-to-peak pressure amplitudes of 0.35, 0.70 or 1.0 MPa were employed. The range of duty cycles varied from 10% to 100% (continuous wave) and the pulse repetition frequency was fixed at 1.7 KHz.
For rt-PA alone, the mass loss increased monotonically as a function of rt-PA concentration up to approximately 0.050 mg/ml. With ultrasound and rt-PA exposure, clot mass loss increased by as much as 104% over rt-PA alone. Ultrasound without the presence of rt-PA did not significantly enhance thrombolysis compared to control treatment. The ultrasound-mediated clot mass loss enhancement increased with the square root of the overall treatment duration.
Both 120-kHz and 1-MHz pulsed and CW ultrasound enhanced rt-PA thrombolysis in a porcine whole blood clot model in vitro. No clear dependence of the observed thrombolytic enhancement on ultrasound duty cycle was evident. The lack of duty cycle dependence suggests a more complex mechanism that could not be sustained by merely increasing the pulse duration.
Ultrasound-enhanced thrombolysis; Stroke therapy; Fibrinolysis; Therapeutic ultrasound; Stable cavitation
Tissue plasminogen activator (t-PA) causes fibrinogen proteolysis when alpha 2-antiplasmin levels fall, and this may contribute to t-PA-induced hemorrhage. Because clot-bound plasmin is protected from alpha 2-antiplasmin inhibition, we tested the possibility that alpha 2-antiplasmin supplementation would block t-PA-induced fibrinogenolysis and bleeding without affecting thrombolysis. When added to human or rabbit plasma, alpha 2-antiplasmin inhibits t-PA-induced fibrinogenolysis, but hat little effect on the lysis of 125I-fibrin clots. To examine its effect in vivo, rabbits with preformed 125I-labeled-jugular vein thrombi were randomized to receive t-PA, t-PA and alpha 2-antiplasmin, or saline. alpha 2-Antiplasmin infusion produced a modest decrease in t-PA-induced thrombolysis (from 40.2% to 30.1%, P = 0.12), but reduced fibrinogen consumption from 87% to 27% (P = 0.0001), and decreased blood loss from standardized ear incisions from 5,594 to 656 microliter (P < 0.0001). We hypothesize that alpha 2-antiplasmin limits t-PA-induced hemorrhage by inhibiting fibrinogenolysis and subsequent fragment X formation because (a) SDS-PAGE and immunoblot analysis indicate less fragment X formation in alpha 2-antiplasmin treated animals, and (b) when added to a solution of fibrinogen and plasminogen clotted with thrombin in the presence of t-PA, fragment X shortens the lysis time in a concentration-dependent fashion. These findings suggest that fragment X incorporation into hemostatic plugs contributes to t-PA-induced bleeding. By blocking t-PA-mediated fibrinogenolysis, alpha 2-antiplasmin supplementation may improve the safety of fibrin-specific plasminogen activators.
Substantial enhancement of recombinant tissue plasminogen activator (rt-PA) thrombolysis can be achieved with ultrasound, suggesting its use as an adjunctive treatment in thrombolytic therapy for stroke. A microscopic visualization method was used to measure the lysis of human whole-blood clots treated with human fresh frozen plasma (HFFP), rt-PA, and 120-kHz ultrasound for 30 min at T = 37 ° C. The clot–plasma interface was imaged using an inverted optical microscope and the thrombolytic front analyzed as a function of time. Ultrasound treatment significantly enhanced the mean lytic rate from 0.5 to 3.4 μm/min (a 580% change) compared with rt-PA treatment alone.
Fibrinogen concentrations were determined in normal plasma and in plasma from patients with high and low levels. There was a good correlation between the results of a rapid semi-quantitative fibrinogen titre technique and those of a quantitative assay of coagulable fibrinogen. In normal subjects fibrinogen levels were not significantly influenced by taking blood into epsilon aminocaproic acid (EACA) or by the addition of protamine to plasma. In patients with the defibrination syndrome in whom increased plasma fibrinolysis was not detected, fibrinogen levels were not affected by taking blood into EACA but considerably increased levels were observed after the addition of protamine to plasma. In patients undergoing thrombolytic therapy the fibrinogen levels measured were increased both in blood taken into EACA and in plasma containing protamine. It is suggested that EACA acted by preventing lysis in vitro whilst protamine counteracted abnormal fibrin polymerization. The pattern of results may be of diagnostic importance.
