Method of lysing thrombi

This invention relates to the prevention and treatment of thrombi using pminogen activators, including t-PA, urokinases, and streptokinases, in conjunction with intermittent (pulsed mode) ultrasound. A preferred modality interposes liquid-containing interface between the skin of the patient and the transducer of the ultrasound generator.

FIELD OF THE INVENTION 
This invention relates to the prevention and treatment of thrombi using 
plasminogen activators in conjunction with intermittent (pulsed mode) 
ultrasound. A preferred modality interposes liquid-containing interface 
between the skin of the patient and the transducer of the ultrasound 
generator. 
BACKGROUND OF THE INVENTION 
Plasminogen activating agents including streptokinase, urokinase, tissue 
plasminogen activator (t-PA), and their analogues have been administered 
as lytic agents for treatment of arterial and venous thrombosis. Although 
such agents as t-PA are efficacious for lysis of coronary thrombi and 
pulmonary emboli, relatively high concentrations are required. (Marder V. 
J., Sherry S.; "Thrombolytic Therapy: Current Status" N Engl J Med 
318:1512-20 (1988). Marder V. J., Sherry S.; "Thrombolytic Therapy: 
Current Status", N Engl J Med 318:1585-95 (1988). Vaughan D. E., Goldhaber 
S. Z., Kim J., Loscalzo J.; "Recombinant Tissue Plasminogen Activator in 
Patients Pulmonary Embolism . . . " Circulation 75:1200-03 (1987).) The 
need for high concentrations may be due, in part, to the paucity of fibrin 
binding sites for t-PA on the surface of whole blood clots. In the absence 
of binding to fibrin, t-PA has greatly reduced efficacy in activating 
plasminogen and inducing clot lysis. In clinical practice, t-PA can be 
directly injected into the site of thrombus with percutaneous transluminal 
catheters rather than infused systemically. Presumably this approach 
improves lysis by increasing the surface area of fibrin available for t-PA 
binding. 
Ultrasound has been used without exogenous t-PA to disrupt peripheral 
arterial and venous thrombi in animal models and to open atherosclerotic 
occlusions of peripheral arteries in selected patients. To achieve these 
effects, ultrasound was used at a frequency (20 kHz) and intensity that 
caused cavitation, thereby disrupting tissues with low elasticity, such as 
atheromas and thrombi. The ultrasound was delivered through catheters to 
prevent damage to normal tissues near the sites of the occlusions. 
Kudo and co-workers are the first to report the use of noninvasive 
ultrasound to increase the efficacy of systemic t-PA. (Kudo S., Furuhata 
H., Hara M., Maie K., Hamano K., Okamura T. "Noninvasive Thrombolysis with 
Ultrasound"; Circulation, 80:supp 1I-345 (1989) (abstract); Kudo S. 
"Thrombolysis with Ultrasound Effect", Tokyo Jikeikai Med J., 
104:1005-1012 (1989); and Hamano K., Fujinaga T., Muto M., Yoshizawa S., 
Kudo S., Hara M., Okamura T., Furuhata H., "Thrombolysis by Transcutaneous 
Ultrasonic irradiation, Circulation, 82:III-309 (1990) (Abstract).) It was 
found that transcutaneous ultrasound that was delivered in a continuous 
mode at a frequency of 200 kHz could enhance t-PA-induced fibrinolysis in 
a canine model of femoral arterial thrombi. The instantly disclosed and 
claimed invention varies from the methods taught in those publications, 
since the inventive method uses intermittent (pulsed mode) ultrasound 
and/or a fluid interface between the skin of the patient and the 
transducer of the ultrasound generator. 
Lower extremity deep venous thrombosis (DVT) incidence in the United States 
is greater than 250,000 cases annually. Pulmonary embolism is the primary 
cause of at least 50,000 deaths annually. The incidence of clinical DVT is 
1 in 500 in general hospital patients. In unselected patients undergoing 
elective major general or orthopedic surgery without prophylaxis the 
incidence of DVT is reported to be 20% to 30%. 
Heparin is often used to prevent thrombus propagation. Plasminogen 
activators (PA's) are used to lyse thrombi, but DVT are often resistent to 
both of these therapies. Complete thrombolysis is difficult to achieve. 
Plasminogen activators are particularly effective thrombolytic agents when 
incorporated into a forming thrombus. It is believed that limited surface 
binding and penetration into the thrombus by PA may explain difficulties 
in achieving complete thrombolysis. 
Raising the dosage of t-PA administered beyond 100 mgm for lysis of acute 
thrombosis increases the number of hemorrhagic complications and, for this 
reason, dosing above this level is not desirable. 
Most investigation of ultrasound bioeffects have been directed to safe 
exposure of human for diagnostic purposes. However, therapeutic 
applications to treat injuries to soft tissue and for ultrasonic 
aspiration have proven useful in neurosurgery and soft tissue resections. 
