SummaryWe have investigated the ability of serum from uremic patients to modify the thrombogenic properties of the endothelium. The effects of uremic medium on the morphology of endothelial cells (ECs), and their resistance to flow was analyzed. The influence of uremic media on the reactivity of the extracellular matrix (ECM) generated by ECs towards normal platelets was evaluated in a parallel-plate perfusion chamber. Exposure of ECs to uremic medium resulted in abnormal cell morphology and signs of an accelerated growth. Detachment of ECs exposed to circulating blood was increased when cells had been grown with media supplemented with uremic serum (21% vs. 14% non exposed). Platelet deposition was significantly elevated on ECMs generated in the presence of uremic media (uremicECMs) (p<0.01 vs. control studies). Effects of uremic serum were not observed at short incubation periods (5 h) but were evident after 24 or 72 h of incubation. Northern blot analysis revealed increased expression of tissue factor (TF) mRNA in ECs exposed to uremic conditions. Immunocytochemical methods detected an augmented expression of TF antigen on uremic ECMs. Incubation of ECMs with an antibody to human tissue factor prevented the increase in platelet deposition observed in uremic ECMs, suggesting that the presence of TF in ECM could be responsible for the enhanced platelet deposition. Results from our study indicate that uremic medium impairs the antithrombotic functions of cultured endothelial cells.

The hemostatic effect of normal platelets in type 3 vWD seems to be related to the platelet vWF in the transfused platelets.

Uraemic patients suffer from haemorrhagic disorders and accelerated atherosclerosis. To evaluate the possible role of the vessel wall in these haemostatic alterations associated with uraemia, we investigated the effect of a uraemic milieu on human endothelial cell (EC) cultures and the reactivity of the extracellular matrices (ECM) generated by these cells towards platelets. EC cultures were exposed to a pool of sera (20% in the culture medium) obtained either from uraemic patients or from normal donors, and the following parameters were evaluated: (1) EC viability (trypan blue exclusion test); (2) von Willebrand factor (vWF) levels in supernatants and associated with ECM; (3) the reactivity of EC and EC-derived ECM towards platelets, measured 'ex vivo' under flow conditions (5 min, wall shear rate 800 s-1); and (4) ultrastructure of the ECM. The viability of EC cultured in the presence of uraemic sera was similar to controls. Platelet interaction with ECM generated by EC exposed to uraemic sera was significantly reduced (P < 0.05). This decrease was mainly related to a reduction in platelet adhesion (9.8 +/- 1.9% vs 16.7 +/- 1.8% in controls, P < 0.02). VWF levels in supernatants and associated with ECM were similar to controls. Ultrastructural analysis of the ECM generated by EC exposed to uraemic sera revealed a deficient matrix. An increased removal of EC was observed in experiments in which EC cultured in the presence of uraemic sera were perfused with citrated blood. These results indicate that a uraemic milieu induces quantitative and qualitative changes in the vascular subendothelium, characterized by a less intrincate network of fibrils, as well as a decreased attachment of EC and reduced thrombogenicity to the ECM. These changes may represent another mechanism which contributes to the haemostatic dysfunction observed in uraemic patients.

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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,ll-