Separation of important new platelet glycoproteins (GPIa, GPIc, GPIc*, GPIIa and GMP-140) by f.p.l.c. Characterization by monoclonal antibodies and gas-phase sequencing.
A large number of membrane glycoproteins (around 40) are present on the surface of human blood platelets. Some of these glycoproteins are expressed in relatively small amounts, and their functions, as well as their structure, remain to be elucidated. The aim of the present study was to separate rapidly, under non-denaturing conditions, and characterize minor glycoproteins such as Very Late Antigens (VLA) (GPIa, GPIc, GPIc* and GPIIa) and GMP-140 (also known as PADGEM). VLAs and GMP-140 are respectively members of the integrin and selectin families. Platelet membrane glycoproteins were separated by wheat-germ agglutinin lectin affinity and Mono Q anion-exchange f.p.l.c. Peaks bearing isolated glycoproteins were electrophoresed on one- or two-dimensional SDS/polyacrylamide gels, Western blotted on to Immobilon poly(vinylidene difluoride) membranes and gas-phase-sequenced. The identity of isolated glycoproteins was also obtained by the use of monoclonal or polyclonal antibodies and tryptic peptide maps. Five minor [GPIa, GPIc, GPIc*, GPIIa and GMP 140 (PADGEM)], as well as a major (GPIIIb) glycoprotein, were eluted at low salt concentrations. GPIIb-IIIa and GPIb were eluted at high salt concentrations. The N-terminal sequence of platelet GPIa was identical with that obtained by Takada & Hemler [(1989) J. Cell Biol. 109, 397-407]. However, the N-terminal sequence of platelet GPIc + Ic* and GPIIa were found to differ from those deduced from cDNA sequences isolated from human placenta or umbilical-vein endothelial-cell cDNA libraries. The combined use of f.p.l.c. and gas-phase sequencing techniques provides a very powerful tool to separate and characterize rapidly platelet or other cellular proteins for structural, immunological and functional studies.
Catimel
B
B
INSERM Unité 331, Faculté de Médecine Alexis Carrell, Lyon, France.
Parmentier
S
S
Leung
L L
LL
McGregor
J L
JL
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*
D046911:Macromolecular Substances; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins