Endothelial Cell Activation Mediates Platelet Response to Pulsatile Shear Stress

Abstract

Thrombin plays critical roles in both hemostasis and thrombosis. Many studies were carried out to examine the effects of different thrombogenic surfaces on thrombin generation. In the present study, how activated endothelial cells affected platelet thrombin generation under dynamic shear stress was investigated. Human platelets were exposed to pulsatile shear stress (physiological, low or elevated) in a cone-and-plate shearing device for 30 minutes in the presence of quiescent or activated human coronary artery endothelial cells. Coagulation factors including Factor II, Factor Xa and calcium were immediately available. Thrombin generation rates were measured using a modified prothrombinase assay. Platelet expression of surface P- selectin, glycoprotein Ibα (GPIbα or CD42b), integrin αIIb (CD41a), and phosphatidylserine was measured using flow cytometry. Endothelial cell expression of intracellular adhesion molecule 1 (ICAM-1) and von Willebrand factor was measured using a solid-phase ELISA and fluorescence microscopy, respectively. Platelet adhesion to the endothelial cells was measured using fluorescence microscopy. The results indicated that the activation state of endothelial cells, but not the pulsatile shear stress, had a significant effect in thrombin generation rates. The activation state of endothelial cells also affected platelet surface GPIbα and P-selectin expression, which may have contributed to the overall change in thrombin generation rates.