Standardized ultrasound as a new method to induce platelet aggregation: Evaluation, influence of lipoproteins and of glycoprotein IIb/IIIa antagonist tirofiban

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 (Δ W=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 m 2 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.