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Blood, 15 May 2008, Vol. 111, No. 10, pp. 4986-4996. Prepublished online as a Blood First Edition Paper on February 27, 2008; DOI 10.1182/blood-2007-08-108472.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Identification of a major GpVI-binding locus in human type III collagen1 Departments of Biochemistry and 2 Haematology, University of Cambridge, Cambridge, United Kingdom
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,  1(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.
Platelet adhesion to collagen is a key initial step in arterial thrombosis.1 Collagen interacts directly with platelets via glycoprotein VI (GpVI), which mediates platelet activation, and integrin 2β1, which supports platelet adhesion to collagen.2
Collagen is an extracellular matrix protein containing the repeating motif (GLY-X-X')n. Glycine (GLY, G) in every third position and a high imino acid content (proline [PRO, P]) in the X position and hydroxyproline [HYP, O] in the X' position) promote triple-helix formation.3 Receptor interactions with collagen are dependent on this triple-helical structure4 and the primary sequence, as shown by the differential platelet reactivity of peptides prepared by cyanogen bromide fragmentation of collagen.5–8 The
Subsequently, the synthetic collagen-related peptide (CRP), consisting of repeating GPO motifs, was shown to induce platelet activation9 in a GpVI-dependent manner.10,11 Platelets deficient in GpVI respond weakly to collagen10,12,13 and not at all to CRP.14 By contrast, peptides consisting of repeating GPP motifs do not support platelet binding or activation,15 indicating an important contribution of the hydroxyl group to the affinity of CRP for GpVI. Recent work using peptides comprising both GPP and GPO triplets shows that platelet and GpVI binding increases with GPO content, such that maximum interaction requires at least 4 contiguous GPO triplets.16 Although GpVI is critical in the response of platelets to collagen,17 there are no extensive stretches of OGP/GPO in collagen, unlike CRP. To explore the interaction of collagen receptors with native sequences, we synthesized a series of 57 peptides representing the 1029 residue triple-helical (COL1) domain of human type III collagen (Toolkit-III).18 Twenty percent of this COL1 domain is OGP/GPO motifs, although the longest discrete motif is GPOGPOGPO (Figure 1). Of the Toolkit-III peptides, 3 have no OGP/GPO motif and 23 contain a single HYP residue within an OGP/GPO motif. One contains 5 HYP residues and 5 contain 4 HYP residues within OGP/GPO motifs. We have identified a major GpVI-binding locus that contains 3 HYP residues within OGP/GPO motifs. These data indicate that GpVI interacts with specific loci in collagen and that motifs other than OGP/GPO contribute to these binding sites.
Approval was obtained for these studies from the Human Biology Research Ethics Committee at the University of Cambridge. Informed consent was obtained from volunteers in accordance with the Declaration of Helsinki. Materials
Fc receptor gamma chain–deficient (FcR Human platelet preparation Citrated blood from healthy donors at the National Blood Service (Cambridge, United Kingdom) was centrifuged (240g, 15 minutes) to obtain platelet-rich plasma (PRP). PGE1 (2 µM) was added to the PRP, which was centrifuged (125g, 10 minutes) to remove residual erythrocytes. Red cell–free PRP was centrifuged (640g, 15 minutes) and the platelet pellet resuspended in 10 mL calcium-free Tyrode (CFT) (mM): NaCl, 137; NaHCO3, 11.9; NaH2PO4, 0.4; KCl, 2.7; MgCl2, 1.1; glucose, 5.6 (pH 7.4). Platelet counts were adjusted to 1.25 x 108/mL for the static adhesion assay and 2.0 x 108/mL for aggregation using a Beckman Coulter Z2 particle analyzer (High Wycombe, United Kingdom). Citrated PRP was used for aggregation assays. Murine platelet preparation
Platelets were obtained from adult male and female WT and FcR Peptide synthesis, polarimetry, and cross-linking The synthesis of Toolkit peptides and their sequences have been previously published.18 The sequences of modified peptides are shown in Table 1.
