Subendothelial matrix of cultured endothelial cells contains fully processed high molecular weight von Willebrand factor.
Von Willebrand factor (vWf) is an adhesive glycoprotein composed of identical subunits linked by disulfide bonds to form multimers of varying sizes. VWf is found in platelets and plasma, where it functions in the adhesion of platelets to exposed subendothelium. Subendothelial matrix contains vWf, but the multimeric composition of matrix vWf, its binding site in matrix, and the mechanism by which it is delivered to matrix are unknown. Using human umbilical vein endothelial cell (HUVE) cultures, we have partially characterized subendothelial matrix vWf. Matrix from HUVE was solubilized in sodium dodecyl sulfate and electrophoresed on 1.25% agarose gels. The extracted vWf was composed of extremely high molecular weight (HMW) multimers of vWf not normally found in plasma. The vWf content of HUVE supernatant, extract, and matrix was quantitated and characterized by radioimmunoassay, agarose gel electrophoresis, and densitometry. The matrix-bound or -associated vWf represented 4% to 18% of total HUVE vWf. Western blot analysis of matrix vWf after reduction showed a subunit species of 220 kd. Long-term incubation of HUVE with phorbol 12-myristate 13-acetate (PMA), a compound that causes release of HMW vWf from Weibel-Palade bodies in HUVE, resulted in a marked decrease in matrix vWf (0.2% to 1% of the total vWf). The multimeric pattern of the remaining matrix vWf continued to show predominantly HMW multimers.(ABSTRACT TRUNCATED AT 250 WORDS)
Tannenbaum
S H
SH
Clinical Pathology Department, Clinical Center, National Institutes of Health, Bethesda, MD 20892.
Rick
M E
ME
Shafer
B
B
Gralnick
H R
HR
D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000032:analysis*; D006487:Hemostasis; D006801:Humans; D008970:Molecular Weight; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014841:von Willebrand Factor / Q000032:analysis*
D014841:von Willebrand Factor; D013755:Tetradecanoylphorbol Acetate