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J. Biol. Chem., Vol. 275, Issue 32, 24560-24564, August 11, 2000

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Micromolar Ca²⁺ Concentrations Are Essential for Mg²⁺-dependent Binding of Collagen by the Integrin ₂₁ in Human Platelets^*

David J. Onley^§, C. Graham Knight, Danny S. Tuckwell^¶, Michael J. Barnes, and Richard W. Farndale

From the  Department of Biochemistry, University of Cambridge, Building 0, Downing Site, Cambridge CB2 1QW, United Kingdom and the ^¶ School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom

Received for publication, May 15, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Integrin receptor ₂₁ requires micromolar Ca²⁺ to bind to collagen and to the peptide GPC(GPP)₅GFOGER(GPP)₅GPC (denoted GFOGER-GPP, where O represents hydroxyproline), which contains the minimum recognition sequence for the collagen-binding ₂ I-domain (Knight, C. G., Morton, L. F., Peachey, A. R., Tuckwell, D. S., Farndale, R. W., and Barnes, M. J. (2000) J. Biol. Chem. 275, 35-40). Platelet adhesion to these ligands is completely dependent on ₂₁ in the presence of 2 mM Mg²⁺. However, we show here that this interaction was abolished in the presence of 25 µM EGTA. Adhesion of Glanzmann's thrombasthenic platelets, which lack the fibrinogen receptor _IIb3, was also inhibited by micromolar EGTA. Mg²⁺-dependent adhesion of platelets was restored by the addition of 10 µM Ca²⁺, but millimolar Ca²⁺ was inhibitory. Binding of isolated ₂₁ to GFOGER-GPP was 70% inhibited by 50 µM EGTA but, as with intact platelets, was fully restored by the addition of micromolar Ca²⁺. 2 mM Ca²⁺ did not inhibit binding of isolated ₂₁ to collagen or to GFOGER-GPP. Binding of recombinant ₂ I-domain was not inhibited by EGTA, nor did millimolar Ca²⁺ inhibit binding. Our data suggest that high affinity Ca²⁺ binding to ₂₁, outside the I-domain, is essential for adhesion to collagen. This is the first demonstration of a Ca²⁺ requirement in ₂₁ function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The widely expressed integrin receptor family mediates many cell-cell and cell-matrix interactions. Each receptor comprises a heterodimeric, non-covalent complex of an  and a  subunit (1). The N-terminal region of the  subunits has been modeled as a seven-bladed -propeller (2), containing EF-hand-like cation-binding motifs (3). Nine  subunits, including ₁ and ₂, contain an inserted domain (I-domain) of ~200 amino acids, homologous to the von Willebrand Factor A-domain, located between blades 2 and 3 of the proposed -propeller structure (4). The isolated I-domain of ₂ is capable of ligand binding (4-6). The I-domain within ₂₁ is crucial for collagen binding of the entire integrin. I-domain crystal structures reveal the presence of a single metal ion-dependent adhesion site (MIDAS)¹ (7-9), which is thought to be occupied by Mg²⁺ in vivo.

The platelet integrin ₂₁ is a collagen receptor that plays an important role in hemostasis. Injury to the endothelium of a blood vessel results in the exposure of collagen fibers to circulating platelets, resulting in their adhesion and activation, leading to platelet aggregation and clot formation (10, 11). Integrin ₂₁ is essential for the recognition of collagen by platelets under flow conditions (10, 12) and platelets lacking functional integrin ₂₁ do not respond to stimulation by collagen (13), resulting in bleeding disorders. The sequence GFOGER,² within a triple-helical structure, was recently identified as the minimum binding motif within collagen I for ₁ and ₂ I-domains (14) and has been co-crystallized with the ₂ I-domain, verifying its interaction with the ₂ MIDAS (15). The availability of this peptide allows the properties of ₂₁ to be resolved from those of other, non-integrin, platelet receptors for collagen (11, 16).

The affinity of some integrins depends upon Ca²⁺ (17-20). Micromolar Ca²⁺ is required to stabilize the heterodimeric structure of the platelet fibrinogen receptor, _IIb3 (21), which is necessary for ligand binding (22). In addition, fibrinogen binding to _IIb3 is supported by millimolar levels of either Ca²⁺ or Mg²⁺ (23). In marked contrast, millimolar Ca²⁺ inhibits Mg²⁺-dependent ligand binding of ₂₁ (24, 25). Together, this evidence suggests that Ca²⁺ has a crucial role in integrin function.

