6] Measurement of platelet interaction with endothelial monolayers

Abstract

Publisher Summary Under physiologic conditions, the vascular endothelium remains nonthrombogenic. Several factors synthesized in the endothelium prostacyclin (PGI), endothelial-derived relaxing factor (EDRF), heparin-like glycosaminoglycans, and thrombomodulin may contribute to the nonthrombogenic properties of the vascular endothelium. The chapter presents the methods utilizing the primary cultures of endothelium from the human umbilical vein, umbilical artery, and bovine aorta for the study of the interaction of platelets and endothelium. The interaction of platelets with these confluent monolayers is measured using a modification of the in vitro model system. Both adherence of activated platelets to endothelial monolayers and the effect of released materials from each cell type on the other are measured. For the adherence of preformed platelet aggregates to a cell monolayer, an incubation mixture and 0.5 mL of 51 Cr-labeled platelets are used. A Beckman DP 5000 gamma counter is used to count 51 Cr radioactivity in a tube. The amount of radioactivity remaining with the cell monolayer is determined by solubilizing the cell monolayer and attached platelets with 1.5 mL of 19 mM Na 2 CO 3 in 0.1 N NaOH.