Reports of studies of ultrasound by Kudo on dogs do not discuss some of 
the problems related to temperature increases arising during exposure for 
therapeutic uses. No disclosure of use of ultrasound to prevent formation 
of thrombosis has previously been disclosed. 
A model frequently used in assessing thrombosis has been the rabbit with 
jugular vein thrombosis. Previous studies with this model indicate that 
the rate and extent of thrombolysis are related and that complete lysis of 
thrombus is very difficult to achieve. Ultrasound that is used in 
physiotherapy is delivered at a frequency of 1 MHz, which can cause 
acoustic streaming, or wave-media interactions that promote agitation of 
solute and micro-particulate matter without inducing cavitation or tissue 
damage. This property of ultrasound suggests that it may have the 
potential for gentle perturbation of a clot, thereby exposing additional 
fibrin for binding to the plasminogen activator. 
Ultrasound travels through fluid and soft tissue by wave propagation. The 
number of ultrasound waves per unit area is a measure of power which is 
usually expressed as watts/cm.sup.2. As ultrasound travels through soft 
tissue it pushes fluids in the direction of the beam. Another effect is 
the momentary absorption of ultrasound energy by dissolved gases in fluid 
which results in expansion and then redissolution of the gas. These 
effects are referred to as acoustic microstreaming and cavitation. The end 
result of ultrasound interactions with soft tissue is attenuation of the 
beam and generation of heat. Attenuation increases as frequency increases, 
which results in a decreasing penetration into soft tissue with increasing 
ultrasound frequencies. The 1 MHz frequency used in these experiments will 
maintain 30% of original energy after traversing 10 cm of soft tissue. At 
power levels of 3.0 watts/cm.sup.2 (average power), harmful effects of 
ultrasound have been noted only under conditions which generate enough 
heat to cause thermal injury. 
Clinical experience with plasminogen activators have resulted in findings 
similar to those noted in rabbit studies. The greatest success has been 
attained in treatment of coronary artery thrombi, but challenges remain: 
1) to decrease the mean time required for restoration of coronary artery 
flow following administration of the plasminogen activating agents and 2) 
in the setting of acute myocardial infarction or venous thrombosis, to 
increase the percent of patients receiving plasminogen activators who will 
have successful restoration of blood flow. Raising dosage of plasminogen 
activators increases the number of hemorrhagic complications and, for this 
reason, dosing at high levels is not a routine option to improve results 
of therapy. 
SUMMARY OF THE INVENTION 
It is the purpose of this invention to provide improved methods for 
prevention or treatment of thrombosis, including deep venous thrombosis 
(DVT) and acute arterial thrombosis such as coronary artery thrombosis, 
whilst avoiding damage to the surrounding tissue. The method of the 
invention comprises administration of plasminogen activators in 
conjunction with ultrasound in a manner that will provide improved 
protection of the surrounding tissue. The methods of the invention include 
administration of intermittent (pulse mode) ultrasound and the imposition 
of a fluid-containing barrier between the ultrasound source and the 
patient's skin. 
It is also a purpose of this invention to provide a means for preventing 
formation of thrombi by administration of small doses of plasminogen 
activating agents along with intermittent ultrasound to patients who are 
likely to suffer from formation of thrombi. Particularly at risk are older 
patients that are immobilized or patients whose injuries have caused 
decreased circulation in the extremities.

DETAILED DESCRIPTION OF THE INVENTION 
The invention described herein teaches means of effectively treating 
patients suffering from thrombosis, particularly deep venous thrombosis 
whilst avoiding injury to the surrounding tissue. The invention also 
provides means of preventing formation of thrombi using minimal dosages of 
plasminogen activators. The most commonly used plasminogen activators are 
strepokinase, urokinase, and t-PA. Additional agents that have been tested 
for use as plasminogen activators include analogues produced by known 
means including acylation of the plaminogen moienty or attaching to the 
molecule of the plasminogen activators targeting antibodies, including 
antifibrin antibodies. An example of such an analog is the p-anisoylated 
derivative of Lys-Plaminogen-Streptokinase activator complex. The 
plasminogen activators have been made by recombinant bioengineering 
methods. The term "plasminogen activating agents" or the particular 
compounds named herein are to be considered to include such analogues of 
the named compounds. The analogues may be administered by the inventive 
method taught herein. Because the amounts of plasminogen activating agents 
required to prevent formation of thrombi are much lower than amounts 
required to treat patients who have thrombosis, patients who are likely to 
suffer from formation of thrombi may be beneficially treated 
prophylactically. The use of intermittent ultrasound and of the fluid 
barrier between the ultrasound source and the skin avoid exposure of the 
tissues to excessive heat and injury during treatment. 
