Conformation-dependent platelet adhesion to collagen involving integrin alpha 2 beta 1-mediated and other mechanisms: multiple alpha 2 beta 1-recognition sites in collagen type I.

164

188

Only three recognition motifs, GFOGER, GLOGER, and GASGER, all present in type I collagen, have been identified to date for collagen-binding integrins, such as ␣ 2 ␤ 1 . Sequence alignment was used to investigate the occurrence of related motifs in other human fibrillar collagens, and located a conserved array of novel GER motifs within their triple helical domains. We compared the integrin binding properties of synthetic triple helical peptides containing examples of such sequences (GLSGER, GMOGER, GAOGER, and GQRGER) or the previously identified motifs. Recombinant inserted (I) domains of integrin subunits ␣ 1 , ␣ 2 , and ␣ 11 bound poorly to all motifs other than GFOGER and GLOGER. Similarly, ␣ 2 ␤ 1 -containing resting platelets adhered well only to GFOGER and GLOGER, while ADP-activated platelets, HT1080 cells and two active ␣ 2 I domain mutants (E318W, locked open) bound all motifs well, indicating that affinity modulation determines the sequence selectivity of integrins. GxO/SGER peptides inhibited platelet adhesion to collagen monomers with order of potency F > L > M > A. These results establish GFOGER as a high affinity sequence, which can interact with the ␣ 2 I domain in the absence of activation and suggest that integrin reactivity of collagens may be predicted from their GER content.Collagen, the most abundant structural protein of the vertebrate organism, currently has 27 reported family members (1). As either a mechanical support or as a bioactive surface, collagen plays a crucial role in processes as diverse as morphogenesis, wound repair, inflammation, tumor metastasis, hemostasis, and thrombosis. The tensile strength of the fibrillar collagens, types I-III, V, XI, and XXVII, is crucial to the function of connective tissues including the blood vessel wall. The non-fibrillar collagens, such as types IV and VI, provide a flexible support for endothelial and epithelial cell attachment and development. Integrins, heterodimeric adhesion molecules, form an important subgroup of receptors, which mediate interaction between collagen and cells. Four ␣-subunits, ␣ 1 , ␣ 2 , ␣ 10 , and ␣ 11 , which associate non-covalently with ␤ 1 , constitute the native collagen-binding integrin family (2).Integrin ␣ 2 ␤ 1 is a well characterized and widespread receptor for collagen, laminin, and other non-matrix ligands among nucleated cells including epithelial and endothelial cells, smooth muscle cells, fibroblasts, leukocytes, and mast cells (3, 4), mediating a wide range of cellular activities. ␣ 2 ␤ 1 is the only collagen binding integrin in platelets, and is crucial for deposition on collagens exposed in damaged arterial walls (5, 6). Accordingly, the expression levels of ␣ 2 ␤ 1 have been associated with myocardial infarction and stroke (7). Recently, knockout studies suggested that the platelet activatory collagen receptor glycoprotein VI (GPVI) is mandatory for the initiation of integrin-mediated adhesion (8), but this concept was challenged by further mouse studies (9). Moreover, in vitro studies suggest that ...

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Integrin Activation State Determines Selectivity for Novel Recognition Sites in Fibrillar Collagens

Siljander¹,

Hamaia²,

Peachey³

et al. 2004

164

188

“…Both blood and concentrate were obtained from consenting donors of the National Blood Service Cambridge, Long Road, Cambridge, UK, in accordance with the Helsinki protocol. Monomeric type I collagen for use in solid phase adhesion assays was purified from bovine skin, following limited pepsin digestion, as described previously (21,22). Collagen fibers were a gift from Ethicon Corp., Somerville, NJ.…”

Section: Methodsmentioning

confidence: 99%

Structural Basis for the Platelet-Collagen Interaction

Smethurst

Onley

Jarvis

et al. 2007

116

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...

“…On the basis of inhibition of platelet adhesion to this collagen by short, linear (non-triple-helical) peptides, an ␣ 2 ␤ 1 -binding site has been assigned to the sequence DGEA, which corresponds to residues 435-438 of the ␣1(I) chain and is found in the CNBr-derived fragment ␣1(I)CB3 (10). 2 However, others have observed no inhibition of ␣ 2 ␤ 1 -mediated cell adhesion to collagen by DGEAcontaining peptides (9,(11)(12)(13)(14)(15). Platelet adhesion to ␣1(I)CB3 is ␣ 2 ␤ 1 -dependent (9,16), but the fragment exhibits little platelet aggregatory activity (17).…”

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confidence: 99%

“…␣ 2 ␤ 1 is a platelet receptor that may be important in the activation of platelets by collagen in hemostasis, an event that may be expressed pathologically as thrombosis (6 -8). Fragmentation studies have indicated the presence in collagen I of several platelet ␣ 2 ␤ 1 -binding sites whose recognition is dependent on collagen triple-helical conformation (9). On the basis of inhibition of platelet adhesion to this collagen by short, linear (non-triple-helical) peptides, an ␣ 2 ␤ 1 -binding site has been assigned to the sequence DGEA, which corresponds to residues 435-438 of the ␣1(I) chain and is found in the CNBr-derived fragment ␣1(I)CB3 (10).…”

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confidence: 99%

The Platelet Reactivity of Synthetic Peptides Based on the Collagen III Fragment α1(III)CB4

Morton¹,

Peachey²,

Knight³

et al. 1997

The platelet-reactive collagen III-derived fragment ␣1(III)CB4 has been synthesized as seven overlapping peptides, each as a homotrimeric triple-helical species covalently linked at the C terminus. Additional Gly-ProHyp triplets were introduced at each end of the peptide sequence to ensure a stable triple-helical conformation at 20°C, the temperature at which cell reactivity was measured. A Cys-containing triplet was included at each end to allow intermolecular cross-linking. All seven peptides in triple-helical, cross-linked form were able to cause platelet aggregation. Peptide 6, the most reactive species, was more aggregatory than collagen fibers. Platelet adhesion occurred to all peptides immobilized on plastic in monomeric form. Adhesion was integrin ␣ 2 ␤ 1 -independent except in the case of peptide 6, adhesion to which was partially reduced by anti-integrin ␣ 2 ␤ 1 monoclonal antibodies. The presence of an ␣ 2 ␤ 1 recognition site in peptide 6 was confirmed using HT 1080 cells, which express ␣ 2 ␤ 1 as their major or sole collagen receptor. HT 1080 adhesion to both peptide 6 and collagen was strongly inhibited by anti-integrin ␣ 2 ␤ 1 monoclonal antibodies. These cells did not adhere to any of the other peptides. Comparison of the structure of peptide 6 with that of adjacent peptides indicates that the sequence Gly-Gly-Pro-Hyp-Gly-Pro-Arg, residues 522-528 of the collagen ␣1(III) chain, represents the minimum structure required for the recognition of ␣ 2 ␤ 1 . Our findings support the view that the collagen triple helix possesses an intrinsic platelet reactivity that can be expressed independently of integrin ␣ 2 ␤ 1 and the precise level of which is governed by the exact nature of the primary sequence. Sequences such as those recognizing ␣ 2 ␤ 1 may potentiate the activity, whereas others may have the opposite effect.Cell interaction with collagens is mediated by specific cellsurface receptors, important among which are the integrins ␣ 1 ␤ 1 and ␣ 2 ␤ 1 (1). Integrins recognize discrete amino acid sequences, the best known of which, perhaps, is the RGD 1 sequence, which serves as a cell-binding site in several matrix proteins and is recognized by a number of integrins, including the ␤ 1 -integrins ␣