Figure 2.
Interaction of Affimer M17, D22, and D18 with GPVI monomer and dimer analyzed by ELISA, MST, and flow cytometry. ELISA: (A) M17 bound GPVI monomer (blue circles) at KD of 11 ± 1 nM, and GPVI dimer (red squares) at KD of 3.6 ± 0.2 nM. (B) D22 bound GPVI monomer at KD of 53 ± 11 nM, and GPVI dimer at KD of 5.3 ± 2.5 nM. (C) No binding was observed for D18 and GPVI monomer. D18 bound GPVI dimer at KD of 0.14 ± 0.02 nM. KD values were obtained through fitting data with Hill equation. MST: (D) M17 bound GPVI monomer at KD of 105 ± 31 nM and GPVI dimer at KD of 4 ± 2 nM. (E) D22 bound GPVI monomer at KD of 171 ± 36 nM and GPVI dimer at KD >1 μM. (F) D18 bound GPVI dimer at KD of 0.5 ± 0.2 nM. No binding was observed for D18 to GPVI monomer. KD values were obtained through fitting data with Hill equation. For ELISA and MST, data and KD were presented as mean ± SD; n ≥ 3. Flow cytometry: binding of Alexa Fluor 488 labeled Affimers scaffold, M17, D22, and D18 to washed platelets was analyzed by comparing the mean fluorescence intensity before and after stimulation (Δ MFI) with CRP-XL (G) and ADP (H). D18, but not M17 and D22, bound to activated platelets. Friedman test is used to determine statistical significance (P < .05). Data were presented as mean ± SD; n ≥ 4.

Interaction of Affimer M17, D22, and D18 with GPVI monomer and dimer analyzed by ELISA, MST, and flow cytometry. ELISA: (A) M17 bound GPVI monomer (blue circles) at KD of 11 ± 1 nM, and GPVI dimer (red squares) at KD of 3.6 ± 0.2 nM. (B) D22 bound GPVI monomer at KD of 53 ± 11 nM, and GPVI dimer at KD of 5.3 ± 2.5 nM. (C) No binding was observed for D18 and GPVI monomer. D18 bound GPVI dimer at KD of 0.14 ± 0.02 nM. KD values were obtained through fitting data with Hill equation. MST: (D) M17 bound GPVI monomer at KD of 105 ± 31 nM and GPVI dimer at KD of 4 ± 2 nM. (E) D22 bound GPVI monomer at KD of 171 ± 36 nM and GPVI dimer at KD >1 μM. (F) D18 bound GPVI dimer at KD of 0.5 ± 0.2 nM. No binding was observed for D18 to GPVI monomer. KD values were obtained through fitting data with Hill equation. For ELISA and MST, data and KD were presented as mean ± SD; n ≥ 3. Flow cytometry: binding of Alexa Fluor 488 labeled Affimers scaffold, M17, D22, and D18 to washed platelets was analyzed by comparing the mean fluorescence intensity before and after stimulation (Δ MFI) with CRP-XL (G) and ADP (H). D18, but not M17 and D22, bound to activated platelets. Friedman test is used to determine statistical significance (P < .05). Data were presented as mean ± SD; n ≥ 4.

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