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.
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 °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)×103 J/kg·K and 0.55±0.13 W/m·K, respectively, and for human clotted blood are (3.5±0.8)×103 J/kg·K and 0.59±0.11 W/m·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.
Background and Purpose
Currently, the only FDA-approved therapy for acute ischemic stroke is the administration of recombinant tissue plasminogen activator (tPA). Echogenic liposomes (ELIP), phospholipid vesicles filled with gas and fluid, can be manufactured to incorporate tPA. Also, transcranial ultrasound-enhanced thrombolysis can increase the recanalization rate in stroke patients. However, there is little data on lytic efficacy of combining ultrasound, echogenic liposomes, and tPA treatment. In this study, we measure the effects of pulsed 120-kHz ultrasound on the lytic efficacy of tPA and tPA-incorporating ELIP (t-ELIP) in an in-vitro human clot model. It is hypothesized that t-ELIP exhibits similar lytic efficacy to that of rt-PA.
Blood was drawn from 22 subjects after IRB approval. Clots were made in 20-μL pipettes, and placed in a water tank for microscopic visualization during ultrasound and drug treatment. Clots were exposed to combinations of [tPA] = 3.15 μg/ml, [t-ELIP] = 3.15 μg/ml, and 120-kHz ultrasound for 30 minutes at 37 °C in human plasma. At least 12 clots were used for each treatment. Clot lysis over time was imaged and clot diameter was measured over time, using previously developed imaging analysis algorithms. The fractional clot loss (FCL), which is the decrease in mean clot width at the end of lytic treatment, was used as a measure of lytic efficacy for the various treatment regimens.
The fractional clot loss FCL was 31% (95% CI: 26-37%) and 71% (56-86%) for clots exposed to tPA alone or tPA with 120 kHz ultrasound. Similarly, FCL was 48% (31-64%) and 89% (76-100%) for clots exposed to tELIP without or with ultrasound.
The lytic efficacy of tPA containing echogenic liposomes is comparable to that of tPA alone. The addition of 120 kHz ultrasound significantly enhanced lytic treatment efficacy for both tPA and t-ELIP. Liposomes loaded with tPA may be a useful adjunct in lytic treatment with tPA.
Acute ischemic stroke; Tissue plasminogen activator; Ultrasound; Echogenic liposomes
The relation between coronary thrombolysis and coagulation variables after administration of anistreplase (anisoylated plasminogen streptokinase activator complex, APSAC) was studied in patients with an acute myocardial infarction. Fifty eight consecutive patients with acute myocardial infarction were given 30 U of anistreplase intravenously within 4 hours of the onset of symptoms. A fall in the plasma concentration fibrinogen to less than 1.0 g/l 90 minutes after administration of anistreplase was considered to reflect a systemic lytic state. Coronary angiography was performed 48 hours after thrombolytic treatment. The overall patency rate was 74% (43/58). Patency rates were significantly different in patients with a systemic lytic (83% (43/52)) and a systemic non-lytic state (0% (0/6)). The absence of a systemic lytic state after anistreplase administration seemed to be highly predictive of the failure of coronary thrombolysis. Coagulation studies showed evidence of inhibition of anistreplase induced fibrinolytic activity which may explain the failure of thrombolytic treatment in patients with evidence of a systemic non-lytic state.
AIMS—To investigate if it was possible to lower the dose of streptokinase and maintain an effective thrombolysis by adding pulsed low energy ultrasound.
Methods—53 retinal veins in 27 rabbits were occluded by rose bengal enhanced laser treatment. Six rabbits were treated with streptokinase (50 000 IU/kg), 10 rabbits were treated with a low dose of streptokinase (25 000 IU/kg), and 11 rabbits were treated with a low dose of streptokinase (25 000 IU/kg) and pulsed ultrasound during 1 hour. Fluorescein angiography was performed immediately before the thrombolytic treatment and after 12 hours.
RESULTS—In the group treated with streptokinase (50 000 IU/kg) all vessels were open. In the group that was given streptokinase (25 000 IU/kg), 21% of the vessels were open. In the group that was treated with streptokinase (25 000 IU/kg) and ultrasound, 64% of the vessels were open. The difference between groups 2 and 3 is statistically significant (p= 0.011)
CONCLUSION—Adding pulsed low energy ultrasound makes it possible to lower the dose of streptokinase while maintaining a good thrombolytic effect.
