OC 29.4 - Identification of Segment Aα340-420 within the Fibrinogen αC-region Responsible Platelet GPVI Interaction

Background: Blood coagulation is mediated by the interaction between activated platelets and fibrin. Previous studies have shown that fibrinogen can bind GPVI with high affinity as confirmed by e.g. surface plasmon resonance (SPR). We recently demonstrated that the αC-region is a key area of fibrinogen involved in binding GPVI. However, the precise binding site of GPVI on the fibrinogen αC-region, which is almost 400 residues long, is not yet known.

Aims: To identify more closely which segment of the fibrinogen αC-region is involved in GPVI binding for the identification of the molecular mechanism underpinning the interaction between platelet GPVI and the fibrin network.

Methods: We generated two new truncation fragments of recombinant fibrinogen αC-region: Aα221-340 and Aα221-610[∆340-420]. Recombinant full length fibrinogen αC-region (Aα221-610) was used as control. Sizes of the recombinant fragments were confirmed by sequencing and SDS-PAGE. Interactions between GPVI and the truncation fragments were identified using pull-down assay and SPR. Competition between GPVI, α2-antiplasmin and FXIII for binding to the fibrinogen αC-region were tested using pull-down assay.

Results: Our data show that GPVI only bound to Aα221-610, but not to the Aα221-610[∆340-420] or the Aα 221-340 truncation fragments, indicating that the GPVI binding site is located to residues Aα340-420 in the fibrinogen αC-region. We also found that the presence of α2-antiplasmin, but not FXIII or FXIIIa, inhibited the binding of GPVI to fibrinogen in solution based binding studies using pull-down assay.

Conclusion(s): Our data show that the platelet GPVI binding site on fibrinogen is located at Aα340-420. There was competition between α2-antiplasmin and GPVI for binding to the fibrinogen αC-region. These findings highlight a new segment of the fibrinogen αC-region that is responsible for GPVI interaction, which could support future efforts to target this interaction as a novel therapeutic approach for thrombosis prevention.