Functional characterization of a nanobody-based glycoprotein VI-specific platelet agonist

Abstract

Background: Glycoprotein (GP)VI is a platelet-specific collagen receptor required for platelet activation during hemostasis. Platelet reactivity toward collagen is routinely assessed during diagnostic workup of platelet disorders. GPVI can be activated by inducing receptor clustering with suspensions of fibrillar collagen or synthetic cross-linked collagen-related peptide (CRP-XL). However, these suspensions are poorly standardized or difficult to produce. Nanobodies are small recombinant camelid-derived heavy-chain antibody variable regions. They are highly stable, specific, and ideal candidates for developing a stable GPVI agonist for diagnostic assays.

Objectives: Develop a stable nanobody-based GPVI agonist.

Methods: Nanobody D2 (NbD2) was produced as dimers and purified. Tetramers were generated via C-terminal fusion of dimers with click chemistry. Nanobody constructs were functionally characterized with light transmission aggregometry (LTA) in platelet-rich plasma and whole blood flow cytometry. Diagnostic performance was assessed in patients with inherited platelet function disorders with LTA and flow cytometry.

Results: NbD2 was specific for human platelet GPVI. Dimers did not result in platelet activation in LTA or flow cytometry settings and fully inhibited CRP-XL-induced P-selectin expression and fibrinogen binding in whole blood and attenuated collagen-induced platelet aggregation in platelet-rich plasma. However, NbD2 tetramers caused full platelet aggregation, as well as P-selectin expression and fibrinogen binding. NbD2 tetramers were able to discriminate between inherited platelet function disorder patients and healthy controls based on fibrinogen binding, similar to CRP-XL.

Conclusion: Nanobody tetramers to GPVI induce platelet activation and can be used to assess the GPVI pathway in diagnostic assays.

Keywords: diagnostic tests; glycoprotein; nanobodies; platelet activation; platelet function tests.

Figure 1

Antiglylcoprotein (GP)VI nanobody D2 (NbD2) blocks cross-linked collagen-related peptide (CRP-XL)-induced platelet aggregation. (A) Affinity of NbD2 dimers (5, 10, 25, 50, and 100 nM) to soluble GPVI was studied with surface plasmon resonance (n = 3). Colored lines indicate binding data. Solid black lines indicate fitted data. (B) Binding of NbD2 dimers was assessed in a flow cytometer. Whole blood from healthy donors (n = 3) was incubated for 10 minutes with AlexaFluor (AF)647-conjugated NbD2 dimer. Data are expressed as median fluorescent intensity (MFI). (C) Human whole blood from healthy donors was stimulated with either 1 μM NbD2 dimer, 1 μg/mL CRP-XL, or buffer for 10 minutes at 37 °C (n = 3). P-selectin expression and fibrinogen binding were assessed using flow cytometry. (D) Platelet-rich plasma from healthy donors was preincubated with either 1 μM NbD2 dimer alone or 1 μM NbD2 dimer, followed by stimulation with 4 μg/mL collagen or 1 μg/mL CRP-XL (n = 3) or 400 nM NbD2 monomer followed by 5 μg/mL collagen (n = 6) in a light transmission aggregometer. Aggregation was monitored for 15 minutes at 37 °C at 900 rpm. Bar graphs represent the maximum amplitude of aggregation. Representative traces of a single donor are shown. (E) sGPVI was incubated with 2.5 nM NbD2 in the presence of CRP-XL (0 to 20 μg/mL), and residual NbD2 binding was assessed (n = 3). Optical density (OD) was measured at 450 nm and plotted against CRP-XL concentration. (F) Human-washed platelets (n = 3) were preincubated with either 1 μM NbD2 dimer, 500 μM D-Arg-Gly-Asp-Trp (dRGDW), or 300 μM Gly-Pro-Arg-Pro acetate (GPRP), followed by stimulation with 50 μg/mL fibrin in a light transmission aggregometer. Aggregation was monitored for 15 minutes at 37 °C at 900 rpm. Bar graphs represent the maximum amplitude of aggregation. Representative traces of a single donor are shown. (G, H) Whole blood from healthy donors (n = 3) was preincubated with NbD2 dimers as indicated, followed by stimulation with 1 μg/mL CRP-XL. (G) Platelet P-selectin expression and (H) fibrinogen binding were assessed in a flow cytometer, and MFIs were plotted as a function of NbD2 dimer concentration. Statistical analyses were performed with a 1-way analysis of variance (anova) with Šidák correction. Error bars represent mean ± SD.