Maximum routine dosage used for t-PA in humans is 100 mg. in 24 hrs. Often 
half the dosage is administered as an intravenous bolus and the remainder 
given over the next 4 to 12 hours. The plasma half-life of t-PA is less 
than 15 minutes, but its clinical effectiveness is often noted to lag 
beyond the time of expected peak plasma levels. Routine measurement of 
t-PA is not performed during therapy, but a local plasma activity of 300 
IU/ml (as determined by calculation rather than clinical testing) could be 
maintained for 8-10 hours by continuous local infusion of the standard 
dose, with peak systemic plasma activities to 5000 IU/ml. probably 
occurring during the acute administration of a t-PA bolus. 
It was found, in in vitro testing, that ultrasound as administered by the 
inventive method shortened the time for 50% clot lysis to as little as 
half the time required without the ultrasound and that absolute lysis was 
increased by 30% to 500% by use of intermittent ultrasound. 
The imposition of a liquid-containing interface between the transducer of 
the ultrasound generator and the patient may be accomplished in any manner 
which allows the resonance to be transmitted to the skin. A bladder of 
fluid within an enclosure wherein the enclosure has openings positioned in 
such a manner that the membrane surrounding the liquid is exposed at two 
points: (1) at the area where the ultrasonic effects are to be delivered 
to the patient and (2) at the area wherein the ultrasonic generator 
surface would in contact with the liquid-filled bladder membrane. FIG. 1 
shows an example of use of such an array wherein a ultrasound generator 
(1) with a timer (4) has a transducer (2) with a contact surface (11) that 
contacts a membrane (9) and (7) that encloses a liquid (6). The membrane 
is in an enclosure (5). The mammal is placed with the thrombus (8) against 
the membrane. The intravenous fluid for infusion (3) may contain the 
plasminogen activating agent. A temperature monitor may be used to assess 
thermal effect on the tissues near the thrombus. The liquid-containing 
interface may also be provided by using a bladder containing fluid without 
an enclosure around it. It is also possible to provide the fluid interface 
by using a gel on the body at the point of contact with the ultrasound 
generator transducer. 
The bladder containing liquid can be made specifically to fit over the 
contact surface of the transducer of ultrasound generator. Such an 
arrangement is shown in FIG. 2 wherein the ultrasound transducer (2) has 
attached thereto a liquid containing bladder (14) in an enclosure (12) 
which is clipped into place with fastening means (15) and wherein one 
membrane of the bladder (9) is against the contact surface of the 
transducer (11) and is held in place by fastening means (15). Fastening 
means may be any means known in the art which will prevent a gap between 
the contact surface of the transducer and the membrane from the bladder 
containing liquid. For example, the enclosure may have a rim which will 
clip onto a complimentary ridge on the transducer. The preferred range of 
frequency for purposes of practicing this invention is 0.1-3MHz with a 
preferred power range of 0.5-3 watts/cm.sup.2. The frequency of ultrasound 
determines the potential depth of penetration through fluid and soft 
tissue. Ultrasound travels very efficiently through water, with 95% of 
initial power conserved after traveling 10 cm. When traveling through soft 
tissue, only 30% of initial power is conserved after traveling 10 cm. Loss 
of ultrasound power or attenuation is the consequence of ultrasound 
interaction with the media. The end result of all interactions is the 
generation of heat. Using the methods of the invention, ultrasound 
treatment did not raise soft tissue temperatures in the region of 
ultrasound effect. In the rabbit model, serum CPK values were not altered 
with ultrasound treatment and histologic examination of the veins did not 
disclose change in the inflammatory response with ultrasound treatment. 
The ability of noninvasive ultrasound, delivered at a frequency of within 
the range taught herein in an intermittent mode to enhance t-PA activity 
was evaluated in vitro and in vivo utilizing the rabbit jugular vein 
thrombosis model. 
Materials and Methods 
Materials: 
Single chain human recombinant tissue plasminogen activator (t-PA, lot 
number N9102, specific activity 580,000 IU/mg) was purchased from 
Genentech, Inc., South San Francisco, Calif.; thrombin (2200 NIH U/mg) was 
from Sigma Chemical Company, St. Louis, Mo. Lysine Sepharose and Sephadex 
G-25 were from Pharmacia Fine Chemicals, Uppsala, Sweden; iodogen was from 
Pierce Chemical, Rockford Ill., and .sup.125 I was purchased from Amersham 
Corp, Arlington Heights, Ill. Human serum albumin was from Miles Inc, 
Elkhart, Ind. Plasminogen was purified from human fresh frozen plasma by 
affinity chromatography on a lysine-Sepharose column. Plasminogen was in 
the glu form as assessed by SDS-gel electrophoresis and had a specific 
activity of 20 CTA U/mg. 
Preparation of .sup.125 I-Fibrinogen: 
Fibrinogen was purified from fresh human plasma by glycine precipitation 
and was radiolabeled with .sup.125 I by the iodogen technique. Two ml of 
fibrinogen (1.8 mg/ml) in 0.05M Tris-HCl, 0.10M NaCl, 0.025M sodium 
citrate, pH 6.8 was mixed with 400 .mu.Ci .sup.125 I at 22.degree. C. for 
2 to 3 minutes in a scintillation vial pre-coated with 20 .mu.g iodogen. 
