Distinct Specificity and Single-molecule Kinetics Characterize the Interaction of Pathogenic and Non-pathogenic Antibodies against Platelet Factor 4-Heparin Complexes with Platelet Factor 4

Litvinov, Rustem I.; Yarovoi, Serge; Rauova, Lubica; Barsegov, Valeri; Sachais, Bruce S.; Rux, Ann H.; Hinds, Jillian L.; Arepally, Gowthami M.; Cines, Douglas B.; Weisel, John W.

doi:10.1074/jbc.m113.481598

Cited by 24 publications

(42 citation statements)

References 37 publications

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“…17–20,23,37,45–47 We found the rupture force values for the complex of α IIb β 3 with cRGDFK were comparable to those we observed for the α IIb β 3– γ C-12 complex, indicating that the strength of the complexes is comparable. Further, the on-rate and off-rate of cRGDFK binding were slightly greater and smaller, respectively, than the corresponding parameters for γ C-12, resulting in a slightly greater affinity constant, although this difference did not reach statistical significance.…”

Section: Discussionsupporting

confidence: 82%

Mechanistic Basis for the Binding of RGD- and AGDV-Peptides to the Platelet Integrin αIIbβ3

et al. 2017

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Binding of soluble fibrinogen to the activated conformation of the integrin αIIbβ3 is required for platelet aggregation and is mediated exclusively by the C-terminal AGDV-containing dodecapeptide (γC-12) sequence of the fibrinogen γ chain. However, peptides containing the Arg-Gly-Asp (RGD) sequences located in two places in the fibrinogen Aα chain inhibit soluble fibrinogen binding to αIIbβ3 and make substantial contributions to αIIbβ3 binding when fibrinogen is immobilized and when it is converted to fibrin. Here, we employed optical trap-based nanomechanical measurements and computational molecular modeling to determine the kinetics, energetics, and structural details of cyclic RGDFK (cRGDFK) and γC-12 binding to αIIbβ3. Docking analysis revealed that NMR-determined solution structures of cRGDFK and γC-12 bind to both the open and closed αIIbβ3 conformers at the interface between the αIIb β-propeller domain and the β3 βI domain. The nanomechanical measurements revealed that cRGDFK binds to αIIbβ3 at least as tightly as γC-12. A subsequent analysis of molecular force profiles and the number of peptide-αIIbβ3 binding contacts revealed that both peptides form stable bimolecular complexes with αIIbβ3 that dissociate in the 60–120 pN range. The Gibbs free energy profiles of the αIIbβ3–peptide complexes revealed that the overall stability of the αIIbβ3-cRGDFK complex was comparable with that of the αIIbβ3–γC-12 complex. Thus, these results provide a mechanistic explanation for previous observations that RGD- and AGDV-containing peptides are both potent inhibitors of the αIIbβ3–fibrinogen interactions and are consistent with the observation that RGD-motifs, in addition to AGDV, support interaction of αIIbβ3 with immobilized fibrinogen and fibrin.

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Section: Discussionsupporting

confidence: 82%

Mechanistic Basis for the Binding of RGD- and AGDV-Peptides to the Platelet Integrin αIIbβ3

et al. 2017

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“…Binding of a non-HIT antibody RTO to PF4 monomers prevents PF4 tetramerization and inhibits KKO and human HIT IgG-induced platelet activation/aggregation in vitro, and thrombus progression in vivo. The probability and the interaction force of KKO binding to PF4 are much greater than those of RTO, while KKO/ PF4 dissociation rate was approximately 10-fold slower than RTO/PF4 [62,63], indicating that KKO binds stronger than RTO and KKO/PF4 complexes are more stable than RTO/PF4. KKO interacts with PF4/H complexes coated platelets with ~4-fold higher forces than with PF4/H complexes coated on a solid phase, while RTO shows only a minor change [64].…”

