VON WILLEBRAND FACTOR MULTIMERS IN CULTURED HUMAN ENDOTHELIAL CELLS: COMPARISON BETWEEN CELLULAR STORAGE POOL AND SUPERNATANT
The multimeric composition of von Willebrand factor (vWf) from cultured human endothelial cells (h.e.c.) has been compared with the multimeric composition of vWf from h.e.c. culture supernatant, human normal platelets and plasma. H.e.c. were derived from umbilical cord veins by collagenase digestion, seeded in culture flasks and grown to confluence in TC 199 culture medium, supplemented with 20% foetal calf serum (FCS). At confluency, cells were harvested by rubber policeman, resuspended in 500 |o.l HBSS with protease inhibitors and stored with culture media until assay. H.e.c. and platelet lysates were obtained by freezing-and-thawing (5 times) and by the addition of 1% Sodium Dodecyl Sulphate (SDS) before centrifugation at 10,000 x g for 20 min at 20°C. Plasma, culture supernatants and cell lysates supernatants were run under the same conditions in an SDS 1.4%, LGT-agarose electrophoresis system using a discontinous buffer. Multimeric patterns were shown by radiolabelled affinity purified rabbit anti-human polyclonal antibody. In all the experiments cultured h.e.c. supernatants exhibited all the set of multimers of vWf usually observed in normal plasma. When vWf from cultured h.e.c. extracts was analyzed, a set of multimers with higher molecular weight was shown, with a pattern very similar to platelet's. We conclude that vWf stored in h.e.c. compartments is characterized by higher molecular weight multimers than culture supernatants. This behaviour recalls the differences recorded between plasma and platelet vWf.