Unbound .sup.125 I was separated from labeled fibrinogen with a Sephadex 
G-25 column. The radiolabeled fibrinogen was 88% clottable and had a 
specific activity of 3.0.times.10.sup.7 cpm/mg. 
Ultrasound: 
A 1MHz ultrasound generator (Amrex Synchrosonic U/50, Amrex-Zetron, Inc., 
Hawthorne, Calif.) was used to provide continuous mode, or intermittent 
mode ultrasound output. For intermittent ultrasound, the "on" interval was 
two seconds, followed by a rest interval of two seconds. Ultrasound was 
tested at two different intensities (expressed as spatial-average 
temporal-average intensities) of 0.375 watt/cm.sup.2 and 1.75 
watt/cm..sup.2 Ultrasound was delivered through water from a planar 
transducer (surface area, 23 cm.sup.2), which was 6 cm from the thrombus 
for both in vitro and in vivo experiments. At this distance, ultrasound 
was delivered as a cylindrical beam. The attenuation was less than 1% in a 
6-cm path in water. In tissue, the intensity was reduced by 11% in a 1-cm 
path. 
In Vitro Thrombolysis: 
Blood was obtained by venipuncture from one normal volunteer under a 
protocol approved by the Human Use Committee of the Walter Reed Army 
Institute of Research. Whole blood clots were prepared by adding thrombin 
(50 .mu.l, 15 NIH U) to a mixture of 3 ml freshly obtained blood and 300 
.mu.l .sup.125 I-fibrinogen (0.7 .mu.Ci) in a 10.times.75 parafilm tube. 
After incubation at 22.degree. C. for two hours, the clots were added to 
two beakers that were in a water bath that was maintained at 37.degree. C. 
The beakers each contained 100 ml supernatant. In one of the two beakers, 
the ultrasound transducer was placed into direct contact with the 
supernatant, and the clot was at the bottom of the conical beaker or 6 cm 
from the face of the transducer. The surface area of each beaker was 43 
cm..sup.2 The walls of the pyrex beakers were 2 mm in thickness and the 
angle between each beaker and the external perpendicular support was 
23..degree. Conical beakers were used so that thrombus would not drift out 
of the range of the ultrasound beam. The temperature of the supernatant 
was monitored throughout the experiment with a thermometer that was 
accurate to 0.1.degree. C. Each paired experiment (at a given t-PA 
concentration and buffer composition) consisted of two clots, one of which 
was exposed to ultrasound. The supernatant was either 100 ml Tris-albumin 
buffer (0.15M NaCl, 0.02M Tris-HCl, pH 7.40 containing 1% human serum 
albumin) or 100 ml human plasma obtained as fresh frozen plasma. In the 
six paired experiments using fresh frozen plasma, six units of plasma, 
each from a different donor was used; plasma from a single unit was used 
for each paired experiment. After the addition of the clot to the 
supernatant, fibrinolysis was initiated by adding t-PA at final 
concentrations ranging from 3 to 3000 IU/ml. 
Fibrinolysis was monitored by determining the radioactivity of the whole 
clot in a gamma counter (Searle Model 1185, Searle Diagnostics, Inc., Des 
Plains, Ill.) before addition to the supernatant. Immediately after the 
addition of the clot to the beaker and before the addition of the t-PA, a 
200 .mu.l aliquot of the supernatant was tested for radioactivity, 
followed by serial sampling throughout the 200 min experiment. Sampling 
intervals ranged from 10-15 min during the initial phase of the lysis to 
30-40 min after the plateau was achieved. The residual radioactivity in 
the clot was determined at 200 min. 
Percent lysis was calculated according to the following formula: 
##EQU1## 
In Vivo Thrombolysis: 
The experimental protocol, which was approved by the Walter Reed Army 
Institute of Research Animal Committee, used male New Zealand White 
rabbits weighing between 3.0-3.5 kg. 
Thrombosis Model: 
The rabbit jugular vein thrombosis model was chosen because of its 
established value in the testing of t-PA for clinical use and the relative 
simplicity of the required surgical procedure and animal care. Anesthesia 
was induced with the intramuscular injection of 50 mg/kg Ketamine 
(Vetalar, Parke-Davis, Morris Plains, N.J.) and 0.1 mg/kg xylazine 
(Rompun, Miles Lab. Inc., Shawnee, Kans.). The animals were intubated and 
anesthesia was maintained with 1.5-2.5% halothane (Halocarbon Lab. Inc., 
Hackensack, N.J.) administered through a model 50122 Bickford vaporizer 
(A.M. Bickford Inc., Wales Center, N.Y.). 