Peptide melting temperatures (Tms) were determined by polarimetry to confirm triple-helical peptide conformation. Optical rotation measurements were obtained as the peptide sample was heated up. Tms were calculated by fitting the data to a theoretic equation by nonlinear regression.11
All peptides formed triple helices at room temperature except the Peptides (3 mM) dissolved in 0.1 M NaHCO3 were cross-linked with 1.5 molar equivalents of SPDP (3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester) at room temperature for 1 hour followed by dialysis against 0.01 M acetic acid for 4 hours.22 Cross-linked peptides were used only in aggregation studies. Static platelet adhesion assay Platelet adhesion was measured by detecting alkaline phosphatase present in platelet lysates.23 Test substrate (100 µL; 10 µg/mL in 0.01 M acetic acid) was added to 96-well plates overnight at 4°C. Excess ligand was discarded and wells were blocked with 175 µL 5% bovine serum albumin (BSA) in CFT for 1 to 2 hours, after which the wells were washed 3 times with 175 µL 0.1% BSA in CFT. Platelets (50 µL; 1.25 x 108/mL) were incubated in each well at room temperature for 1 hour. Excess platelets were discarded and the wells washed 3 times. Citrate lysis buffer (150 µL; mM: p-nitrophenyl phosphate, 3.53; trisodium citrate, 71.4; citric acid, 28.55; Triton X-100, 0.1% [vol/vol]; pH = 5.4) was added to each well for 1 hour at room temperature. Then, 100 µL 2 M NaOH was added to each well and absorbance at 405 nm was determined using a Fluostar plate reader (BMG Labtech, Aylesbury, United Kingdom).
In addition to peptides, we measured adhesion to Horm, Ethicon, and PDI collagens. CRP and the Adhesion of GpVI ectodomains (D1D2) Receptor binding to ligands was determined using recombinant ectodomains of GpVI (D1D2) coexpressed with calmodulin. Murine and human D1D2 constructs were prepared as previously described and detected using a horseradish peroxidase (HRP)–conjugated calmodulin-binding peptide, N9A.15 The 96-well plates were coated with test substrate as described above in "Static platelet adhesion assay." Excess ligand was discarded and wells were blocked for 2 hours at room temperature with 175 µL 5% BSA in Tris-buffered saline (TBS) (mM): NaCl, 150; TrisHCl, 50; CaCl2, 1. The wells were washed 3 times with wash buffer (0.1% BSA in TBS) and then 50 µL D1D2 (500 µg/mL in wash buffer) was added to each well for 2 hours at room temperature. Excess D1D2 was discarded and the plate washed 4 times. HRP-conjugated N9A (100 µL) was added to each well for 1 hour at room temperature. This was discarded and the wells were rewashed 4 times. HRP substrate (100 µL, KPL TMB Sure Blue Reserve; Insight Biotechnology, Wembley, United Kingdom) was added to each well and incubated for 3 minutes at room temperature. The reaction was terminated with 50 µL H2SO4 (2.5 M) and the absorbance of the wells measured at 450 nm using a Fluostar plate reader. Aggregation Platelet aggregation was carried out as previously described24 using Chronolog (Labmedics, Manchester, United Kingdom) and BioData PAP-4 (Alpha Laboratories, Eastleigh, United Kingdom) turbidimetric aggregometers. Aggregation of human platelets was performed in citrated PRP, and in washed platelets for murine platelets. Data analysis To determine which peptides supported platelet or D1D2 binding, data from each experiment was resolved into responders and nonresponders as follows. A probability density function for each dataset was calculated using 2 models: (1) a single normal distribution with 2 parameters (mean and SD of the dataset); and (2) two independent normal distributions with 5 parameters (2 means, 2 SDs, and a parameter defining the proportion of the data in each of these 2 distributions). The likelihood for each model was maximized by modifying the parameter values and a likelihood ratio test performed to determine whether model no. 1 or no. 2 best described the data.25 The test statistic is – 2ln(L1/L2), where L1 and L2 are the maximized likelihoods for model no. 1 and no. 2, and follows a chi-squared distribution. A P value was obtained using the test statistic with 3 degrees of freedom (5 minus 2 parameters). Where model no. 2 described the data best, the probabilities for each data point for each of the 2 distributions were compared and the data point was determined to belong to the distribution with the highest probability. Figure 2 shows one set of data and the representations of model no.1 and no. 2. Analysis was performed using Microsoft Excel (Redmond, WA).
Prior to analysis of inhibition data, equality of variances was assessed using the Levene statistic. All the raw datasets showed significant heteroscedasticity and so data were transformed as follows: y' = ((y  – 1)/) + , where y = raw datum, y' = transformed datum, and = power of the transformation. When = 1, y' = y; when = 0, y' = ln(y).
