Introduction: Structure/function studies have shown 3 major conformations of αIIbβ3, bent-closed, extended-closed, and extended-open. We recently noted the interaction of K514 in the thigh domain of αIIb with E500 in the I-EGF2 domain of β3 in the bent, closed conformation because we produced a mAb (R21D10) that 1) disrupts this interaction, 2) traps a semi-extended conformation in which the K514-E500 is lost and a new interaction is created between αIIb R516 and β3 E475, 3) partially inhibits fibrinogen binding and platelet aggregation. The K514-E500 interaction must be broken in order for αIIbβ3 to adopt the extended, open high-affinity ligand binding conformation, but we did not know if breaking this interaction is sufficient for the receptor to adopt this conformation. Therefore, we tested the effect of mutations designed to break the K514-E500 interaction on activation-independent, (i.e., constitutive), fibrinogen binding.

Methods: αIIb K514A, K514E and β3 E500A, E500K mutations were generated by the QuickChange XL Site-directed mutagenesis kit (Agilent). The wildtype (WT) αIIb and β3 and the mutants were transfected into HEK293T cells using lipofectamine 3000 to produce cells expressing αIIb WT/β3 E500A (β3 E500A), αIIb WT/β3 E500K (β3 E500K), αIIb K514A/β3 WT (αIIb K514A), αIIb K514E/β3 WT (αIIb K514E), αIIb K514A/β3 E500A (K514A/E500A), and αIIb K514E/β3 E500K (K514E/E500K). As a positive control, the αIIb F992A/F993A (FF) mutant, which is known to bind fibrinogen constitutively, was also tested. Expression of αIIbβ3 was measured by the binding of Alexa488-labeled mAb 10E5. Constitutive activation of αIIbβ3 was assessed by the binding of Alexa488-labeled fibrinogen to the cells in the absence of the activating mAb PT25-2. Background fibrinogen binding to the cells, assessed by adding 10 mM EDTA, was subtracted from each determination. The geometric mean fluorescence intensity (GMFI) of fibrinogen binding was normalized to the corresponding GMFI of 10E5 to obtain the normalized fibrinogen binding (NGMFI). The data are expressed as mean ± SD.

Results: The transfection efficiency of αIIbβ3 WT and the mutants was similar based on the percentage of 10E5 positive cells (WT 47%; mutants ranged from 34 to 63%). The αIIbβ3 expression levels were measured as GMFI of Alexa488-labeled 10E5 and normalized to the GMFI of the WT value. The relative expression levels are shown below (mean ± SD; student t-test vs WT).

Relative Expression Compared to WT (GMFI)

WT, 100

β3 E500A, 68 ± 13, n=3, p < 0.01,

β3 E500K, 45 ± 5, n=5, p < 0.0001,

αIIb K514A, 112 ± 11, n=3, p = 0.14,

αIIb K514E, 106 ± 16, n=3, p = 0.58,

K514A/E500A, 119 ± 18, n=3, p = 0.15,

K514E/E500K, 36 ± 7, n=3, p < 0.0001,

FF, 33 ± 4, n=6, p < 0.0001,

Little fibrinogen bound constitutively to WT αIIbβ3, but fibrinogen constitutively bound to the positive control αIIb FF mutant and all the K514-E500 mutants. The charge reversal mutant (K514E/E500K) also bound fibrinogen constitutively, indicating that it did not restore the interaction between the αIIb thigh and β3 I-EGF2 domains.

Fibrinogen binding (NGMFI, mean ± SD) without PT25-2 compared to WT

WT, 0.16 ± 0.05, n=6,

β3 E500A, 1.48 ± 0.60, n=3, p < 0.05,

β3 E500K, 2.17 ± 0.21, n=5, p < 0.0001,

αIIb K514A , 1.14 ± 0.21, n=3, p < 0.001,

αIIb K514E, 0.81 ± 0.19, n=3, p < 0.01,

K514A/E500A, 1.60 ± 0.33, n=3, p < 0.001,

K514E/E500K, 3.83 ± 0.20, n=3, p < 0.0001,

FF, 2.90 ± 0.29, n=3, p < 0.0001,

mAb PT25-2, which primes αIIbβ3 to bind ligand, induced a significant increase of fibrinogen binding to WT and some mutants.

Fibrinogen binding (NGMFI, mean ± SD) with PT25-2 compared to no PT25-2

WT, 1.79 ± 0.13, n=6, p <0.0001,

β3 E500A, 1.55 ± 0.07, n=3, p = 0.92,

β3 E500K, 2.63 ± 0.24, n=3, p = 0.19,

αIIb K514A, 2.76 ± 0.53, n=3, p = 0.05,

αIIb K514E, 3.58 ± 0.57, n=3, p < 0.05,

K514A/E500A, 3.76 ± 0.80, n=3, p = 0.07,

K514E/E500K, 5.19 ± 0.15, n=3, p < 0.01,

FF, 4.99 ± 0.48, n=6, p < 0.01,

Conclusion: Our data support the hypothesis that the αIIb K514-β3 E500 interaction that links the αIIb thigh and β3 I-EGF2 domains plays a vital role in maintaining the receptor in the bent, closed, inactive conformation and that loss of this interaction leads to spontaneous adoption of a conformation(s) that can bind ligand.

Disclosures

Coller:CeleCor Therapeutics: Consultancy, Current equity holder in private company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Chugai Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Accumetrics/Instrumentation Laboratories: Patents & Royalties; Scholar Rock: Current equity holder in publicly-traded company.

This content is only available as a PDF.
2024
Sign in via your Institution