Analysis of the Interaction of Platelet Collagen Receptor Glycoprotein VI (GPVI) with Collagen

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Collagen peptides have been used to identify binding sites for several important collagen receptors, including integrin ␣ 2 ␤ 1 , glycoprotein VI, and von Willebrand factor. In parallel, the structures of these collagen receptors have been reported, and their interactions with collagen peptides have been studied. Recently, the three-dimensional structure of the intact type I collagen fiber from rat tail tendon has been resolved by fiber diffraction. It is now possible to map the binding sites of platelet collagen receptors onto the intact collagen fiber in three dimensions. This minireview will discuss these recent findings and their implications for platelet activation by collagen. Structural Architecture of Fibrillar CollagensCollagen is the most abundant protein in the human body, where it is a vital component of the extracellular matrix and connective tissue, including blood vessels. There are 29 collagen types, of which seven (types I-III, V, XI, XXIV, and XXVII) are fibrillar, able to assemble as stable triple helices, which then form a complex higher order three-dimensional fibrous superstructure. At the simplest level, the collagen polypeptide sequence is composed of GxxЈ triplets containing glycine followed by variable residues that often include proline at position x and hydroxyproline at position xЈ. The three polypeptide chains that assemble to form a tropocollagen triple helix can be identical gene products, as for collagen III (three ␣1 chains), or may differ, as for collagen I (two ␣1 chains and one ␣2 chain). The tropocollagen triple helices assemble to form a supermolecular fiber. Studies over the past 3 decades (1-7) have established that the basic repeating unit within the collagen fiber contains five tropocollagens packed side-by-side in a pseudohexagonal arrangement, with a gap along the fiber axis between the end of one triple helix and the start of the next. This regularly repeated gap region gives rise to the characteristic banding pattern of the intact collagen fiber, with a spacing D of 67 nm between successive bands corresponding to the offset between adjacent tropocollagen molecules, the classical "quarter-stagger." The densely packed (pseudohexagonal) regions extend over nearly half (0.46D) of a single D period, whereas the gap regions occupy the remaining length of the D period (0.54D). Each collagen molecule can be divided into five D periods (Fig. 1A). Collagen fibers have a circular cross-section, suggested by fiber diffraction data to be composed of concentric layers of fibrils with a thickness of ϳ40 Å (8).In 2006, Orgel et al. (9) published the three-dimensional structure obtained using fiber diffraction of an intact collagen type I fiber from rat tail tendon. This structure revealed the complicated interdigitated arrangement of the triple helices within the collagen fiber. Each unit cell (corresponding to one D period in the mature collagen fiber) measured 678 Å long, 27 Å wide, and 40 Å deep and contained segments of five tropocollagen triple helices. As expected, a cross-s...

Section: Three-dimensional Mapping Of Receptor-binding Sites On the Cmentioning

confidence: 81%

Structural Insights into the Interactions between Platelet Receptors and Fibrillar Collagen

Herr

Farndale

2009

108

100

“…However, Christou et al used monomeric recombinant CLEC-2 with a His tag and podoplanin-coated sensor chips, whereas we used dimeric recombinant CLEC-2 with Fc2, and the sensor chips were coated with this dimer form; we made dimeric CLEC-2, as it has been suggested that CLEC-2 exists as a dimer form on cell surfaces (22,33). Because dimer forms of receptors are known to have much higher affinity for their ligands than single forms in the case of GPVI (34), this apparent discrepancy in the affinity of CLEC-2 for ligands may be due to the different experimental conditions. Alternatively, recombinant CLEC-2 fixed on the surface may assume a conformation that has higher homophilic activity than the natural CLEC-2 molecule on resting platelets, thereby giving a smaller K d in a surface resonance study.…”

Section: Discussionmentioning

confidence: 99%

Essential in Vivo Roles of the C-type Lectin Receptor CLEC-2

Suzuki‐Inoue

Ito

Ding

et al. 2010

243

110

CLEC-2 has been described recently as playing crucial roles inClec-2 ؉/؉ embryos, we were able to demonstrate that CLEC-2 is involved in thrombus stabilization in vitro and in vivo, possibly through homophilic interactions without apparent increase in bleeding tendency. We propose that CLEC-2 could be an ideal novel target protein for an anti-platelet drug, which inhibits pathological thrombus formation but not physiological hemostasis.

“…The enhanced reactivity of GPP4GPO2 suggests that two linked GpVI receptors at the platelet surface interact with the separate clusters of GPO triplets in each of these peptides. Some of us used such evidence to predict the occurrence of GpVI as a dimer (31), whereas Miura et al (32) found dimeric Fc fusion constructs, but not monomeric forms, of GpVI could bind collagen in vitro. The crystal structure of GpVI presented by Horii et al (13) indeed contained a dimeric structure, maintained by D2-D2 interaction in the absence of CRP, such that the first domain of each points in opposite directions along the same axis, although the occurrence of this GpVI dimer conformation on the platelet surface has yet to be verified.…”

Section: Table 2 Effectiveness Of Cross-linked Peptides In Promoting mentioning

confidence: 99%

Structural Basis for the Platelet-Collagen Interaction

Smethurst

Onley

Jarvis

et al. 2007

117

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 ectodomain, 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.Damage to the blood vessel endothelium results in the exposure of underlying extracellular matrix proteins, including the fibrillar collagens I and III, both abundant in that location. Interaction of circulating platelets with collagen is a multistage process involving several receptors in which the activatory collagen receptor glycoprotein VI (GpVI) 2 plays a central role (1-3). GpVI has been identified as an anti-thrombotic target (4), and for this potential to be exploited, an understanding of the molecular mechanism by which GpVI recognizes and is activated by collagen is essential.Each fibrillar collagen strand contains ϳ1000 amino acid residues arranged in the triple helical COL domain, comprised of (G-X-XЈ) repeats, where X is often proline and XЈ, hydroxyproline (O). Glycine must occur at every third residue to allow the structure to fold as a triple helix (5). Collagen-related peptide (CRP) (GCO-(GPO) 10 -GCOG) readily adopts this triple helical structure (6) but is a highly potent platelet agonist only after cross-linking (CRP-XL) that introduces quaternary structure (7-9). CRP-XL has been shown to interact with GpVI (10, 11) and has been widely adopted as a specific tool with which to investigate the platelet-collagen interaction. In contrast, soluble monomeric CRP is at best a weak agonist (12).GPO triplets are abundant in collagens, forming ϳ10% of the fibrillar collagen COL domain, although very few contiguous runs of GPO triplets occur; exceptions are the N-terminal domain of collagens I (␣1) and III (␣1), where four and three GPO triplets are present, respectively, and the C-terminal domain of collage...