The jugular vein was isolated as previously described with ligation of all 
side branches except the facial vein. A venotomy in the facial vein then 
allowed a 0.038 O.D. inch polyethylene catheter (Intramedic tubing, Clay 
Adams, Parsippany, N.J.) to be introduced and a one ml aliquot of blood to 
be withdrawn. The blood was then mixed with 100 .mu.l (1 .mu.Ci) .sup.125 
I-fibrinogen (human) and kept on ice to prevent clotting. Next the jugular 
vein was atraumatically occluded proximal and distal to the facial vein 
with microvascular clamps (Scanlan Int., Inc., St. Paul, Minn.). A 4-0 
silk thread soaked in human thrombin solution was placed in a small 
polyethylene catheter which was inserted into the venotomy in the facial 
vein and guided into the jugular vein. The radiolabeled blood (0.5 ml) was 
introduced into the jugular vein through the facial vein to create a 
thrombus around the 4-0 silk thread. The catheter in the facial vein was 
removed, the facial vein ligated around the thread, and the end of the 
thread was brought out to the skin through a small puncture to facilitate 
percutaneous removal at the completion of the experiment. The 
microvascular clamps were removed and the skin was closed. 
A temperature probe (2400 series temp. probe, Electromedics Inc., 
Englewood, Colo.) to be used for temperature monitoring during the 
experiment was inserted into the neck through a separate skin puncture. 
The animal to receive ultrasound was positioned on a specially designed 
treatment table with an enclosed water bath maintained at 
36.5.degree.-38.5.degree. C. (FIG. 1). The other animal was positioned in 
a similar fashion on a circulating water warming blanket also maintained 
at 36.5.degree.-38.5.degree. C. 
Experimental Protocol: 
To determine the effect of ultrasound on t-PA induced fibrinolysis, a 
series of paired experiments were performed in which both animals received 
t-PA and one of each pair received ultrasound in addition to the t-PA. 
Intermittent ultrasound (1 MHz, 1.75 watt/cm.sup.2 with a two-second "on" 
interval and a two-second rest interval) was delivered throughout the 
experiment to the neck of the animal from a fixed transducer through a six 
cm depth water bath (FIG. 1). The distance was chosen so that the 
ultrasound beam could provide adequate coverage of the site of potential 
thrombosis in a human model. Controls for the rabbits receiving t-PA and 
ultrasound were four rabbits that received ultrasound and no t-PA. 
In six paired experiments animals received t-PA at a dose of 1 mg delivered 
intravenously during the first 30 min of the experiment. An additional six 
pairs of animals received t-PA at a dose of 2 mg. In this group, 1 mg t-PA 
was administered during the first 30 minutes followed by 1 mg from 35 to 
185 min. One control animal in this group died of post-operative 
respiratory insufficiency; therefore, only five paired studies are 
reported. 
All animals received maintenance fluid and t-PA through a marginal ear vein 
cannula ipsilateral to the jugular vein thrombus. One mg t-PA was diluted 
in 5 ml of 0.3M NaCl, 0.01% Tween 80 (Aldrich Chemical Co., Arlington 
Heights, Ill.) and administered with a constant infusion pump (Sage 
Instruments, Cambridge, Mass.). Fibrinolysis was monitored by obtaining a 
2-min count of radioactivity over the neck of each animal at time zero 
(before the infusion of t-PA) with a calibrated gamma ratemeter (model 
2221 scaler ratemeter, Ludlum Measurements Inc., Sweetwater, Tex.). This 
was followed by serial counts throughout the experiment, with background 
counts obtained as well at each time point. 
Percent lysis was calculated according to the following formula: 
##EQU2## 
Plasma fibrinogen and serum plasminogen were measured with functional 
assays as described by Hassett, et al. (Thromb Res 43: 313-323 (1986)). 
Toxicity Studies: 
To determine the extent of tissue injury sustained due to insonation, the 
levels of creatinine phosphokinase (CPK, Encore Chemistry System, Baker 
Instruments Inc., Allentown, Pa.) were measured in serum at time 0, 200 
min, 24 hours and seven days in the pairs of rabbits receiving 1 mg t-PA 
in the presence and absence of ultrasound. 
A blinded histologic examination for pulmonary emboli was performed in four 
rabbits treated only with t-PA and in eight rabbits that received 
combination t-PA and ultrasound. 
The effect of insonation on jugular veins in the absence of thrombosis was 
studied histologically in three pairs of animals. Each pair underwent a 
sham operation to include catheterization of the jugular vein. Then one 
animal in each pair received 200 minutes of intermittent ultrasound (1MHz, 
1.75 watt/sec.sup.2) directed at the jugular vein. Seven days later the 
animals were killed with a 5 ml intravenous injection of sodium 
pentobarbital (Euthanasia-6). The jugular vein segments and lungs were 
removed and immersion-fixed in neutral buffered 10% formalin. Tissue was 
subjected to blinded histologic evaluation for inflammatory changes in 
vein segments. 