Keywords: retinal vein occlusion; ultrasound; thrombolytic treatment; streptokinase
Targeted delivery of thrombolytics to the site of occlusion is an attractive concept, with implications for the treatment of many thrombo-occlusive diseases. Ultrasound enhances thrombolysis, which can be augmented by the addition of a contrast agent. We have previously reported development of echogenic liposomes (ELIP) for targeted highlighting of structures with potential for drug and gene delivery. This study evaluated the potential of ELIP for thrombolytic loading, and the effect of ultrasound exposure of thrombolytic-loaded ELIP on thrombolytic efficacy.
Materials and methods
Tissue-plasminogen activator (tPA) was loaded into ELIP. Echogenicity was assessed and reported as mean grayscale values. Whole porcine clots were treated with plasma, free tPA, tPA+Optison® (echocontrast agent), or tPA-loaded ELIP, with and without ultrasound (1 MHz, continuous wave, 2 W/cm2, for 2 min). Clots were weighed before and after a 30-min treatment period, and results reported as percent clot mass loss.
tPA entrapment into ELIP was feasible with 50% entrapment, and retention of echogenicity. Treatment with tPA-loaded ELIP resulted in effective clot lysis with an effect similar to treatment with free tPA. Ultrasound exposure of tPA-loaded ELIP resulted in enhanced thrombolysis (49.5% relative improvement vs. no ultrasound). Much of the ultrasound effect appeared to be related to drug release from the tPA—ELIP complex.
We have demonstrated entrapment of tPA into ELIP with effective clot lysis and drug release using ultrasound. Our tPA-loaded ELIP has potential for specific highlighting of clots to confirm agent delivery and help focus ultrasound therapy for targeted ultrasound-facilitated thrombolysis.
Ultrasound; Thrombolysis; Echogenic; Liposomes; Contrast agent
The effect of ultrasound on the rate of fibrinolysis has been investigated using an in vitro system. Plasma or blood clots containing a trace label of 125I fibrin were suspended in plasma containing plasminogen activator and intermittently exposed to continuous wave 1-MHz ultrasound at intensities up to 8 W/cm2. Plasma clot lysis at 1 h with 1 microgram/ml recombinant tissue plasminogen activator (rt-PA) was 12.8 +/- 1.2% without ultrasound and was significantly (P = 0.0001) increased by exposure to ultrasound with greater lysis at 1 W/cm2 (18.0 +/- 1.4%), 2 W/cm2 (19.3 +/- 0.7%), 4 W/cm2 (22.8 +/- 1.8%), and 8 W/cm2 (58.7 +/- 7.1%). Significant increases in lysis were also seen with urokinase at ultrasound intensities of 2 W/cm2 and above. Exposure of clots to ultrasound in the absence of plasminogen activator did not increase lysis. Ultrasound exposure resulted in a marked reduction in the rt-PA concentration required to achieve an equivalent degree of lysis to that seen without ultrasound. For example, 15% lysis occurred in 1 h at 1 microgram/ml rt-PA without ultrasound or with 0.2 microgram/ml with ultrasound, a five-fold reduction in concentration. Ultrasound at 1 W/cm2 and above also potentiated lysis of retracted whole blood clots mediated by rt-PA or urokinase. The maximum temperature increase of plasma clots exposed to 4 W/cm2 ultrasound was only 1.7 degrees C, which could not explain the enhancement of fibrinolysis. Ultrasound exposure did not cause mechanical fragmentation of the clot into sedimentable fragments, nor did it alter the sizes of plasmic derivatives as demonstrated by SDS polyacrylamide gel electrophoresis. We conclude that ultrasound at 1 MHz potentiates enzymatic fibrinolysis by a nonthermal mechanism at energies that can potentially be applied and tolerated in vivo to accelerate therapeutic fibrinolysis.
A range of tests of coagulation and fibrinolysis was measured in "normal" dogs and compared with values obtained in "normal" humans by the same methods. The hematocrit platelet count, fibrinogen and plasminogen were similar in dogs and in humans. The prothrombin and partial thromboplastin times were considerably shorter in the dog than in man but the thrombin clotting time was comparable. Fibrinolysis was more active in dogs but the levels of fibrin degradation products were low, suggesting that there was no significant fibrin deposition and lysis occurring in vivo.