Transformed data were analyzed by univariate ANOVA in which treatment groups were fixed factors and experiments random factors. Posthoc analysis was carried out using the Waller-Duncan method with a type I/type II error ratio of 100. This analysis was performed using SPSS 13.0 for Windows (Chicago, IL). Concentration-response data were analyzed and EC50 values determined as previously described.26
Adhesion of human platelets to 1(III)CB4 peptides
Cyanogen bromide cleavage of bovine collagen III yields fragments with differing properties. Fragment
HYP  ALA substitutions in p8 generated 4 new peptides (Table 1). Platelet adhesion to these peptides was attenuated. Replacing the single GPOGPA (p8-A534) and the doublet GPOGPOGPAGPA (p8-A546/549) abolished binding, whereas replacing either of the doublet GPOGPA (p8-A546 and p8-A549) only attenuated binding. Adhesion of human platelets to Toolkit peptides Platelet adhesion to Toolkit peptides was determined using platelets from 6 donors. Figure 4A shows adhesion of human platelets plus or minus EDTA (2 mM). Maximal adhesion was observed to CRP. Six Toolkit peptides supported binding in all 6 experiments (% control CRP binding): III-30 (94%), III-04 (92%), III-31 (72%), III-40 (69%), III-07 (45%), and III-32 (35%). Three bound in 5 of 6 experiments: III-01 (57%), III-22 (35%), and III-23 (25%). Two bound in 4 of 6 experiments: III-09 (20%) and III-49 (19%). Of the remaining 46 peptides, 34 did not bind in any experiment, 9 bound in 1 of 6 experiments, and 3 bound in 2 of 6 experiments. CRP, GFOGER, Ethicon, Horm, and PDI bound platelets in 6 of 6 experiments. GPP did not bind platelets in any.
EDTA-treated platelets bound maximally to CRP at 56% of control levels. Adhesion to CRP was observed in 5 of 6 experiments. None of the Toolkit peptides bound in all 6 experiments. III-30 bound in 5 of 6 experiments (73% of EDTA-treated platelet binding to CRP) and III-23 bound in 4 of 6 experiments (11%). III-01 (24%), III-04 (12%), and III-40 (8%) bound in 3 of 6 experiments. Of the remaining 52 peptides, 43 did not bind in any experiment, 7 bound in 1 of 6 and 2 bound in 2 of 6 experiments. EDTA-treated platelets bound to Ethicon and Horm in 6 of 6 experiments but did not bind GFOGER, PDI, and GPP in any.
Inhibition of binding by EDTA is consistent with roles for GpVI dependency of human platelet binding The anti-GpVI scFv 10B12 abolished binding of human platelets to III-01, III-30, III-40, and CRP (P < .05). 10B12 did not inhibit adhesion to III-07, III-22, III-23, III-31, III-32, III-57, or to GFOGER, PDI, Ethicon, or Horm. There was a small but significant effect of both 10B12 and the negative control anti–HLA-A2 2D415 on adhesion to III-04. 2D4 had an incomplete inhibitory effect on binding to III-40 (Figure 4B).
Role of
Adhesion of human platelets to III-04, III-31, and III-32 is mediated by integrin Adhesion of murine platelets to Toolkit peptides
Adhesion was measured using platelets from 10 WT and 10 FcR
Maximal adhesion of FcR  –/– platelets was to GFOGER, which was 30% of WT adhesion to CRP. III-15 (40% of binding to GFOGER) bound platelets in all 10 experiments. III-04 (23%) and III-44 (14%) bound in 9 of 10 experiments. Of the remaining 54 peptides, 46 did not bind in any experiment, while 6 bound in 1 of 10 and 1 bound for each of 2 and 4 of 10 experiments. GFOGER bound FcR–/– platelets in all experiments. CRP and GPP did not support any binding.
The Adhesion of recombinant GpVI ectodomains to Toolkit peptides Adhesion of human GpVI ectodomains (hD1D2) to Toolkit peptides and controls was measured on 6 occasions (Figure 6A). Maximal binding was observed to CRP. Nine peptides supported binding in 6 of 6 experiments (% CRP binding): III-16 (84%), III-30 (69%), III-40 (67%), III-49 (42%), III-01 (42%), III-09 (40%), III-04 (34%), III-57 (21%), and III-23 (18%). Of the remaining 48 peptides, 34 showed no binding, 7 bound in 1; 4, in 2; 2, in 3; and 1 (III-13), in 4 experiments. hD1D2 bound to CRP, Horm, Ethicon, and GPP in 6 of 6 and to PDI in 5 of 6 experiments. Binding to GPP was unexpected and is discussed later.