Statistical Methods: 
Summary thrombolysis data are reported as mean values and corresponding 
standard errors of the mean (SEM). Analysis of variance (ANOVA) was used 
to determine the statistical significance of differences in clot lysis due 
to effects of : (1) ultrasound (alone or in combination with t-PA) (2) 
concentration of t-PA, and (3) time of measurement. This analysis included 
time as a repeated measures factor and took into account the paired nature 
of the experimental design for comparing clots receiving ultrasound and 
t-PA with those receiving t-PA alone. This feature results because each 
experimental run at a given t-PA concentration included two clots studied 
in parallel--one receiving ultrasound and t-PA, the other t-PA alone. The 
50 minute and 200 minute time points (the repeated measures factor) were 
analyzed to determine early and late effects of insonation (reflecting 
rate and extent of fibrinolysis) for all in vitro studies. Similar 
analyses were used for the in vivo data at 50 and 100 minutes, a time when 
fibrinolysis had reached a plateau. The t-test (paired or unpaired as 
appropriate) was used when only two groups were compared. Observed 
significance levels (p-values), involving effects of ultrasound derived 
from t-tests or from 1 degree of freedom F tests (F (1, df), were two 
sided. A p value &lt;0.05 was considered to be significant. Analyses based on 
transformed data (logs) yielded similar results. The ordinary (Pearson's) 
correlation coefficient was used as a measure of correlation between two 
variables. 
Results: 
Comparison of Different Ultrasound Modes on T-PA Induced Clot Lysis in 
Vitro 
The optimal intensity of ultrasound for enhancement of clot lysis was 
determined by exposing blood clots in Tris-albumin buffer containing t-PA 
(300 IU/ml) to ultrasound at a frequency of 1 MHz and an intensity of 1.75 
watt/cm.sup.2 or 0.375 watt/cm.sup.2 for 200 min. Intermittent or 
continuous ultrasound at an intensity of 0.375 watt/cm.sup.2 did not 
enhance thrombolysis. Continuous ultrasound at an intensity of 1.75 
watt/cm.sup.2 increased the temperature of the medium from 37.0.degree. C. 
to 39.0.degree. C. within ten minutes of initiation and maximally by 
5.degree. C. during the course of the 200 min experiment. This mode was 
therefore not evaluated further. However, intermittent ultrasound at an 
intensity of 1.75 watt/cm.sup.2 caused significant enhancement of lysis at 
200 min (64.+-.10% versus 42.+-.5%, p&lt;0.05) without increasing the 
temperature of the surrounding medium by more than 1.degree. C. Therefore, 
intermittent ultrasound at an intensity of 1.75 watt/cm.sup.2 and a 
frequency of 1MHz was chosen for all further studies. 
Effect of Intermittent Ultrasound (1MHz, 1.75 watt/cm.sup.2) on Clot Lysis 
Induced with Different Doses of T-PA 
Studies were conducted in the absence and presence of intermittent 
ultrasound and t-PA at four different concentrations (3, 30, 300 and 3000 
IU/ml) to determine if ultrasound affected the rate and extent of t-PA 
induced fibrinolysis. Differences in mean clot lysis due to effects of 
ultrasound and t-PA concentration were determined using repeat measures 
ANOVA, with the 50 min time point reflecting the rate of fibrinolysis and 
the 200 min time point reflecting the extent of fibrinolysis. The overall 
differences in mean clot lysis among the four t-PA concentrations were 
significant (F(3,10)=8.3;p&lt;0.005) with the extent of fibrinolysis 
increasing in a dose-related manner for thrombi receiving ultrasound or no 
ultrasound. 
The overall difference in mean clot lysis between thrombi receiving 
ultrasound and no ultrasound was highly significant (F(1,10)=52.2;p&lt;0.001) 
with differences in time course consistent across all four t-PA 
concentrations. There was also a significant difference in the two groups 
(ultrasound versus no ultrasound) with respect to the rate of lysis as 
measured at 50 min (p&lt;0.001) and extent of clot lysis as measured at 200 
min (p&lt;0.001), when analyzed separately. At the lowest concentration of 
t-PA (3 IU/ml), ultrasound caused a 100% enhancement of lysis as 
determined at 200 min, and an approximate 50% increase at the higher t-PA 
concentrations. 
Effect of Plasma and Plasminogen on Fibrinolysis Induced by t-PA and 
Ultrasound (1MHz, 1.75 watt/cm.sup.2) 
In paired experiments, clots were incubated with fresh frozen plasma to 
which t-PA had been added at a final concentration of 300 or 3000 IU/ml, 
and one clot in each pair was exposed to intermittent ultrasound. 
Ultrasound induced a significant increase in the extent of thrombolysis as 
determined with the repeat measures ANOVA utilizing 50 and 200 minute data 
at both tPA concentrations (F(1,4)=36.9;p=0.004;). In all six paired 
experiments (three pairs at 300 IU/ml and three pairs at t-PA 3000 IU/ml) 
the thrombus exposed to ultrasound and t-PA underwent greater lysis than 
the thrombus incubated with t-PA alone. 
