Comparative lytic efficacy of rt-PA and ultrasound in porcine versus human clots

Abstract

Introduction: Porcine thrombi are employed routinely in preclinical models of ischemic stroke. In this study, we examined the differential lytic susceptibility of porcine and human whole blood clots with and without the use of microbubbles and ultrasound (US) as an adjuvant.

Materials and methods: An in vitro system equipped with time-lapse microscopy was used to evaluate recombinant tissue-plasminogen activator (rt-PA) lysis of porcine and human clots in the same species or cross species plasma. Human and porcine whole blood clots were treated with rt-PA and an echo contrast agent, Definity®, and exposed to intermittent 120 kHz US.

Results and conclusions: The rt-PA lytic efficacy observed for porcine clots in porcine plasma was 22 times lower than for human clots in human plasma reported previously. Further, porcine clots did not exhibit increased lysis with adjuvant Definity® and US exposure. However, the rt-PA lytic susceptibility of the porcine clots in human plasma was similar to that of human clots in human plasma. Human clots perfused with porcine plasma did not respond to rt-PA, but adjuvant use of Definity® and US enhanced lysis. These results reveal considerable differences in lytic susceptibility of porcine clots and human clots to rt-PA. The use of porcine clot models to test new human thrombolytic therapies may necessitate modulation of coagulation and thrombolytic factors to reflect human hemostasis accurately.

Fig 1. The experimental set-up for the in vitro flow system.

In a water tank held at 37°C, the clot is mounted within a capillary tube through which plasma is drawn from an upstream plasma reservoir to a downstream syringe pump. Acoustic absorber was used to line the 4 vertical walls of the water tank (not shown) and the bottom of the tank (shown), with approximately 3 cm of the capillary tube visible through a window in the acoustic absorber.

Fig 2. Thrombolysis of human and porcine clots.

(a) Fractional clot loss (FCL) for porcine clots in porcine plasma (n = 5, black), human clots in porcine plasma (n = 12, light grey), and porcine clots in human plasma (n = 12, dark grey) exposed to plasma alone, rt-PA (3.15 μg/mL), and rt-PA (3.15 μg/mL) with Definity^® and intermittent 120 kHz ultrasound (US). Statistically significant differences in FCL (p<0.05) across treatments are denoted by (*). No difference (p>0.05) in FCL was observed for porcine clots in porcine plasma exposed to rt-PA without or with the use of Definity^® and US as an adjuvant compared to plasma alone. (b) Average lytic rate (ALR) for the same clots and treatments shown in 2(a). Statistically significant differences in ALR (p < 0.05) across treatments are denoted by (*). ^† Data with human clots in human plasma (n = 12, white) was reproduced from [22].

Fig 3. Measured ultraharmonic and broadband emissions for trials involving US exposure.

Cavitation dose is shown for porcine clots in porcine plasma (black), human clots in porcine plasma (light grey), and porcine clots in human plasma (dark grey) when treated with rt-PA, Definity^®, and 120 kHz US. ^† Data with human clots in human plasma (dashed line) was reproduced from [22].

Fig 4. Representative human (a) and porcine (b) clots with standard H&E staining.

Fig 5. Representative human (a, c, e) and porcine (b, d, f) SEM images of clot surfaces.

Images are at 800x magnification (a, b; bar = 25 μm), 3500x magnification (c, d; bar = 5 μm), and 7000x magnification (e, f; bar = 2.5 μm)

Fig 6. Clot fibrin fiber diameter.

Box plots showing the range, median, 25th quartile, and 75th quartile of measured fiber diameter for human (n = 130) and porcine (n = 140) clots as measured by blinded observers from SEM images. Outliers are represented by the symbol "+" and statistically significant differences are denoted with a "*".