The synergy site of fibronectin is required for strong interaction with the platelet integrin alphaIIbbeta3.
Integrins are a class of cell adhesion molecules that bind to ligands containing the RGD peptide sequence. There is increasing evidence that peptide sites other than the RGD site are required for optimal binding of integrins with their ligands. We have examined the sites on the protein fibronectin that are needed for optimal binding to the platelet integrin alphaIIbbeta3 using a strategy of site directed mutagenesis. Single amino acids near the RGD site or near the synergy site of fibronectin were mutated and the resultant proteins were expressed in a bacterial expression system. The purified protein was coated onto glass cover slips. Platelets, expressing alphaIIbbeta3 were perfused over the surface at physiologically relevant shear rates and the extent of adhesion was quantified. We found that the single amino acid substitution of the aspartic acid in the RGD sequence, D1495A, completely abolished adhesion. Surprisingly, the mutants R1445A and R1448Q that are near the RGD site also abolished adhesion of platelets under flow. Additionally, the synergy site mutants R1371A, R1374Q, or R1379A displayed only minimal adhesion of platelets. These results show that the binding site for alphaIIbbeta3 on fibronectin extends over a considerable distance from the RGD site and that these distant sites are required for optimal attachment of cells in the presence of physiologically relevant shear stress.
Chada
Diwakar
D
Department of Chemical, Biological, and Materials Engineering, University of Oklahoma, Norman, 73019-1004, USA.
Mather
Timothy
T
Nollert
Matthias U
MU
D000911:Antibodies, Monoclonal; D001665:Binding Sites / Q000235:genetics; D001698:Biomedical Engineering; D001792:Blood Platelets / Q000378:metabolism*; D005353:Fibronectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008855:Microscopy, Electron, Scanning; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015539:Platelet Activation; D010973:Platelet Adhesiveness; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D005353:Fibronectins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins