Activated, But Not Unactivated, αIIbβ3 Can Mediate Adhesion to Fibrinogen Lacking the γ Chain Dodecapeptide
Authors: Zafar Hina , Li Jihong , David George A , Coller Barry S. 
Published: October 2019

The interaction of the fibrinogen γ chain dodecapeptide (HHLGGAKQAGDV; γ12) with the αIIbβ3 binding pocket is required for fibrinogen binding to αIIbβ3 and the binding of other αIIbβ3 ligands that contain RGD sequences such as von Willebrand factor and fibronectin. Since the strength of fibrinogen binding to αIIbβ3 increases over time, it is possible that there are additional sites of interaction between αIIbβ3 and fibrinogen, and several studies have proposed potential candidate ancillary sites on fibrinogen, including γ316-322 and γ370-381 (Podolnikova et al. JBC 289;2371,2014 and Remijn et al. Br J Haematol 117;650,2002). If such fibrinogen sites interact with αIIbβ3 at regions separate from the γ12 (RGD) binding site, these αIIbβ3 sites may be attractive targets for developing pure αIIbβ3 antagonists that are specific for fibrinogen. To identify potential additional interaction sites on fibrinogen and αIIbβ3, we studied adhesion of HEK293 cells expressing normal αIIbβ3 (αIIbβ3-HEK) to either fibrinogen or the D98 plasmin cleavage fragment of fibrinogen (which lacks γ12) in the presence of 2 mM Ca2+/1 mM Mg2+. The D98 fragment did not contain the γ12 peptide as judged by both immunoblotting with mAb 7E9 (anti-γ12) and mass spectroscopy. We also studied HEK293 cells expressing αIIbβ3 containing: 1. The double αIIb F992A + F993A mutations (αIIb-FF), which causes constitutive activation and ligand binding 2. Other activating mutations of αIIbβ3, including αIIb truncation at 991, β3 N339S, and β3 truncation at 717, 3. The β3 D119A mutation, which disrupts the β3 metal ion-dependent adhesion site (MIDAS) and eliminates adhesion of cells expressing normal αIIbβ3 to fibrinogen, or 4. Combined αIIb FF and β3 D119 mutations (FF+D119).

αIIbβ3-HEK and αIIb-FF both bound to immobilized fibrinogen (10 µg/ml coating concentration). αIIbβ3-HEK did not adhere to immobilized D98 (10 µg/ml coating concentration), whereas αIIb-FF did adhere [αIIbβ3-HEK: 201 ± 295 vs. αIIb-FF: 8,221 ± 1,585 arbitrary fluorescence intensity units (AFU); n=7; p=0.003]. HEK cells expressing the other activating mutations also adhered to both fibrinogen and D98. HEK cells expressing the D119 mutation did not adhere to fibrinogen or D98 and adding the D119 mutation to the αIIb FF mutant led to loss of adhesion to both fibrinogen and D98. Adhesion of αIIb-FF to D98 was inhibited by mAb 10E5, which inhibits fibrinogen binding to αIIbβ3 and binds to the αIIb cap domain (89% ± 18%; n=7; p=0.003) and by mAb 7E3, which inhibits fibrinogen binding and binds to the β3 subunits of both αIIbβ3 and αVβ3 (95% ± 10%; n=7; p=0.006), but not by mAb 7E9 (28% ± 29%; n=7; p=0.299).

Since cells expressing activated, but not unactivated αIIbβ3 were able to adhere to D98, our data are consistent with a model in which the initial interaction between αIIbβ3 and fibrinogen is mediated by γ12 binding to the αIIbβ3 ligand binding site followed by a conformational change that exposes additional site(s) on αIIbβ3 for another region or regions of fibrinogen.

Refrences:

Podolnikova NP, Yakovlev S, Yakubenko VP, Wang X, Gorkun OV, Ugarova TP. The interaction of integrin alphaIIbbeta3 with fibrin occurs through multiple binding sites in the alphaIIb beta-propeller domain. J Biol Chem. 2014;289:2371-2383.

Remijn JA, Ijsseldijk MJ, van Hemel BM, Galanakis DK, Hogan KA, Lounes KC, Lord ST, Sixma JJ, de Groot PG. Reduced platelet adhesion in flowing blood to fibrinogen by alterations in segment gamma316-322, part of fibrin-specific region. Br J Haematol 2002;117:650-657.

Disclosures

No relevant conflicts of interest to declare.