Selection and optimization strategy for Rap1-targeting single-domain antibodies as platelet activation markers

Rap1 is critical for platelet activation, functioning as a key node of the platelet activation pathways.

This study aimed to develop VHH-Fc (minibodies) against Rap1 for the purpose to quantify active Rap1 levels in platelets.

We have produced the first generation of VHHs against active Rap1 through a series of negative and positive screenings of a synthetic phage display library, utilizing both inactive and active Rap1B. We performed random mutagenesis, followed by yeast 2-hybrid screening to optimize variants.

Among 122 VHH clones, 2 with the highest redundancy were subcloned as VHH-Fc. Both selectively detected active Rap1B G12V in HeLa cells but failed to recognize the inactive Rap1B S17N isoform. They successfully captured Rap1 from platelet lysates incubated with GTPγS and from thrombin receptor activator peptide 6-stimulated platelets. Further optimization yielded 2 superbinder VHH clones: VHH-Fc B89 and VHH-Fc B14. VHH-Fc B89 exhibited a K _D of 4.4 nM and showed enhanced capacity to capture active GTP-bound Rap1 compared with the original VHH-Fc. Additionally, it was able to detect overexpressed active Rap1B G12V in HeLa cells. An ELISA setup combining VHH-Fc B14 and a commercial monoclonal antibody targeting total Rap1 was highly effective in detecting both GTPγS-bound Rap1 and endogenous active Rap1 in platelets.

This study identifies, that accurately capture active Rap1 in platelets, thereby establishing them as promising tools for future research. We also developed a reliable ELISA test that can facilitate clinical studies to monitor platelet Rap1 activation in various medical contexts.