Abstract Number: OC 37.3
Meeting: ISTH 2021 Congress
Background: GPVI plays a key role in collagen-induced platelet aggregation. Recent findings show that it also contributes to platelet aggregation and thrombus growth through interaction with fibrin(ogen). Our recent work has shown that this interaction is mediated by the fibrinogen αC-region (Xu et al., ATVB). Affimers are small conformational proteins that bind to their target molecules with high affinity and specificity, and are an alternative for antibodies as tools to study protein function.
Aims: Our aim was to screen and characterise GPVI-binding Affimers, investigate whether they can modulate GPVI interactions with collagen or fibrinogen, and test their effects on GPVI-mediated platelet aggregation.
Methods: GPVI-binding Affimers were isolated by phage display. Binding to monomeric and dimeric GPVI was tested by phage ELISA, pull-down assays and western blots. Binding affinities of Affimers to dimeric GPVI and competitive inhibition of dimeric GPVI binding to CRP-XL or the fibrinogen αC-region by Affimers were determined by ELISA. The effect of Affimers on CRP-XL induced platelet aggregation was tested by aggregation assays.
Results: Among the isolated Affimers, 8 bound GPVI stronger than the rest. Of those 8 Affimers, we observed that Affimers 17 and 22 interact with GPVI with the highest affinity (KD=4±0.1 and 13±1.2 nM, respectively). Both showed strong inhibition of GPVI interaction with CRP-XL in competitive ELISA. Affimer 17 also inhibited GPVI interactions with fibrinogen αC-region, whereas 22 was observed to strengthen GPVI binding to fibrinogen. Inhibition of CRP-XL induced platelet aggregation was observed for both Affimers.
Conclusions: We show that GPVI-collagen/fibrinogen interaction and platelet aggregation can be modulated by Affimers. Distinct effects of Affimers 17 and 22 on GPVI interactions with CRP-XL and αC indicate discrete binding sites for collagen and fibrinogen. It therefore may be possible to develop further Affimers that inhibit GPVI-αC but not GPVI-CRP-XL interaction to study their effects on thrombus growth.
To cite this abstract in AMA style:Xu R-, Cheah LT, Adams L, Tiede C, Gauer JS, Slater A, Duval C, Tomlinson D, Naseem K, Herr AB, Watson SP, Ariëns RAS. Affimers Modulate GPVI Interactions with Collagen/Fibrinogen and GPVI-mediated Platelet Aggregation [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/affimers-modulate-gpvi-interactions-with-collagen-fibrinogen-and-gpvi-mediated-platelet-aggregation/. Accessed August 16, 2022.
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