PLATELET-ADHESION TO CULTURED VASCULAR ENDOTHELIAL-CELLS - A QUANTITATIVE MONOLAYER ADHESION ASSAY

Abstract

A sensitive in vitro method was developed for measuring the adherence of platelets to cultured vascular cells. Human platelets, in citrated plasma, were radiolabeled with [3H]adenine to achieve a specific activity 100-1000 times greater than that obtainable with 51C or [14C]serotonin. After exposure of cultured cell monolayers to radiolabeled PRP [platelet-rich plasma], nonadherent platelets were removed by a standardized wash procedure and the number of adherent platelets was calculated from liquid scintillation measurements. Significant release of platelet-associated radioactivity was not detectable after challenge with standard aggregating agents, and uptake of plasma (unincorporated) [3H]adenine by cultured cells was minimized by the addition of unlabeled adenine during the adhesion assay. Scanning electron microscopy, performed in parallel, allowed visual discrimination between platelet adhesion and aggregation as well as morphologic examination of the interacting platelets and cells. After incubation with PRP for 30 min, primary cultures of umbilical vein HEC [human endothelial cells] bound < 0.05% of added platelets (< 1 platelet/cell). Virally transformed HEC showed increased platelet adhesion that was directly proportional to cultured cell density. Visual counts of the number of adherent platelets in scanning electron micrographs showed good agreement with calculated radiometric data. Apparently, suspensions of washed, radiolabeled human platelets can also be utilized in this monolayer adhesion assay. This in vitro model system should facilitate study of the mechanisms of thromboresistance of vascular endothelium and its pathophysiologic alterations.