Adhesion of murine D1D2 was measured on 4 occasions (Figure 6B). Overall, binding of mD1D2 was lower than that of hD1D2. Maximal binding was observed to CRP. No Toolkit peptide supported binding in every experiment. Three peptides supported binding in 3 of 4 experiments (% CRP binding): III-30 (33%), III-01 (16%), and III-57 (13%). Three peptides showed binding in 2 of 4 experiments: III-40 (15%), III-49 (7%), and III-26 (4%). Of the remaining 51 peptides, 37 showed no binding and 14 showed binding in 1 experiment. mD1D2 bound to CRP in 4 of 4; GFOGER and GPP, in 3 of 4; Horm and Ethicon, in 2 of 4; and PDI, in 1 of 4 experiments. Platelet adhesion to modifications of III-01 and III-30
III-30 was the best GpVI substrate for both human and mouse platelets. It contains 3 HYP residues within OGP/GPO motifs. Peptide III-01 has the most HYP residues within such motifs, namely, 5. However, there was no detectable binding of murine platelets to III-01, and human platelet binding was heavily dependent on
For human platelets, single HYP
For mouse platelets, the HYP PRO substitutions in III-30 had substantial effects. Binding to III-30c was abolished, whereas substantial binding remained to III-30b in all experiments. Adhesion to III-30a was attenuated and variable: in 5 of 10 experiments, WT platelets bound to this peptide. Binding of WT platelets was the same to III-01a and CRP. Both III-01a and III-01b bound platelets in 10 of 10 experiments, although binding was lower to III-01b. WT platelets did not bind to III-01c. FcR–/– platelets did not bind to any III-01 or III-30 derivatives (Figure 7B). D1D2 binding to modifications of peptides III-01 and III-30
For III-30, the HYP For III-01, binding increased with III-01a, most notably with mD1D2, which bound at levels similar to CRP. The same was true for hD1D2. Binding of hD1D2 to III-01b was similar to III-01a and CRP, whereas mD1D2 binding to III-01b was reduced. Binding of hD1D2 and mD1D2 to III-01c was comparable with peptide III-01 (Figure 7C). Platelet aggregation induced by cross-linked peptides
Cross-linking of peptides (-XL) is necessary for proaggregatory activity.9 Several peptides were cross-linked to determine their ability to induce aggregation (Figure 8). Non–cross-linked peptides did not induce aggregation (data not shown). At 100 µg/mL, CRP-XL, III-30-XL, III-30b-XL, and III-01a-XL induced aggregation of human platelets and CRP-XL, III-30-XL, and III-30b-XL induced aggregation of murine platelets. III-01b-XL induced weak and inconsistent aggregation of human platelets. Although III-01a-XL induced full aggregation of human platelets, the rate of aggregation was lower than for the other peptides, indicating less potent activation (Figure 8B). Concentration-response curves (Figure 8A) revealed CRP-XL as the most potent agonist (EC50 = 20 ng/mL in human platelets) compared with III-30-XL (EC50 = 2.8 µg/mL) and III-30b-XL (EC50 = 12.7 µg/mL). The difference between III-30-XL and III-30b-XL was statistically significant (P = .03). An estimate for the EC50 of III-01a-XL was not obtained as the concentration-response curve did not level off (Figure 8A). CRP-XL was less potent in murine compared with human platelets (EC50 = 321 ng/mL). However, III-30-XL and III-30b-XL were as potent in the mouse (EC50 = 1.8 and 2.3 µg/mL, respectively) as III-30-XL was in human platelets. U46619
[GenBank]
induced aggregation in FcR
Of the 1(III)CB4 peptides, p8-XL induced aggregation at 20 µg/mL and p7-XL at concentrations higher than 200 µg/mL. None of the others induced aggregation at concentrations higher than 200 µg/mL. The HYPALA-substituted 1(III)CB4 peptides did not induce aggregation at concentrations higher than 500 µg/mL (data not shown).
Collagen has long been recognized as a platelet activator.28,29 The identification of the 1(III)CB4 fragment following cyanogen bromide cleavage of collagen as a platelet agonist was an early step toward locating platelet-binding sites within collagen. Similar studies resulted in the development of CRP,9,11 a ligand for GpVI, the major activatory collagen receptor on platelets.17 We extended this strategy by synthesizing the collagen Toolkit,18 identifying novel binding loci for 2β118 and von Willebrand factor (VWF).30 Although CRP is a selective GpVI agonist, and collagen-induced platelet activation is GpVI dependent,12 collagen itself does not contain continuous regions of GPO like CRP. We therefore used the Toolkit to continue the work begun with cyanogen bromide fragmentation to identify native GpVI-binding motifs within collagen.
