The Role of Cavitation in Enhancement of rt-PA Thrombolysis

We have investigated the synergistic effect of rt-PA and 120-KHz ultrasound on thrombolysis using in vitro porcine and human whole blood clot models. In our ongoing studies, we have demonstrated that significant enhancement of thrombolysis correlates with the presence of stable cavitation and this type of gentle bubble activity can be sustained using an intermittent infusion of a contrast agent. In addition, we have shown that inertial cavitation, which elicits broadband acoustic emissions, is counter-productive for enhanced thrombolysis. Rather, the most effective form of bubble activity is stable cavitation, which elicits ultrasonic subharmonic generation. These data strongly support the central hypothesis that ultrasound enhances thrombolysis primarily via mechanical mechanisms.

Related References:
Datta et al. 2006, Ultrasound in Medicine and Biology,32:1257–1267
Datta et al. 2008, Ultrasound in Medicine and Biology,34:1421–1433
Holland et al. 2008, Thrombosis Research,121:663-673
Datta S., PhD Dissertation, 2007

Immunofluorescent staining of mature porcine whole-blood clot.
The clot is located to the left of each panel. Panels A and B are stained with goat-antihuman rt-PA with a secondary of donkey-anti-goat conjugated to FITC. Panels C and D are stained with mouse anti-plasminogen and donkey-anti-mouse conjugated to Rhodamine. Panels A and C are treated with rt-PA in plasma alone and panels B and D were treated with rt-PA in plasma and 120-kHz pulsed ultrasound. These data show that ultrasound treatment dramatically increased rt-PA penetration into the clot.

Ultrasound-enhanced stroke therapy in an ex-vivo artery model

Previous studies showed enhanced lysis in the presence of ultrasound mediated stable cavitation in a static system. This stable bubble activity needs to be optimized and sustained during therapy. Studies were conducted in excised porcine carotid arteries kept alive in an ex-vivo tissue bath system. Occlusive porcine clots in the arteries were treated with plasma alone, plasma containing rt-PA (7.1 ± 3.8 µg/mL) or plasma with rt-PA and Definity® ultrasound contrast agent (0.79 ± 0.47 µL/mL) with and without 120-kHz continuous wave ultrasound at a peak-to-peak pressure amplitude of 0.44 MPa. An ultrasound pulsing scheme was used to maximize the stable cavitation throughout the treatment by incorporating a quiescent period to allow the contrast agent (Definity®) to refill the lumen. Thrombolytic efficacy was determined by the clot mass loss measured before and after the treatment. A significant improvement in clot mass loss was observed in clots treated with rt-PA, 120kHz ultrasound and Definity® (83%) compared to clots treated with rt-PA alone (30%).

Related References:

Hitchcock et al. 2010, Stroke,41: S50-S53
Hitchcock et al. 2011, Ultrasound in Medicine and Biology,37:1240–1251
Hicthcock,K. E., PhD Dissertation, 2010

Mean percent clot mass loss by treatment group.
Stable cavitation optimized treatment of clots resulted in >80% lysis of the clot mass compared to 30% lysis from treatment with rt-PA alone.

Clot Mass Loss vs. D-Dimer Production as a Metric for Assessing Thrombolytic Efficacy

Ultrasound (US) has been shown to be an effective adjuvant to recombinant tissue plasminogen activator (rt-PA) to accelerate thrombolysis. The amount of the fibrin degradation product, D-dimer, has previously been shown to correlate well with clot dissolution. To further explore this relationship in the context of US-enhanced thrombolysis, the correlation between clot mass loss and D-dimer production was investigated using an in vitro clot thrombolysis model. D-dimer concentration correlated with clot mass loss in this venous clot model. In future studies we will develop this biochemical assay as a metric for thrombolytic efficacy in a porcine hemorrhagic stroke model.