Characterization of GMP-140 (P-selectin) as a circulating plasma protein.

Abstract

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.

Increased Plasma P-Selectin and Decreased Thrombomodulin in Pulmonary Arterial Hypertension Were Improved by Continuous Prostacyclin Therapy

“…Measuring soluble P-selectin, which originates from both platelets and endothelial cells, is proposed as a marker of increased membrane-bound P-selectin expression, attributable to vascular dysfunction and/or platelet activation, and may provide information on in vivo interactions among vascular and circulating cells. 6,7,10 In vitro experiments revealed that P-selectin induced tissue factor expression in human circulating monocytes and suggest that an increase in plasma P-selectin may have as a consequence the induction of a hypercoagulable state. 19 Plasma TM level appears to be initially increased with acute vascular injury, perhaps through cleavage from the cell surface, and is then decreased with subsequent downregulation of production after chronic vessel injury.…”

“…6 Several reports have demonstrated that a soluble form of P-selectin is present in the normal circulation. 7 Previous investigations related to thrombotic, inflammatory, and atherosclerotic disorders have suggested that plasma P-selectin may reflect endothelial cell injury and platelet activation. 8 -10 There is, however, no report concerning the relationship between PH and P-selectin.…”

Mouse Model of Femoral Artery Denudation Injury Associated With the Rapid Accumulation of Adhesion Molecules on the Luminal Surface and Recruitment of Neutrophils

“…27 Sections were deparaffinized, rinsed in xylene, and rehydrated. They were blocked with 3% H 2 O 2 , washed in water, treated with 2% ovalbumin in PBS, washed in PBS, and incubated with the primary antibodies at 37°C for 2 hours.…”

Soluble P-selectin levels during normal pregnancy: a longitudinal study

“…Upon activation of platelets or endothelial cells, P-selectin is quickly expressed on the surface membrane, where it mediates both the interaction of activated platelets with leucocytes, and leucocyte rolling on the activated endothelium 10 . Circulating degranulated platelets rapidly shed surface P-selectin 11 , producing the circulating plasma protein soluble P-selectin (sP-selectin) 12 . Although P-selectin is present in both platelets and endothelial cells, several authors have concluded that platelets are the major source of sP-selectin in plasma 13 -15 .…”