Preliminary study of the effect of hemolysis on platelet aggregation through microscopic observation under physiological shear flow

Hemolysis during mechanical circulatory support has been suggested as a possible trigger for non-hemorrhagic stroke. The purpose of this study was to investigate the hypothesis that slight hemolysis enhances thrombus formation through platelet aggregation under physiological shear stress in humans. Using sodium citrate-treated porcine whole blood from a slaughterhouse, platelet suspensions with a constant platelet density of 20.5 ± 1.3 × 10³ cells/μL with three plasma free hemoglobin (pfHb) concentrations (44.9 ± 15.0, 74.3 ± 18.3, and 130.6 ± 20.3 mg/dL) were prepared through centrifugation. These suspensions were exposed to a physiological shear rate up to 200 (1/s) using our custom-built shear chamber for 0, 5, 10, and 15 min. After exposure to each shear load, microscopic image acquisition was performed and the images were analyzed to count the number of aggregated platelets. It was observed that platelet aggregation increased in an exposure timedependent manner in all suspension fluids. In addition, the samples with the highest mean pfHb concentration of 130.6 mg/dL showed 1.23-and 1.28-fold numerically greater aggregation than those with a pfHb concentration of 74.3 and 44.9 mg/dL, respectively. The Wilcoxon signed-rank sum test showed p-values of 0.028 and 0.047 between 5 vs. 10 min and 10 vs. 15 min under the lowest pfHb concentration, respectively, and a p-value of 0.028 between 0 vs. 5 min under the medium pfHb concentration. However, there was no significant difference in aggregation according to pfHb concentration at the same exposure time. From these results, our data suggest that hemolysis might enhance platelet aggregation under physiological shear conditions.