The relative thrombogenicity of extracellular matrixes (ECMs) produced by cultured human umbilical endothelial cells (ECs) was studied under flow conditions. ECMs were prepared using a number of physical and chemical methods, and their reactivity toward platelets was morphometrically evaluated. von Willebrand factor (vWF), fibronectin (FN), and 13-hydroxy-9-cis,11-trans-octadecadienoic acid (13-HODE) were also determined. We found that platelet adhesion to ECMs differed significantly, both quantitatively and qualitatively, with the method of ECM preparation. Mechanically prepared ECM exposed a less thrombogenic surface compared with ECM prepared by chemical methods (platelet-covered surface of 20% and 50%, respectively). Evaluation of the ECM components vWF, FN, and 13-HODE showed significant changes, both in their concentrations and distribution patterns, depending on the method of ECM preparation. The decrease measured in the levels of ECM-associated vWF (from 108 to 9.2 ng/10(4) cells) and the minor changes observed in the distribution pattern of subendothelial FN did not appear to be sufficient to explain the altered platelet adhesion observed in our model. This suggests that the amount of 13-HODE probably associated to the remaining ECs present in the mechanically exposed ECM could be one factor that specifically contributed to the nonthrombogenic state of these preparations. We conclude that the degree of ECM reactivity toward platelets is dependent on the method of ECM preparation and that this is related to the removal of specific EC/ECM components that modulate their thromboresistant/thrombogenic properties. This fact should be taken into account when ECMs produced by cultured ECs are used in platelet adhesion studies.