Resolving two-dimensional kinetics of the integrin αIIbβ3-fibrinogen interactions using binding-unbinding correlation spectroscopy.
Using a combined experimental and theoretical approach named binding-unbinding correlation spectroscopy (BUCS), we describe the two-dimensional kinetics of interactions between fibrinogen and the integrin αIIbβ3, the ligand-receptor pair essential for platelet function during hemostasis and thrombosis. The methodology uses the optical trap to probe force-free association of individual surface-attached fibrinogen and αIIbβ3 molecules and forced dissociation of an αIIbβ3-fibrinogen complex. This novel approach combines force clamp measurements of bond lifetimes with the binding mode to quantify the dependence of the binding probability on the interaction time. We found that fibrinogen-reactive αIIbβ3 pre-exists in at least two states that differ in their zero force on-rates (k(on1) = 1.4 × 10(-4) and k(on2) = 2.3 × 10(-4) μm(2)/s), off-rates (k(off1) = 2.42 and k(off2) = 0.60 s(-1)), and dissociation constants (K(d)(1) = 1.7 × 10(4) and K(d)(2) = 2.6 × 10(3) μm(-2)). The integrin activator Mn(2+) changed the on-rates and affinities (K(d)(1) = 5 × 10(4) and K(d)(2) = 0.3 × 10(3) μm(-2)) but did not affect the off-rates. The strength of αIIbβ3-fibrinogen interactions was time-dependent due to a progressive increase in the fraction of the high affinity state of the αIIbβ3-fibrinogen complex characterized by a faster on-rate. Upon Mn(2+)-induced integrin activation, the force-dependent off-rates decrease while the complex undergoes a conformational transition from a lower to higher affinity state. The results obtained provide quantitative estimates of the two-dimensional kinetic rates for the low and high affinity αIIbβ3 and fibrinogen interactions at the single molecule level and offer direct evidence for the time- and force-dependent changes in αIIbβ3 conformation and ligand binding activity, underlying the dynamics of fibrinogen-mediated platelet adhesion and aggregation.
Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
Rustem I
RI
Litvinov
Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
Andrey
A
Mekler
Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
Henry
H
Shuman
Department of Hematology-Oncology Division of the Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
Joel S
JS
Bennett
Department of Chemistry, University of Massachusetts, Lowell, Massachusetts 01854. Electronic address: Valeri_Barsegov@uml.edu.
Valeri
V
Barsegov
Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104. Electronic address: weisel@mail.med.upenn.edu.
John W
JW
Weisel
D001792:Blood Platelets / Q000737:chemistry / Q000378:metabolism; D005340:Fibrinogen / Q000737:chemistry* / Q000378:metabolism; D006487:Hemostasis / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000378:metabolism; D013927:Thrombosis / Q000378:metabolism
D046912:Multiprotein Complexes; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D005340:Fibrinogen