Development of a new clot formation protocol for standardized in vitro investigations of sonothrombolysis

Agreement about the most suitable clot formation protocol for sonothrombolysis investigations is lacking. Lysis rates vary strongly owing to different test conditions and, thus, cannot be compared. We aim to establish a simple but physiologically grounded protocol for in vitro coagulation to enable standardized sonothrombolysis investigations. Clots were generated from platelet-rich plasma (PRP) obtained by centrifugation (10min, 180×g) of human venous blood (VB). PRP was mixed with the boundary layer formed between the supernatant and the erythrocyte layer. To achieve clots with different platelet counts, PRP was gradually substituted with platelet-free plasma (PFP), harvested from the supernatant of VB after centrifugation (10min, 2570×g). Clot types were examined for histological appearance, hydrodynamic resistance under physiological flows, and lysis rate measured by weight loss after a 2-h treatment with recombinant tissue plasminogen activator (rt-PA) (60kU/ml). Lysis rates of the most suitable clot were measured after a 1-h treatment with rt-PA (60kU/ml), and combined treatment with rt-PA and 2-MHz transcranial color-coded sonography (TCCS) (0.179W/cm2) or 2-MHz transcranial Doppler (TCD) (0.457W/cm2). With increased platelet count, the hydrodynamic resistance of the artificial clots increased, their histological appearance became more physiological, and lysis rates decreased. The most suitable clots consisted of 1.5-ml PRP, 2.0-ml PFP, and 0.5-ml boundary layer. Their lysis rates were 36.7±7.8% (rt-PA), 40.8±8.6% (rt-PA+TCCS), and 40.4±8.3% (rt-PA+TCD). These systemic investigations were conducted for the first time. This protocol should be used for standardized sonothrombolysis investigations.