Abstract No. 347: Minimizing tissue plasminogen activator (TPA) concentration while retaining maximal ultrasound-microbubble sonothrombolysis in vitro

Abstract

Establish the optimal combination of ultrasound (intensity, frequency, and duty factor) and microbubbles (MBs: diameter and elasticity) required to retain maximal in vitro sonothrombolysis (STBL) with minimal tPA concentration. Clots formed from fresh rabbit blood (72 h old) were placed into a Mylar flow chamber (37.0 C) that was infused with rabbit serum containing tPA (0.0–0.2 mg/ml). The tPA concentration and ultrasound intensity (0.1–2.0 W/cm2), frequency (0.7–4 MHz), and duty factor (0.05–100%) were adjusted to achieve maximal STBL (% clot mass loss/min). Uniformly sized MBs (0.5 to 5.0 μm diameter) were added to the rabbit serum and their concentration and elasticity were adjusted to maintain maximal STBL while minimizing tPA concentration. An FDA-approved tissue permeabilizer, cyclopentadecanolide (CPDL), was tested to determine if it could enhance STBL while further reducing or eliminating tPA. Maximal STBL with just ultrasound and tPA was observed with a tPA concentration of 0.1 mg/ml at every ultrasonic frequency, intensity, and duty factor: Higher tPA concentrations produced no additional clot lysis. Using MBs (1.0–2.5E8/ml) maintained maximal STBL while reducing tPA concentrations to 0.006–0.01 mg/ml. Different diameter MBs were required at different frequencies to accomplish this, e.g. 3 μm diameter MBs at 1 MHz and 1 μm diameter MBs at 3 MHz. STBL with MBs improved while increasing the duty factor up to 80%, but the pulse repeat frequency also needed to be increased proportionately. Adding CPDL (0.001% w/V) to the MB suspension increased STBL fourfold without the need for tPA. Using MBs with ultrasound permitted reducing the tPA concentration required for maximal STBL at all ultrasonic frequencies and intensities by more than ten fold. Thus, maximal STBL might be achievable in vivo while reducing the bleeding risk associated with tPA substantially. Results with MB-ultrasound STBL in a rabbit stroke model support this con-tention. Adding the tissue permeabilizer CPDL to MB-ultrasound STBL resulted in even more effective STBL without the need for tPA.