RESUMO
Glycoprotein (GP)VI plays a key role in collagen-induced platelet aggregation. Affimers are engineered binding protein alternatives to antibodies. We screened and characterized GPVI-binding Affimers as novel tools to probe GPVI function. Among the positive clones, M17, D22 and D18 bound GPVI with the highest affinities (KD in the nM range). These Affimers inhibited GPVI-CRP-XL/collagen interactions, CRP-XL/collagen induced platelet aggregation and D22 also inhibited in vitro thrombus formation on a collagen surface under flow. D18 bound GPVI dimer but not monomer. GPVI binding was increased for D18 but not M17/D22 upon platelet activation by CRP-XL and ADP. D22 but not M17/D18 displaced nanobody2 (Nb2) binding to GPVI, indicating similar epitopes for D22 with Nb2 but not for M17/D18. Mapping of binding sites revealed that D22 binds a site that overlaps with Nb2 on the D1-domain, while M17 targets a site on the D2-domain, overlapping in part with the glenzocimab binding site, a humanized GPVI antibody Fab-fragment. D18 targets a new region on the D2-domain. We found that D18 is a stable non-covalent dimer and forms a stable complex with dimeric GPVI with 1:1 stoichiometry. Taken together, our data demonstrate that Affimers modulate GPVI-ligand interactions and bind different sites on GPVI D1/D2-domains. D18 is dimer-specific and could be used as a tool to detect GPVI dimerization or clustering in platelets. A dimeric epitope regulating ligand binding was identified on the GPVI D2-domain, which could be used for the development of novel bivalent antithrombotic agents selectively targeting GPVI dimer on platelets.
RESUMO
BACKGROUND: Robust platelet activation leads to the generation of subpopulations characterized by differential expression of phosphatidylserine (PS). Prostacyclin (PGI2 ) modulates many aspects of platelet function, but its influence on platelet subpopulations is unknown. OBJECTIVES AND METHODS: We used fluorescent flow cytometry coupled to multidimensional fast Fourier transform-accelerated interpolation-based t-stochastic neighborhood embedding analysis to examine the influence of PGI2 on platelet subpopulations. RESULTS: Platelet activation (SFLLRN/CRP-XL) in whole blood revealed three platelet subpopulations with unique combinations of fibrinogen (fb) binding and PS exposure. These subsets, PSlo /fbhi (68%), PShi /fblo (23%), and PShi /fbhi (8%), all expressed CD62P and partially shed CD42b. PGI2 significantly reduced fibrinogen binding and prevented the majority of PS exposure, but did not significantly reduce CD62P, CD154, or CD63 leading to the generation of four novel subpopulations, CD62Phi /PSlo /fblo (64%), CD62Phi /PSlo /fbhi (22%), CD62Phi /PShi /fblo (3%), and CD62Plo /PSlo /fblo (12%). Mechanistically this was linked to PGI2 -mediated inhibition of mitochondrial depolarization upstream of PS exposure. Combining phosphoflow with surface staining, we showed that PGI2 -treated platelets were characterized by both elevated vasodilator-stimulated phosphoprotein phosphorylation and CD62P. The resistance to cyclic AMP signaling was also observed for CD154 and CD63 expression. Consistent with the functional role of CD62P, exposure of blood to PGI2 failed to prevent SFLLRN/CRP-XL-induced platelet-monocyte aggregation despite reducing markers of hemostatic function. CONCLUSION: The combination of multicolor flow cytometry assays with unbiased computational tools has identified novel platelet subpopulations that suggest differential regulation of platelet functions by PGI2 . Development of this approach with increased surface and intracellular markers will allow the identification of rare platelet subtypes and novel biomarkers.
