2:18 PM Abstract No. 98 - Influence of thrombin content and fibrin complexity on in vitro sonothrombolysis rate and efficacy

Abstract

Purpose Blood clots that cause stroke and other vascular occlusion diseases vary with regard to fibrin complexity, and rigidity. This study sought to establish protocols for producing blood clots with different levels of fibrin content and rigidity for in vitro experiments to determine how to adjust pulsed ultrasound parameters (intensity, frequency, and duty factor), tPA and microbubbles (MBs) to improve STBL efficacy for more fibrin-rich, rigid and aged clots. Materials and Methods Clots were formed by mixing pooled rabbit plasma with fresh rabbit blood cells. Fibrin content and complexity were adjusted by varying thrombin content, calcium concentration, incubation time at 37 o C (3 h to 72 h), and curing time at 5 o C (24 h to 72 h). In vitro STBL of 7.5-12.0 mg pieces of clot was performed in a flow chamber (37.0 o C) through which fresh bovine serum containing tPA and/or MBs was flowed continuously. STBL efficacy was measured as % of clot mass lysed in 15 min. Clot fibrin content was measured using a D-Dimer assay and complexity determined by scanning electron microscopy. Results Increasing thrombin content, 37 o C incubation time, and 5 o C curing time produced more rigid clots that were increasingly resistant to STBL. Resistance to STBL was maximal with a thrombin concentration of 30 U/mL, a 24 h 37 o C incubation time, and a 24 h 5 o C curing time. Using tPA or MBs improved STBL efficacy, but improvement was less pronounced as clot rigidity increased. Combining tPA with MBs improved STBL efficacy additively in the least rigid clots, but this complementation diminished markedly as clot rigidity increased. Conclusion Varying thrombin concentration, 37 o C incubation time and/or 5 o C cure time provided reproducible control for forming clots with different rigidity and resistance to STBL. Such clots can putatively be used to model the STBL response of different clot types found in human thrombi and how well tPA and MBs can potentiate STBL. The degree to which tPA and MBs complement each other to potentiate STBL decreases with increasing clot rigidity. tPA and MB complementation was additive for thrombin concentrations of 1-12 U/mL but became sub-additive when higher concentrations of thrombin were used to form clots.