Adherence of platelet and fibrin targeted ultrasound contrast bubbles to human blood clots in vitro

The purpose of this study is to assess platelet and fibrin targeted ultrasound contrast microbubbles ability to adhere to fresh platelet and platelet poor clots in vitro with the assistance of radiation force. Whole blood from healthy volunteers (25 ml) was collected and centrifuged at 1100 rpm for 15 min to separate platelet-rich plasma (PRP), which was separated and centrifuged (3000 rpm, 5 min) to get platelet poor plasma (PPP). Calcium and Thrombin from human plasma were added to form fresh blood clots with upregulated receptors. Platelet targeted (MRX44; ImaRx Therapeutics, Inc., Tucson, AZ), fibrin targeted (MRX802; ImaRx Therapeutics) or untargeted, control bubbles were added to the clots. Radiation force was employed, using a single-element transducer with continuous wave ultrasound (2.5-5 MHz, 100 kHz PRF, 30 s exposure, 50.20-123.65 kPa_p-p pressure), to push the bubbles onto the thrombi. The adherence of the microbubbles to the clots was facilitated by the targeting ligands present on the microbubbles that bind to receptors present in blood clots or thrombi. Attachment rates between the groups were determined using a microscope setup and compared using unpaired t-tests. The average number of bubbles attached per 1000 mum² of clot area using MRX44 was 7.0 plusmn 4.21 bubbles and 2.72 plusmn 2.44 bubbles, respectively, in PRP and PPP clots. With MRX802 the corresponding attachment rates were 11.45 plusmn 8.52 and 9.25 plusmn 3.40 bubbles, while typically less than 2 control bubbles adhered per 1000 mum² of clot area in either clot type. The differences in adherence rates were statistically significant for MRX802 compared to MRX44 and to controls in both PRP and PPP clots (p < 0.05). Relative to controls more MRX44 bubbles adhered in PRP clots (p = 0.003) but not in PPP clots (p = 0.07). In conclusion, the in vitro attachment rates of bubbles targeted to human blood clots depend on target availability (i.e., platelet targe- - ted bubbles do better in PRP than in PPP clots). Fibrin targeted bubbles showed the best adherence rates in this study.