Functional and structural correlations of individual alphaIIbbeta3 molecules.
The divalent cation Mn(2+) and the reducing agent dithiothreitol directly shift integrins from their inactive to their active states. We used transmission electron microscopy and laser tweezers-based force spectroscopy to determine whether structural rearrangements induced by these agents in the integrin alphaIIbbeta3 correlate with its ability to bind fibrinogen. Mn(2+) increased the probability of specific fibrinogen-alphaIIbbeta3 interactions nearly 20-fold in platelets, and both Mn(2+) and dithiothreitol increased the probability more than 2-fold using purified proteins. Of 3 alphaIIbbeta3 conformations, closed with stalks touching, open with stalks separated, and globular without visible stalks, Mn(2+) and dithiothreitol induced a significant increase in the proportion of open structures, as well as structural changes in the alphaIIbbeta3 headpiece. Mn(2+) also increased the number of complexes between fibrinogen and purified alphaIIbbeta3 molecules, all of which were in the open conformation. Finally, Mn(2+) induced the formation of alphaIIbbeta3 clusters that resulted from interactions exclusively involving the distal ends of the stalks. These results indicate that there is a direct correlation between alphaIIbbeta3 activation and the overall conformation of the molecule. Further, they are consistent with the presence of a linked equilibrium between single inactive and single active alphaIIbbeta3 molecules and active alphaIIbbeta3 clusters.
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, 421 Curie Blvd, 1054 BRB II/III, Philadelphia, PA 19104-6058, USA.
Rustem I
RI
Litvinov
Chandrasekaran
C
Nagaswami
Gaston
G
Vilaire
Henry
H
Shuman
Joel S
JS
Bennett
John W
JW
Weisel
D001665:Binding Sites; D001792:Blood Platelets / Q000502:physiology; D004229:Dithiothreitol / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D008345:Manganese / Q000494:pharmacology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D011487:Protein Conformation
D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D008345:Manganese; D005340:Fibrinogen; D004229:Dithiothreitol