Characterization of GMP-140 (P-selectin) as a circulating plasma protein.
GMP-140 is a 140-kD granule membrane protein, found in the alpha granules of platelets and the Weibel-Palade bodies of endothelial cells, that is surface expressed on cell activation and mediates neutrophil attachment. Cloning data for GMP-140 from an endothelial library predict a soluble form of the protein, the transcription message for which is also found in platelets. In this study, we report the detection by enzyme-linked immunosorbent assay of soluble GMP-140 in plasma centrifuged for 3 h at 100,000 g (to remove platelet microparticles) and confirm its identity by purification from plasma. Plasma concentrations were found to be 0.251 +/- 0.043 micrograms/ml (means +/- SD, n = 10) in normal male controls and 0.175 +/- 0.063 micrograms/ml (means +/- SD, n = 10) in normal female controls. The purified protein had an identical molecular mass (nonreduced) to platelet membrane GMP-140 (approximately 3 kD lower, reduced) and was immunoblotted by polyclonal anti-GMP-140, and the anti-GMP-140 monoclonal antibodies AK4 and AK6. Analytical gel filtration studies indicated that the plasma GMP-140 eluted as a monomer whereas detergent-free, platelet membrane GMP-140 eluted as a tetramer consistent with plasma GMP-140 lacking a transmembrane domain. Purified plasma GMP-140 bound to the same neutrophil receptor as the membrane-bound form, and when immobilized on plastic, bound neutrophils equivalently to immobilized platelet membrane GMP-140. Since it has been shown that fluid-phase GMP-140 is antiinflammatory and downregulates CD18-dependent neutrophil adhesion and respiratory burst, its presence in plasma may be of major importance in preventing the inadvertent activation of neutrophils in the circulation.
Dunlop
L C
LC
Baker Medical Research Institute, Prahran, Victoria, Australia.
Skinner
M P
MP
Bendall
L J
LJ
Favaloro
E J
EJ
Castaldi
P A
PA
Gorman
J J
JJ
Gamble
J R
JR
Vadas
M A
MA
Berndt
M C
MC
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry; D001792:Blood Platelets / Q000378:metabolism*; D001798:Blood Proteins / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D019007:P-Selectin; D010446:Peptide Fragments / Q000737:chemistry; D015539:Platelet Activation; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry*; D012995:Solubility
D015703:Antigens, CD; D001798:Blood Proteins; D019007:P-Selectin; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins