Advances in laser scanning imaging cytometry for high-content screening.

The advent of high-content screening more than a decade ago remodeled drug discovery workflows by recasting the role of cell-based approaches in target identification, primary screening, lead optimization, and toxicity. The ability to identify and quantify compound effects on multiple cellular functions allows for rapid characterization of chemical libraries. Laser scanning imaging cytometry (LSIC) is one of the technologies that is being applied to a broad range of assays utilizing fluorescent labeling, at throughputs compatible with primary screening campaigns. Cellular resolution is achieved using laser scanning excitation through a specialized F-theta scan lens. This configuration results in rapid whole well scanning and large depth of field. The recent availability of systems equipped with multiple sources of laser excitation and arrays of detectors for spectral analysis has significantly increased its applicability through enabling more fluorescent reagents and higher levels of multiplexing. LSIC is being used most extensively for phenotypic screening especially in areas such as cell health, RNA interference (RNAi) screening, and three-dimensional cell models. This review communicates advances in LSIC and how it is being applied by presenting an overview of the technology and a range of real-world case studies.

Identification of a major GpVI-binding locus in human type III collagen.

We have analyzed the adhesion of human and murine platelets, and of recombinant human and murine GpVI ectodomains, to synthetic triple-helical collagen-like peptides. These included 57 peptides derived from the sequence of human type III collagen and 9 peptides derived from the cyanogen bromide fragment of bovine type III collagen, alpha1(III)CB4. We have identified several peptides that interact with GpVI, in particular a peptide designated III-30 with the sequence GAOGLRGGAGPOGPEGGKGAAGPOGPO. Both human and murine platelets bound to peptide III-30 in a GpVI-dependent manner. III-30 also supported binding of recombinant GpVI ectodomains. Cross-linked III-30 induced aggregation of human and murine platelets, although with a lower potency than collagen-related peptide. Modifications of the peptide sequence indicated that the hydroxyproline residues play a significant role in supporting its GpVI reactivity. However, many peptides containing OGP/GPO motifs did not support adhesion to GpVI. These data indicate that the ability of a triple-helical peptide to bind GpVI is not solely determined by the presence or spatial arrangement of these OGP/GPO motifs within the peptides.

Structural basis for the platelet-collagen interaction: the smallest motif within collagen that recognizes and activates platelet Glycoprotein VI contains two glycine-proline-hydroxyproline triplets.

Collagen-related peptide is a selective agonist for the platelet collagen receptor Glycoprotein VI. The triple helical peptide contains ten GPO triplets/strand (single letter amino acid nomenclature, where O is hydroxyproline) and so over-represents GPO compared with native collagen sequence. To investigate the ability of Glycoprotein VI to recognize GPO triplets in a setting more representative of the collagens, we synthesized a set of triple helical peptides containing fewer GPO triplets, varying their number and spacing within an inert (GPP)n backbone. The adhesion of recombinant human Glycoprotein VI ectodo-main, like that of human platelets, to these peptides increased with their GPO content, and platelet adhesion was abolished by the specific anti-Glycoprotein VI-blocking antibody, 10B12. Platelet aggregation and protein tyrosine phosphorylation were induced only by cross-linked peptides and only those that contained two or more GPO triplets. Such peptides were less potent than cross-linked collagen-related peptide. Our data suggest that both the sequences GPOGPO and GPO.........GPO represent functional Glycoprotein VI recognition motifs within collagen. Furthermore, we propose that the (GPO)4 motif can support simultaneous binding of two glycoprotein VI molecules, in either a parallel or anti-parallel stacking arrangement, which could play an important role in activation of signaling.

Selective blockade of glycoprotein VI clustering on collagen helices.

Platelet activation by collagen relies on the interaction of the receptor glycoprotein VI (GPVI) with collagen helices. We have previously generated two recombinant single chain human antibodies (scFvs) to human GPVI. The first, 10B12, binds to the collagen-binding site on the apical surface between the two immunoglobulin-like domains (D1D2) of the receptor and so directly inhibits GPVI function. The second, 1C3, binds D1D2 independently of 10B12 and has been shown to have a more subtle effect on platelet responses to collagen. Here we have shown that 1C3 potentiates the effect of 10B12 on platelet aggregation induced by collagen and cross-linked collagen-related peptide (CRP-XL). We investigated this by measuring the effect of both scFvs on the binding of D1D2 to immobilized collagen and CRP. As expected, 10B12 completely inhibited binding of GPVI to each ligand in a dose-dependent manner. However, 1C3 inhibited only a proportion of GPVI binding to its ligands, implying that it interferes with another aspect of ligand recognition by GPVI. To further understand the mode of inhibition, we used a unique set of CRPs in which the content of critical glycine-proline-hydroxyproline (GPO) triplets was varied in relation to an "inert" scaffold sequence of GPP motifs. We observed that a stepwise increase in D1D2 binding with (GPO)(2) content was blocked by 1C3. Together these results indicate that 1C3 inhibits clustering of the immunoglobulin-like domains of GPVI on collagen/CRPs, a conclusion that is supported by mapping the 1C3 epitope to the region including isoleucine 148 in D2.

Collagen-platelet interactions: recognition and signalling.

The collagen-platelet interaction is central to haemostasis and may be a critical determinant of arterial thrombosis, where subendothelium is exposed after rupture of atherosclerotic plaque. Recent research has capitalized on the cloning of an important signalling receptor for collagen, glycoprotein VI, which is expressed only on platelets, and on the use of collagen-mimetic peptides as specific tools for both glycoprotein VI and integrin alpha 2 beta 1. We have identified sequences, GPO and GFOGER (where O denotes hydroxyproline), within collagen that are recognized by the collagen receptors glycoprotein VI and integrin alpha 2 beta 1 respectively, allowing their signalling properties and specific functional roles to be examined. Triple-helical peptides containing these sequences were used to show the signalling potential of integrin alpha 2 beta 1, and to confirm its important contribution to platelet adhesion. Glycoprotein VI appears to operate functionally on the platelet surface as a dimer, which recognizes GPO motifs that are separated by four triplets of collagen sequence. These advances will allow the relationship between the structure of collagen and its haemostatic activity to be established.