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.
Baker Medical Research Institute, Prahran, Victoria, Australia.
L C
LC
Dunlop
M P
MP
Skinner
L J
LJ
Bendall
E J
EJ
Favaloro
P A
PA
Castaldi
J J
JJ
Gorman
J R
JR
Gamble
M A
MA
Vadas
M C
MC
Berndt
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