Standardized ultrasound as a new method to induce platelet aggregation

Abstract

Most of the published studies concerning platelet aggregation were performed with chemical stimulation procedures, however, mechanical stimulation might be a better simulation of physiological activation of platelets. In order to evaluate the influence of ultrasound on platelet aggregation in vitro, we developed an ultrasound device in a standardized set-up, and we evaluated the influence of lipoproteins and the glycoprotein IIb/IIIa inhibitor tirofiban on ultrasound induced platelet aggregation. A cylindrical shaped plastic test tube with 1 ml of platelet-rich plasma was placed in an ultrasound bath (35kHz) for 5 s. The ultrasound energy transfer into the sample (ΔW=3.77 J) was calculated using the average temperature increase (averaged by 0.935 °C) of the sample. Platelet aggregation was quantified immediately after stimulation with ultrasound or adenosine diphosphate (ADP 2.1 and 4.2 μM) by the Myrenne Aggregometer PA2 at low (40 s⁻¹) and afterwards at high (2500 s⁻¹) shear. To evaluate the influence of lipoproteins, seven healthy male volunteers were investigated before and after a fat load (50 g fat per m² body surface), and 11 patients suffering from hypercholesterolemia and atherosclerotic disease before and after a single low-density lipoprotein (LDL) apheresis. Platelet aggregation after ultrasound stimulation was well correlated with platelet aggregation after ADP (r between 0.50 and 0.95). However, when exposed to high shear, the low shear-induced platelet aggregates were more stable after ultrasound stimulation compared with ADP stimulation either with or without tirofiban. After the fat load triglyceride concentration increased from 0.86±0.39 to 2.10±1.10 mmol l⁻¹ (P<0.05) resulting in a reduced formation of platelet aggregates after weak (ADP 2.1 μM) but not after strong (ADP 4.2 μM or ultrasound) stimuli. After a single LDL apheresis LDL cholesterol dropped from 3.99±0.90 to 1.06±0.55 mmol l⁻¹ (P<0.005). No changes in platelet aggregation were observed with the exception of a lower aggregation when exposed to high shear after stimulation with 2.1 μM ADP. In conclusion, we found the ultrasound stimulation of platelet-rich plasma easy to perform. The platelet aggregation after ultrasound stimulation correlated well with stimulation after ADP. While a reduction in LDL cholesterol concentration had only slight effects on platelet aggregation, an increase in triglyceride concentration resulted in a reduced formation of platelet aggregates after weak stimulation.