Abstract
Platelet membrane glycoproteins (GP) IIb and IIIa have been identified as platelet aggregation sites. These glycoproteins form a heterodimer complex (GP IIb-IIIa) in the presence of Ca2+. To study the morphology of this glycoprotein complex in membranes, we incorporated GP IIb-IIIa into artificial phospholipid vesicles using a detergent (octyl glucoside) dialysis procedure. Phosphatidylserine-enriched vesicles (70% phosphatidylserine, 30% phosphatidylcholine) incorporated approximately 90% of the GP IIb-IIIa as determined by sucrose flotation. Glycoprotein IIb-IIIa incorporation into the vesicles was unaffected by ionic strength, suggesting a hydrophobic interaction between the glycoprotein and the phospholipid. In both intact platelets or phospholipid vesicles, GP IIb was susceptible to neuraminidase hydrolysis, indicating that most of the glycoprotein complexes were oriented toward the outside of the platelets or vesicles. The morphology of GP IIb-IIIa in the phospholipid vesicles was observed by negative staining electron microscopy. Individual GP IIb-IIIa complexes appeared as spikes protruding as much as 20 nm from the vesicle surface. Each spike consisted of a GP IIb "head," which was distal to the vesicle and was supported by the GP IIIa "tails." The GP IIb-IIIa complex appeared to be attached to the vesicle membrane by the tips of the GP IIIa tails. Treatment of vesicles with EGTA dissociated the GP IIb-IIIa complex. The dissociated glycoproteins remained attached to the phospholipid vesicles, indicating that both GP IIb and GP IIIa contain membrane-attachment sites. These data suggest a possible structural arrangement of the GP IIb-IIIa complex in whole platelets.
Highlights
From the Gladstone Foundation Laboratories for Cardiovascular Disease, Cardiovascular Research Institute, Department of Pathology, University of California, S a n Francisco, California 94140
Platelet membrane glycoproteins (GP) IIb and IIIa Several properties of purified GP’ IIb and GP IIIa have have beenidentified as plateletaggregation sites. been determined
The stability of the GP IIbphology of this glycoprotein complex in membranes, IIIa complex depends on divalent cations; chelating agents we incorporated GP IIb-IIIa into artificial phospho- dissociate the complex into monomeric glycoproteins (1).The lipidvesiclesusing a detergent di- complex is asymmetrical, as indicatedby hydrodynamic meaalysis procedure.Phosphatidylserine-enrichedvesicles surements (1).The asymmetrywas confirmed in the compan
Summary
Upon removal of Triton X-100, the GP IIb-IIIa or phospholipidvesicles, GPIIbwassusceptibleto complexes aggregate by interacting at the tiposf the GP IIIa neuraminidase hydrolysis, indicating thatmost of the tails (15) This finding suggests that the tips of these tails glycoprotein complexeswere oriented toward the out-contain hydrophobic domains, which could be the sites on GP side of the platelets ovresicles. Glycoprotein IIb-IIIa was eluted from the column with 15 mlof a buffer containing 50 mM Tris, 0.5 mM CaC12, 60 mM octyl glucoside, 2 mM NaN3, and0.5 M NaCl (pH 7.5). Platelet Glycoprotein IIb-IIIain Phospholipid Vesicles dialysis into a Tris buffer containing 1mM EGTA and noCaC12.The. I dissociation of G P IIb from G P IIIa was verified by thrombin hydrolysis of[3H]GP IIbm2daesscribed below.
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