Atomic force microscopy-based molecular recognition of a fibrinogen receptor on human erythrocytes.
The established hypothesis for the increase on erythrocyte aggregation associated with a higher incidence of cardiovascular pathologies is based on an increase on plasma adhesion proteins concentration, particularly fibrinogen. Fibrinogen-induced erythrocyte aggregation has been considered to be caused by its nonspecific binding to erythrocyte membranes. In contrast, platelets are known to have a fibrinogen integrin receptor expressed on the membrane surface (the membrane glycoprotein complex alpha(IIb)beta(3)). We demonstrate, by force spectroscopy measurements using an atomic force microscope (AFM), the existence of a single molecule interaction between fibrinogen and an unknown receptor on the erythrocyte membrane, with a lower but comparable affinity relative to platelet binding (average fibrinogen--erythrocyte and --platelet average (un)binding forces were 79 and 97 pN, respectively). This receptor is not as strongly influenced by calcium and eptifibatide (an alpha(IIb)beta(3) specific inhibitor) as the platelet receptor. However, its inhibition by eptifibatide indicates that it is an alpha(IIb)beta(3)-related integrin. Results obtained for a Glanzmann thrombastenia (a rare hereditary bleeding disease caused by alpha(IIb)beta(3) deficiency) patient show (for the first time) an impaired fibrinogen--erythrocyte binding. Correlation with genetic sequencing data demonstrates that one of the units of the fibrinogen receptor on erythrocytes is a product of the expression of the beta(3) gene, found to be mutated in this patient. This work demonstrates and validates the applicability of AFM-based force spectroscopy as a highly sensitive, rapid and low operation cost nanotool for the diagnostic of genetic mutations resulting in hematological diseases, with an unbiased functional evaluation of their severity.
Carvalho
Filomena A
FA
Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, 1649-028 Lisboa, Portugal.
Connell
Simon
S
Miltenberger-Miltenyi
Gabriel
G
Pereira
Sónia Vale
SV
Tavares
Alice
A
Ariëns
Robert A S
RA
Santos
Nuno C
NC
D001792:Blood Platelets / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D000077542:Eptifibatide; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D005260:Female; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D018625:Microscopy, Atomic Force / Q000379:methods*; D009154:Mutation; D010455:Peptides / Q000494:pharmacology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000037:antagonists & inhibitors / Q000378:metabolism; D011485:Protein Binding; D039341:Receptors, Fibrinogen / Q000378:metabolism*; D015203:Reproducibility of Results; D013379:Substrate Specificity; D013915:Thrombasthenia / Q000235:genetics / Q000378:metabolism
D010455:Peptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D039341:Receptors, Fibrinogen; D005340:Fibrinogen; D000077542:Eptifibatide; D002118:Calcium