Abstract
Human umbilical vein endothelial (HUVE) and bovine aortic endothelial (BAE) cells in culture were examined to determine whether membrane proteins similar to human platelet glycoproteins (GP) IIb and IIIa were present. The HUVE and BAE cells were either 125I-surface labeled or metabolically labeled. Triton X-100 lysates of labeled cells were immunoprecipitated with polyclonal antibodies prepared against purified human platelet GP IIb-IIIa complex. Two membrane proteins were detected on both HUVE (Mr = 130,000 and 110,000) and BAE (Mr = 135,000 and 105,000) cells, which were similar to human platelet GP IIb (Mr = 125,000) and GP IIIa (Mr = 108,000). The two membrane proteins from HUVE cells and the two from BAE cells cosedimented in sucrose gradients, indicating that they exist as a complex. Unlike the human platelet GP IIb-IIIa complex, the HUVE and BAE membrane protein complexes were not dissociated by chelation of Ca2+. Platelet GP IIb and GP IIIa and the related membrane proteins on both HUVE and BAE cells showed similar changes in electrophoretic mobility upon disulfide reduction. These data demonstrate that human and bovine endothelial cells synthesize membrane proteins that have properties similar to the platelet membrane GP IIb-IIIa complex.
Highlights
Human umbilical vein endothelial (HUVE) and bovine aortic endothelial (BAE) cells in culture were examined to determine whether membrane proteins similar to human platelet glycoproteins (GP) IIb and IIIa were present
Affinity purified G P IIIa antibody was obtained by incubating the G P IIb-IIIa antisera with nitrocellulose blots of G P IIIa (20) and eluting the antibody from the washed nitrocellulose with 0.1 M glycine, p H 2.8
Cell Culture-The HUVE cells were obtained by incubating umbilical veins for 10 min with Medium 199 that contained 1 mg/ml collagenase (Type I, Worthington) (21)
Summary
Human umbilical vein endothelial (HUVE) and bovine aortic endothelial (BAE) cells in culture were examined to determine whether membrane proteins similar to human platelet glycoproteins (GP) IIb and IIIa were present. Triton X-100 lysates of labeled cells wereimmunopre- cultured in Medium 199 that was supplemented with 20% (v/v) fetal cipitated with polyclonal antibodies prepared against calf serum, 25 mM Hepes buffer (pH 7.4), 100units/ml penicillin and purified human platelet GP IIb-IIIa complex. 105,000) cells, which were similar to human platelet
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