Section: Hit-like Antibodiesmentioning

confidence: 92%

“…KKO has been used to unravel the pathogenesis of HIT and is the basis for a recently FDA approved plasma-based antigen assay (HIT-HemosIL) for detection of PF4/P antibodies [60,61]. KKO mimics the biological activity of human aPF4/P Abs [62] and has been used to understand the binding characteristics of an antibody recognizing PF4/P complexes and activating platelets [62,63]. Binding of a non-HIT antibody RTO to PF4 monomers prevents PF4 tetramerization and inhibits KKO and human HIT IgG-induced platelet activation/aggregation in vitro, and thrombus progression in vivo.…”

Section: Hit-like Antibodiesmentioning

confidence: 99%

Not Only Heparin but Also Antibody Induces Thrombocytopenia

Nguyen¹

2018

Thrombocytopenia

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In the last two decades, heparin was widely used as an anticoagulant. Besides numerous advantages of heparin, some patients with heparin administration suffer from a side effect, the so-called heparin-induced thrombocytopenia (HIT), which can result in thromboses such as deep vein thrombosis, pulmonary embolism, occlusion of a limb artery, acute myocardial infarct, stroke, and a systemic reaction or skin necrosis. The basic on HIT complication have been investigated and led to clinical insights. Recent studies provided detail mechanisms among binding partners in HIT; especially, it has been shown that not only heparin but also a subset of antibody induce thrombocytopenia. In this chapter, insights into both heparin-and antibody-induced thrombocytopenia will be discussed and the novel mechanism of the autoimmune HIT caused by a subset of antibodies will be introduced.

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“…Single-molecule interactions of DNA with an anti-DNA-Ab MRL4 were investigated with an optical trap-based model system described in detail elsewhere [33, 34]. For these studies, two contacting surfaces were coated, each with one type of the interacting molecules.…”

Section: Methodsmentioning

confidence: 99%

Single-Molecule Interactions of a Monoclonal Anti-DNA Antibody with DNA

et al. 2016

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Interactions of DNA with proteins are essential for key biological processes and have both a fundamental and practical significance. In particular, DNA binding to anti-DNA antibodies is a pathogenic mechanism in autoimmune pathology, such as systemic lupus erythematosus. Here we measured at the single-molecule level binding and forced unbinding of surface-attached DNA and a monoclonal anti-DNA antibody MRL4 from a lupus erythematosus mouse. In optical trap-based force spectroscopy, a microscopic antibodycoated latex bead is trapped by a focused laser beam and repeatedly brought into contact with a DNA-coated surface. After careful discrimination of non-specific interactions, we showed that the DNA-antibody rupture force spectra had two regimes, reflecting formation of weaker (20–40 pN) and stronger (>40 pN) immune complexes that implies the existence of at least two bound states with different mechanical stability. The two-dimensional force-free off-rate for the DNA-antibody complexes was ~2.2 × 10−3 s−1, the transition state distance was ~0.94 nm, the apparent on-rate was ~5.26 s−1, and the stiffness of the DNA-antibody complex was characterized by a spring constant of 0.0021 pN/nm, suggesting that the DNA-antibody complex is a relatively stable, but soft and deformable macromolecular structure. The stretching elasticity of the DNA molecules was characteristic of single-stranded DNA, suggesting preferential binding of the MRL4 antibody to one strand of DNA. Collectively, the results provide fundamental characteristics of formation and forced dissociation of DNA-antibody complexes that help to understand principles of DNA-protein interactions and shed light on the molecular basis of autoimmune diseases accompanied by formation of anti-DNA antibodies.

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Distinct Specificity and Single-molecule Kinetics Characterize the Interaction of Pathogenic and Non-pathogenic Antibodies against Platelet Factor 4-Heparin Complexes with Platelet Factor 4

Cited by 24 publications

References 37 publications

Mechanistic Basis for the Binding of RGD- and AGDV-Peptides to the Platelet Integrin αIIbβ3

Mechanistic Basis for the Binding of RGD- and AGDV-Peptides to the Platelet Integrin αIIbβ3

Not Only Heparin but Also Antibody Induces Thrombocytopenia

Single-Molecule Interactions of a Monoclonal Anti-DNA Antibody with DNA

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