When clots were exposed to ultrasound and t-PA at a concentration of 300 
IU/ml, the lysis at 200 min was 75.+-.7% compared to 50.+-.2% when clots 
were incubated with t-PA alone. Furthermore, ultrasound reduced the time 
to reach 50% lysis from 200 min to 96 min. 
For the clots exposed to t-PA at a final concentration of 3000 IU/ml, the 
mean time to reach 50% lysis was reduced from 98 to 48 minutes in the 
presence of ultrasound. Clot lysis at 200 minutes was 91.+-.11% in the 
presence of insonation compared to 62.+-.5% in the absence of ultrasound. 
Fresh frozen plasma derived from a single donor was used in each paired 
experiment. The mean plasminogen concentration in the plasmas (n=6) used 
for these experiments was 3.8.+-.0.2 CTA U/ml. In additional experiments, 
clots were incubated with t-PA (3000 IU/ml) in Tris-albumin buffer 
containing purified plasminogen at a concentration of 1.2 CTA U/ml. The 
same degree of lysis was achieved at the lower concentration of 
plasminogen (Table 1). 
The results of thrombolysis experiments with 3000 IU/ml t-PA in 
Tris-albumin, plasma, and Tris-albumin containing plasminogen are 
summarized in Table 1. In the absence of ultrasound, there was no 
significant difference among the rates of lysis for clots that were 
exposed to t-PA that was in buffer, plasma or buffer containing 
plasminogen (p&gt;0.05). There was also no difference in lysis rates for 
clots that were incubated with these different supernatants in the 
presence of ultrasound, although a trend toward further improved 
thrombolysis with plasma and plasminogen compared to buffer is evident at 
200 minutes (Table 1). 
Effect of Insonation on t-PA-induced Thrombolysis In Vivo: 
Data obtained in in vivo studies using the rabbit jugular vein thrombosis 
model were conducted on paired groups of rabbits that received t-PA in the 
presence or absence of ultrasound, four rabbits received only ultrasound 
and no infusion of t-PA. In these "ultrasound only" controls, clot lysis 
was 9.+-.5% at 50 min. and 6.+-.10% at 100 min. 
Lysis in the rabbits that received 1 mg t-PA and ultrasound was increased 
at 50 min. compared to that of rabbits receiving t-PA alone (41.+-.14% 
versus 20.+-.12% p=0.12) The increase at 100 min., a time when lysis had 
reached a plateau, was sustained (55.+-.11% versus 30.+-.12%, p=0.11). 
Furthermore, four pairs out of six showed improved thrombolysis with 
ultrasound. 
Clot lysis for the rabbits that received 2 mg t-PA and insonation compared 
to those that received only t-PA was 45.+-.7% versus 28.+-.11% (p=0.10) at 
50 min and 50.+-.12% versus 40.+-.12% (p=0.29) at 100 min. Three pairs out 
of the five showed improved thrombolysis with ultrasound. 
Although the overall difference (repeat measures ANOVA using data at 50 and 
100 minutes for both t-PA doses) in mean thrombolysis between rabbits 
receiving insonation and t-PA and those receiving t-PA alone was not 
significant (F (1,9)=2.7; p=0.13;), differences favored insonation at both 
50 and 100 minutes for both doses of t-PA. The contrast between insonation 
and t-PA versus t-PA alone was greatest at 50 minutes (F (1,9)=).13 ;), 
p=0.09) when the combined data for the two doses were analyzed. The extent 
of thrombolysis in the group receiving t-PA at a dose of 2 mg was not 
different from that receiving 1 mg (p=0.73). 
In the rabbits receiving 2 mg t-PA alone there was no significant change in 
the levels of plasminogen and fibrinogen between the beginning and the end 
of the experiment at 200 min. Fibrinogen levels were 2.2.+-.0.2 mg/ml 
initially and then 1.6.+-.0.3 mg/ml (p=0.13) at 200 min. Plasminogen 
levels were 3.9.+-.0.3 CTA U/ml and then 3.7.+-.0.2 CTA U/ml (p=0.6). In 
the rabbits receiving intermittent ultrasound and t-PA, there were 
significant changes in the levels of fibrinogen and plasminogen. The 
fibrinogen level decreased from 2.0.+-.0.1 mg/ml at the beginning of the 
experiment to 1.1.+-.0.3 mg/ml at 200 min (p=0.03). The plasminogen level 
decreased from 3.6.+-.0.4 to 2.6.+-.0.3 CTA U/ml (p=0.03). There was no 
significant change in the levels of fibrinogen of plasminogen in the four 
animals receiving intermittent ultrasound without t-PA. In this group, 
fibrinogen levels at the beginning and end of the experiment were 
1.9.+-.0.1 and 2.0.+-.0.1 mg/ml (p= 0.42), respectively, and the 
plasminogen levels were 4.2.+-.0.3 and 4.0.+-.0.2 CTA U/ml (p=0.33). 
Investigation of potential insonation-induced injury. 