Figure 2

Tetramers of anti-glycoprotein VI nanobody D2 (NbD2) induce platelet aggregation. (A) A C-terminal azido group was introduced into NbD2 dimers with sortagging, followed by the formation of NbD2 tetramers with a DBCO-PEG4-DBCO (DBCO) linker with copper-free click chemistry. (B) Coomassie blue staining of NbD2 dimers (27 kDa) before sortase A treatment and NbD2 tetramers (56 kDa) after sortase A treatment, DBCO-linking and size-exclusion chromatography. (C) Platelet-rich plasma from healthy donors (n = 3) was stimulated with either 18 nM NbD2 tetramers or 1 μg/mL cross-linked collagen-related peptide (CRP-XL). Aggregation was monitored for 15 minutes at 37 °C. Bar graphs represent the maximum amplitude of aggregation. Representative traces of a single donor are shown. (D, E) Whole blood from healthy donors (n = 3) was preincubated with NbD2 tetramers as indicated. (D) Platelet P-selectin expression and (E) fibrinogen binding were assessed in a flow cytometer, and median fluorescent intensities (MFI) were plotted as a function of NbD2 tetramer concentration. (F) Signal transduction in platelet lysates was assessed using Western blot. Platelets were stimulated with 1 μg/mL CRP-XL, 18 nM NbD2 dimers (NbD2-2), or 18 nM NbD2 tetramers (NbD2-4) for 10 minutes at 37 °C with or without 10 μM Src inhibitor dasatinib (Das) or 1 μM SYK inhibitor PRT-060318 (PRT). phosphoSYK (pSYK) and phosphotyrosine (pY) were detected. The arrow indicates SYK. SYK and GAPDH were used as lane loading controls. Statistical analysis was performed with a Student’s t-test. Error bars represent mean ± SD. AF647/488, AlexaFluor 647/488; V_HH, heavy-chain variable domain antibodies. DMSO, dimethyl sulfoxide.

Figure 3

Nanobody D2 (NbD2) tetramer can be used as a glycoprotein VI agonist during the diagnostic follow-up of patients with a suspected platelet function disorder. (A, B) Human whole blood was incubated with 5 μM cangrelor or 200 μM indomethacin for 30 minutes at room temperature. Platelet activation was allowed for 10 minutes at 37 °C with either cross-linked collagen-related peptide (CRP-XL), NbD2 tetramers, or adenosine diphosphate (ADP), and P-selectin expression and fibrinogen binding was assessed with median fluorescent intensity (MFI) of fluorescently labeled nanobodies. Bar graphs represent mean P-selectin expression and fibrinogen binding in healthy donors (n = 9). (C) Platelet-rich plasma from patients with an inherited platelet function disorder (IPFD; n = 21) and healthy controls (n = 50) were stimulated with either 1, 2, or 18 nM NbD2 tetramer or 1 or 4 μg/mL collagen. Aggregation was monitored for 15 minutes at 37 °C. Bar graphs represent the maximum amplitude of aggregation. A correlation plot is shown for maximum aggregation with 2 nM NbD2 tetramer and 1 μg/mL collagen. (D, E) Whole blood from IPFD patients (n = 25) and healthy controls (n = 50) were stimulated with either 18 nM NbD2 tetramer or 1 μg/mL CRP-XL for 10 minutes at 37 °C. (D) P-selectin expression and (E) fibrinogen binding were assessed using flow cytometry. Data are expressed as MFI. Correlation plots for both agonists are shown for P-selectin expression and fibrinogen binding. (F) Area under the receiver operating characteristic for CRP-XL vs NbD2 tetramer in flow cytometric fibrinogen binding in healthy controls and IPFD patients. Statistical analyses were performed with 1-way anova with Šidák correction. Error bars represent mean ± SD. AF647/488, AlexaFluor 647/488.