Quantitative analysis of platelet alpha v beta 3 binding to osteopontin using laser tweezers.
To determine whether platelet adhesion to surfaces coated with the matrix protein osteopontin requires an agonist-induced increase in the affinity of the integrin alpha v beta 3 for this ligand, we used laser tweezers to measure the rupture force between single alpha v beta 3 molecules on the platelet surface and osteopontin-coated beads. Virtually all platelets stimulated with 10 microM ADP bound strongly to osteopontin, producing rupture forces as great as 100 piconewtons (pN) with a peak at 45-50 pN. By contrast, 90% of unstimulated, resting non-reactive platelets bound weakly to osteopontin, with rupture forces rarely exceeding 30-35 pN. However, approximately 10% of unstimulated platelets, resting reactive platelets, exhibited rupture force distributions similar to stimulated platelets. Moreover, ADP stimulation resulted in a 12-fold increase in the probability of detecting rupture forces >30 pN compared with resting non-reactive platelets. Pre-incubating stimulated platelets with the inhibitory prostaglandin E1, a cyclic RGD peptide, the monoclonal antibody abciximab, or the alpha v beta 3-specific cyclic peptide XJ735 returned force histograms to those of non-reactive platelets. These experiments demonstrate that ADP stimulation increases the strength of the interaction between platelet alpha v beta 3 and osteopontin. Furthermore, they indicate that platelet adhesion to osteopontin-coated surfaces requires an agonist-induced exposure of alpha v beta 3-binding sites for this ligand.
Litvinov
Rustem I
RI
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, 19104-6058, USA.
Vilaire
Gaston
G
Shuman
Henry
H
Bennett
Joel S
JS
Weisel
John W
JW
D000077284:Abciximab; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000527:Alprostadil / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001792:Blood Platelets; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D007834:Lasers; D009842:Oligopeptides / Q000494:pharmacology; D053495:Osteopontin; D010973:Platelet Adhesiveness / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000037:antagonists & inhibitors; D011485:Protein Binding; D012795:Sialoglycoproteins / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D039302:Integrin alphaVbeta3; D009842:Oligopeptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D053495:Osteopontin; D000244:Adenosine Diphosphate; C047981:arginyl-glycyl-aspartic acid; D000527:Alprostadil; D000077284:Abciximab