No increases in temperature were observed for animals receiving 
intermittent ultrasound and t-PA compared to those receiving only t-PA. 
There was no difference in the CPK values between rabbits receiving 
intermittent ultrasound and 1 mg t-PA and those receiving 1 mg t-PA alone. 
Pulmonary emboli were not detected histologically in twenty sections from 
the lungs of four rabbits treated only with t-PA. Two out of the forty 
histologic sections from eight rabbits that received combination t-PA and 
ultrasound had a single embolus and each was located in a 1 mm arteriole. 
Intermittent ultrasound did not induce endothelial changes in the rabbits 
that underwent a sham operation with catheterization of the jugular vein, 
and there was no evidence for thrombosis. 
TABLE 1 
__________________________________________________________________________ 
Effect of Intermittent Ultrasound (US, 1 MHz, 1.75 watt/cm.sup.2) 
on Clot Lysis Induced by t-PA (3000 IU/ml) in Buffer, Plasma, and 
Buffer Containing Purified Plasminogen 
% Lysis at 50 minutes 
% Lysis at 200 minutes 
__________________________________________________________________________ 
t-PA 
(3000 IU/ml) 
US No US 
p* US No US p 
__________________________________________________________________________ 
Buffer 45 .+-. 12** 
33 .+-. 7 
0.07 
73 .+-. 12 
62 .+-. 15 
0.03 
Plasma 50 .+-. 9 
33 .+-. 4 
0.05 
91 .+-. 11 
62 .+-. 5 
0.03 
Plasminogen 
54 .+-. 5 
35 .+-. 2 
0.02 
106 .+-. 6 
77 .+-. 4 
0.01 
__________________________________________________________________________ 
US 
Controls 
(No t-PA) 
No US 
p US No US p 
__________________________________________________________________________ 
Buffer 4 .+-. 1 
1 .+-. 1 
0.04 
8 .+-. 1 
3 .+-. 1 
0.02 
__________________________________________________________________________ 
*P-values are those for the given time points at the single dose of tPA. 
The differences in mean clot lysis incubated in buffer, plasma, or buffer 
with plasminogen are not significant (p &gt; 0.05). Three paired experiments 
(ultrasound vs no ultrasound) were performed with each different 
incubation medium. 
**Values are expressed as the mean .+-. SEM. 
TABLE 2 
__________________________________________________________________________ 
Clot Lysis in Rabbits Receiving t-PA Alone or T-PA and Intermittent 
Ultrasound (US, 1 MHz, 1.75 watt/cm.sup.2) 
% Lysis at 50 min % Lysis at 100 min 
US NO US p* US NO US p 
__________________________________________________________________________ 
t-PA (1 mg) 
41 .+-. 14** 
20 .+-. 12 
0.12 
55 .+-. 11 
30 .+-. 12 
0.11 
(n = 6 pairs) 
t-PA (2 mg) 
45 .+-. 7 
28 .+-. 11 
0.10 
50 .+-. 12 
40 .+-. 12 
0.29 
(n = 5 pairs) 
Controls: 
9 .+-. 5 
ND.sup.+ 6 .+-. 10 
ND 
t-PA (0 mg) 
__________________________________________________________________________ 
**Values are expressed as the mean .+-. SEM. 
.sup.+ ND is "not done. 
While the practitioner will adapt dosage of medications given and time and 
frequency of exposure to intermittent ultrasound to meet the particular 
needs of the patients, dosing with the usually prescribed doses of the 
particular plasminogen activating agents will be appropriate. For adult 
humans, the dosage which is usually given for lysis of thrombi in the 
absence of use of ultrasound augmentation is appropriate. (See Table III.) 
However, because of the improved lysis resulting from use of the 
ultrasound, the lysis occurs more quickly. Therefore, the length of time 
that such dosages are used will be greatly decreased. 
TABLE III 
__________________________________________________________________________ 
Streptokinase 
Urokinase t-PA* 
__________________________________________________________________________ 
Systemic use: 
Loading dose 
250,000 U 4,000 U/Kg 8 mg 1-3 min 
(30 min) (30 min) 50-60 mg/57 min. 
100,000 U/Hr 
4,000 U Kg/Hr. 
20 mg/60 min. 
12-72 hr. 12-72 hr. 20 mg/60 min. 
Low dosage: 
5000-7500 U/hr 
15000-21000 U/Hr 
1-15 mg/hr 
Duration 24-72 hr. 12-72 hr. 3 hr. for 
high dosage 
Local* use: 
10,000 U/hr 
400-5000 U/hr. 
Duration 12-72 hrs. 
__________________________________________________________________________ 
*Total dosage of this agent should not exceed 100 mg for one course of 
treatment. 
*"Local" indicates that the active agent was administered into the 
thrombus or near the thrombus in the blood vessel. 
The lower dosages should be tried when the pulsed mode ultrasound is used 
For prophylactic use, even lower dosages (up to 10 fold lower) are 
appropriate.