Platelet adhesion to cultured vascular endothelial cells. A quantitative monolayer adhesion assay

We have developed a sensitive in vitro method 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 to 1000 times greater than that obtainable with chromium-51 or [14C]serotonin. After exposure of cultured cell monolayers to radiolabeled PRP, nonadherent platelets were removed by a standardized wash procedure, and the number of adherent platelets 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 bound less than 0.05% of added platelets (less than 1 platelet per cell). In contrast, 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. Comparative studies indicated that suspensions of washed, radiolabeled human platelets also can 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.