Several peptides bound both human and murine platelets. Murine platelets bound substantially to III-04, III-30, and III-31. We previously reported that III-04 contains an
Human platelets bound to more peptides than murine platelets. In addition to III-04, III-30, and III-31, platelets bound to III-01, III-07, III-09, III-22, III-23, III-32, III-40, and III-49. EDTA attenuated binding, although it remained strongest to III-30. Platelet adhesion to III-30 was abolished by the anti-GpVI antibody 10B12, unaffected by the anti- We also used recombinant GpVI ectodomains (D1D2) to identify binding sites in collagen. hD1D2 bound to III-01, III-04, III-09, III-16, III-30, III-40, III-49, and III-57. The more extensive binding with hD1D2 than platelets may be because the adhesive forces required are lower for isolated GpVI than platelets. The number of peptides to which hD1D2 did not bind was striking. Surprisingly, hD1D2 bound to III-16, containing just one OGP, but not to other peptides containing OGP or GPO. III-40 contains OGPO and OGPOGP motifs and bound hD1D2, whereas III-41 contains OGPOGP, GPOGP, and OGP motifs but did not bind hD1D2. Adhesion of mD1D2 was not as strong as hD1D2. This indicates a lower affinity of mD1D2 for CRP than hD1D2.15 mD1D2 bound best to III-30, although this was less than to CRP. mD1D2 also bound weakly to III-01 and III-57, which both have OGP/GPO motifs. hD1D2 and mD1D2 bound noticeably to GPP, although not to many Toolkit peptides. This is surprising, since Toolkit peptides have (GPP)5 motifs at either end. Although GPP is structurally similar to GPO, and would align with the CRP-binding site on GpVI,32 the interaction is thought to be too weak to sustain binding. However, several D1D2 molecules may align and stack alongside each other, effectively increasing their overall affinity.16 That this may occur on a (GPP)10 sequence but not on shorter (GPP)5 ends of Toolkit peptides is consistent with the recent suggestion that a single GpVI molecule spans approximately 3 GXX' triplets.16,32 Nevertheless, this interaction may be insufficient to support platelet rather than D1D2 binding to (GPP)10. Terminal "fraying" of the triple helix may also reduce the availability of GPP sequence for binding.
Peptide p8 from the Peptide III-30 stands out as the best GpVI-binding substrate. Its specific sequence is GAOGLRGGAGPOGPEGGKGAAGPOGPOGP, containing 3 HYP residues within OGP/GPO motifs (bold type highlights OGP/GPO-containing portions of the peptide). However, it does not have the highest OGP/GPO content. III-01 contains 5 HYPs within GPOGPOGPO and 2 OGP motifs, and III-41 contains 4 HYPs within OGPOGP, GPOGP, and OGP motifs. Other peptides containing 4 HYP residues within OGP/GPO motifs are III-12, III-22, III-40, and III-57. The distribution of OGP/GPO motifs within these peptides does not reveal an obvious pattern for determining GpVI reactivity. The 2 longest stretches of OGP/GPO in the Toolkit are in III-01 (GPOGPOGPO) and III-09 (OGPOGPOGP). These peptides show some human but no murine platelet binding. They show similar levels of binding of hD1D2, which is approximately one third that of CRP. This is consistent with binding of a single hD1D2 across the 3 triplet motif, and CRP binding 3 hD1D2 molecules across its full length. III-04 and III-49 each have an OGPOGP motif and bind similar levels of hD1D2 to III-01 and III-09. However, III-21, III-22, and III-41 also contain OGPOGP motifs and do not bind hD1D2. III-30 and III-40 show hD1D2 binding that is approximately double that of III-01 and III-09 and two-thirds of CRP, perhaps indicating adhesion of 2 hD1D2 molecules, consistent with the presence of 2 OGP/GPO loci present in these peptides. However, other peptides containing comparable groupings (eg, III-22, III-26, III-41) do not support hD1D2 binding. Hence, any hypothesis on the relationship between amino acid sequence and GpVI reactivity must account for more than the level and location of OGP/GPO sequences. Adhesion of hD1D2 to III-16 dramatically reinforces this view.