Disseminated intravascular coagulation causes thrombotic tendency leading to multiple organ failure and occurs in a wide variety of diseases including malignancy. Disseminated intravascular coagulation is a latent complication in people with prostate cancer.
A 51-year-old Japanese man with advanced castration-resistant prostate cancer was admitted to our hospital because of extensive purpura and severe anemia. Prolonged plasma coagulation time, hypofibrinogenemia and normal platelet count suggested that a decrease in fibrinogen induced a bleeding tendency causing purpura. However, elevated plasma levels of thrombin-antithrombin complex, fibrin and/or fibrinogen degradation products and D-dimers, with positive fibrin monomer test, manifested disseminated intravascular coagulation and subsequent fibrinolysis. Plasma levels of thrombin-antithrombin complex, fibrin and/or fibrinogen degradation products and D-dimers decreased after administration of low-molecular-weight heparin. However, low fibrinogen and α2-antiplasmin levels were not improved and plasmin-antiplasmin complex did not decrease, which revealed excessive fibrinolysis complicated with disseminated intravascular coagulation. We suspected that prostate cancer cell-derived urokinase-type plasminogen activator caused excessive fibrinolysis. Administration of tranexamic acid for fibrinogenolysis was added together with high-dose anti-androgen therapy (fosfestrol) for prostate cancer. Thereafter, prostate-specific antigen and plasmin-antiplasmin complex decreased, followed by normalized fibrinogen and α2-antiplasmin levels, and the patient eventually recovered from the bleeding tendency. Immunohistochemical staining of the biopsied prostate tissue exhibited that the prostate cancer cells produced tissue factor, the coagulation initiator, and urokinase-type plasminogen activator.
This patient with rare complications of disseminated intravascular coagulation and excessive fibrinolysis is a warning case of potential coagulation disorder onset in patients with prostate cancer. We propose that combined administration of tranexamic acid and low-molecular-weight heparin together with high-dose anti-androgen therapy is a useful therapeutic option for patients with this complicated coagulation disorder.
Castration-resistant prostate cancer; Disseminated intravascular coagulation; Excessive fibrinolysis; Low-molecular-weight heparin; Tranexamic acid
Ultrasound enhances thrombolysis when combined with a thrombolytic and a contrast agent. This study aimed to evaluate the thrombolytic effect of our tissue plasminogen activator (tPA)–loaded echogenic liposomes (ELIP) in an in vivo clot model, with and without ultrasound treatment.
Methods and Results
The femoral arteries of New Zealand White rabbits (n=4 per group) were cannulated. The abdominal aortas were denuded, and thrombi were created using a solution of sodium ricinoleate plus thrombin. Rabbits were then randomly selected to receive tPA-loaded ELIP (200 μg of tPA/5 mg of lipid) or empty ELIP with or without pulsed (color) Doppler ultrasound (5.7 MHz) for 2 minutes. Thrombus was imaged and echogenicity analyzed before and after ELIP injection. Blood flow velocities were measured at baseline, after clot formation, and serially after treatment up to 15 minutes. tPA-loaded ELIP highlighted thrombus in the abdominal aorta more effectively than empty ELIP (P<0.05). Ultrasound enhanced the thrombolytic effect of tPA-loaded ELIP, resulting in earlier and more complete recanalization rates (P<0.001).
This study demonstrates effective highlighting of clots and thrombolytic effect of tPA-loaded ELIP in an in vivo rabbit aorta clot model. Doppler ultrasound treatment enhances this thrombolytic effect, resulting in earlier and more complete recanalization rates.
arterial thrombosis; contrast echo; Doppler ultrasound; echocardiography; thrombolysis
Consensus regarding the use of thrombolysis to treat acute pulmonary embolism has not yet been reached. There is good evidence that thrombolytic agents dissolve clot more rapidly than heparin. However, proving that this benefit reduces the death rate from pulmonary embolism has been difficult. Each of the 3 thrombolytic agents (tissue type-plasminogen activator, streptokinase and urokinase) is equally efficacious at dissolving clot, but all are associated with an increased risk of major hemorrhage when compared with heparin. One evolving position is that, in addition to patients presenting in circulatory collapse, for whom thrombolysis has been demonstrated to be life-saving, a subgroup of patients may be identified by echocardiography, through its ability to assess right ventricular dysfunction, who should also be considered for thrombolytic therapy. It remains to be seen whether this approach can reduce the death rate associated with pulmonary embolism.