The role of HYP in GpVI-dependent binding was investigated by modifying III-30 with HYP All modified p8 peptides showed reduced platelet binding. p8-A534 (cf III-30c) lost all binding, while p8-A546 (cf III-30b) retained best binding. Although these results reflect those with the III-30 peptides, precise comparisons cannot be drawn, since the substituting amino acid is ALA, allowing greater flexibility of the peptide backbone, compared with PRO. D1D2 binding to modified peptides was comparable for human and mouse. Among the III-30 analogues, binding was lowest to III-30a and highest to III-30c. This suggests that the GPOGPOGP motif in III-30c can bind D1D2. The loss of the single GPO motif abolishes murine but not human platelet binding, highlighting potential differences between murine and human GpVI function. A K59E substitution in human GpVI reduces its affinity for collagen.15 This suggests that negative charges in collagen may coordinate with K59 in hD1D2. Conspicuously, III-30 contains a GLU residue between its 2 OGP/GPO clusters, although there is a LYS in the same region. Peptide III-16, which bound hD1D2 effectively, also has 3 GLU residues close to its OGP motif. Any role for charged residues in the GpVI-binding site can be adequately addressed only by the synthesis of new peptides in conjunction with structural studies. III-30 also has a glycine in the X position between the 2 OGP/GPO clusters. GLY-GLY motifs increase instability of the collagen triple helix,33 resulting in extra flexibility. This could allow GpVI receptors to align more freely, enabling an interaction between them, thereby increasing the overall affinity. Aggregation studies enabled a comparison of the activation of human and murine platelets by the modified cross-linked peptides. CRP-XL was a more potent agonist than III-30-XL, particularly in human platelets. III-30b-XL was slightly less potent than III-30-XL in human platelets, but equipotent in murine platelets. It is striking that III-30b-XL, which contains 2 HYP residues in OGP/GPO motifs, activated murine platelets, whereas III-01a-XL, which contains 6 such HYP residues, did not. The lack of activity of III-01a-XL is unlikely to be due to differences in the extent of cross-linking since it was effective in human platelets, albeit weakly. Furthermore, 2 different preparations of III-01a-XL with visibly different levels of cross-linking generated indistinguishable results. The disparity in activity between III-30a-XL, III-30b-XL, and III-30c-XL is also noteworthy. These data suggest that the particular spatial arrangement generated when collagen clusters GpVI is a significant determinant of the subsequent activation of the platelet and further support the view that the interactions of human and murine GpVI with collagen differ significantly. CRP also interacts with nonplatelet receptors. Leukocyte-associated Ig-like receptor (LAIR-1, also a member of the Ig-like superfamily34) binds collagen and CRP, but not GPP. Although CRP may provide a useful template for the interaction of collagen with Ig-like receptors, the specificity of these interactions is probably not dependent solely on OGP/GPO content.35 Peptide III-30 may prove a valuable tool for modeling and understanding the GpVI-collagen interaction, and provide insights into the differences between human and murine GpVI binding to collagen. As a more physiologically agonist, it may be useful for the investigation of GpVI function in ways that CRP is not. In conclusion, we have identified the major GpVI-binding locus in human collagen type III. III-30 is an effective substrate for both human and murine GpVI. The GpVI-binding site cannot be explained simply by the location and number of OGP/GPO motifs in collagen, but other factors must also contribute. Further work will identify those features of III-30 that contribute to GpVI-binding activity.
Contribution: G.E.J. performed research, designed research and reagents, analyzed data, and wrote the paper; N.R. synthesized peptides; J.P.L., D.J.O., and A.A. performed research; P.A.S. developed bioanalytical reagents; R.W.F. was the group head, attracted funding, designed research and reagents, assisted in writing, and reviewed the paper. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Gavin E. Jarvis, Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, United Kingdom; e-mail: gej1000{at}cam.ac.uk.
We thank Prof Barry Coller for generously supplying the antibody 6F1. This work was supported by the British Heart Foundation (G.E.J., D.J.O., P.A.S.), the Wellcome Trust (peptide synthesis), and the Medical Research Council.
Submitted August 23, 2007; accepted February 13, 2008.
Prepublished online as Blood First Edition Paper, February 27, 2008
DOI: 10.1182/blood-2007-08-108472
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Abstract
Introduction
Significantly different from control within ligand group (P < .05). (C) Effect of 