Against this background, we examined the effects of the Ca²⁺ chelator, EGTA, on adhesion of platelets, of ₂₁, and of recombinant ₂ I-domain to immobilized type I collagen and to the peptide GFOGER-GPP. We demonstrate a requirement for micromolar Ca²⁺ in ₂₁-mediated platelet adhesion and the binding of isolated ₂₁ to these substrates. Both the stimulatory and inhibitory Ca²⁺-binding sites appear to lie outside the I-domain.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- Human platelets were from fresh whole blood, provided by the National Blood Service, Long Road, Cambridge, UK. Platelets lacking _IIb3 were prepared from whole blood, kindly provided by Dr. M. Makris (Royal Hallamshire Hospital, Sheffield, UK), from two type I Glanzmann's patients. Monomeric type I collagen for use in solid phase adhesion assays was purified from bovine skin, following limited pepsin digestion, as described previously (26, 27). The anti-(human integrin ₂ subunit) monoclonal antibody 6F1 (28) was a generous gift from Dr. B. S. Coller (Mount Sinai Hospital, New York, NY). GR144053F was a gift from Glaxo Wellcome (Stevenage, Hertfordshire, UK). Chemicals were from Sigma-Aldrich (Poole, Dorset, UK) unless otherwise stated. Recombinant ₂ I-domain, as a glutathione S-transferase (GST) fusion protein, was produced (5, 29) and used in solid phase binding assays as described previously (5, 29-31). Peptide GPC(GPP)₅GFOGER(GPP)₅GPC (denoted GFOGER-GPP) and collagen-related peptide (CRP; GCO(GPO)₁₀GCOG) were synthesized as described previously (14, 30, 31). The central GFOGER sequence is the minimum recognition sequence for the ₂ I-domain (14) and the flanking GPP sequences stabilize the triple-helical conformation, which is essential for recognition (14, 26). Integrin ₂₁ was purified from solubilized membranes of human platelets by affinity chromatography on collagen-Sepharose (32, 33), biotinylated using an Amersham Pharmacia Biotech ECL biotinylation module, according to the manufacturer's instructions. Purity of the preparation was assessed by separation on SDS-polyacrylamide gel electrophoresis, followed by staining with Gelcode blue stain reagent (Pierce and Warriner, Chester, UK) and image analysis with Leica Q500 (34).

Static Platelet Adhesion Assay-- 96-well plates (Immulon 2, Dynex Technologies, Ashford, Middlesex, UK) were coated with 100 µl per well of monomeric type I collagen or peptides GFOGER-GPP or CRP at 10 µg/ml in 0.01 M acetic acid for 1 h at 20 °C. Platelet-rich plasma was prepared from fresh whole blood after 2 spins for 1 min at 1200 × g. 10% (v/v) of ACD buffer (39 mM citric acid, 75 mM tri-sodium citrate·2H₂O, 135 mM D-glucose, pH 4.5) and prostaglandin E₁ (100 ng/ml final concentration) were added, and the platelet-rich plasma was spun for 6 min at 700 × g. The platelet pellet was resuspended in 6 ml of buffer (5.5 mM D-glucose, 128 mM NaCl, 4.26 mM Na₂HPO₄·2H₂O, 7.46 mM NaH₂PO₄·2H₂O, 4.77 mM tri-sodium citrate·2H₂O, 2.35 mM citric acid, 0.35% bovine serum albumin (BSA), pH 6.5). Prostaglandin E₁ was added as before, and the platelets were spun for 6 min at 700 × g. Platelets were resuspended to 2 × 10⁸ platelets/ml in adhesion buffer (0.05 M Tris-HCl, 0.14 M NaCl, 0.1% BSA, pH 7.4) and treated as appropriate with MgCl₂, CaCl₂, or EGTA and allowed to rest for 15 min at room temperature. Ligand-coated wells were blocked by incubation with 200 µl of blocking buffer (0.05 M Tris-HCl, 0.14 M NaCl, 5% BSA, pH 7.4) for 30 min. The wells were washed three times with 200 µl of adhesion buffer, then 50 µl of platelet suspension (10⁷ platelets) was added to each well and left for 1 h. The wells were emptied and washed three times with 200 µl of adhesion buffer to remove non-adherent platelets. Adherent platelets were lysed by incubation for 1 h with 150 µl per well of lysis buffer (0.07 M tri-sodium citrate, 0.3 M citric acid, 0.1% Triton X-100 (v/v), 5 mM p-nitrophenyl phosphate). The reaction was terminated by the addition of 100 µl of 2 M NaOH to each well. Adhesion was measured colorimetrically as the absorbance of the p-nitrophenol product at 405 nm in a Maxline Emax microplate reader (Molecular Devices Ltd., Crawley, UK). Values were corrected for background by subtraction of readings from BSA-coated wells. In agreement with others (35), the relationship between platelet number and A₄₀₅ was linear up to 3.0 (Fig. 1) in our conditions, and in a typical experiment, adhesion to collagen I resulted in A₄₀₅ ~1 ± 0.3, which corresponds to adhesion of ~20% of the cells applied. For clarity, absorbance values were scaled so that platelet adhesion to collagen I in the presence of 2 mM Mg²⁺ alone resulted in A₄₀₅ = 1, as we have done previously (14).

Integrin ₂₁ and ₂ I-domain Binding Assay-- The assays for ₂₁ (31, 32) and ₂ I-domain (5, 29) adhesion have been described previously. Briefly, 96-well plates (Immulon 2) were coated and blocked as above. After three washes with 200 µl of adhesion buffer, 100 µl of either biotinylated ₂₁ (1 µg/ml) or recombinant GST-₂ I-domain fusion protein (5 µg/ml) (both in adhesion buffer containing 2 mM MgCl₂ and other ions as required), was applied to the wells and incubated for 2 h at room temperature. Wells were then washed as above and incubated for 30 min with 100 µl of either 0.67 µg/ml streptavidin-horseradish peroxidase (Pierce and Warriner), for ₂₁ detection, or 22 µg/ml rabbit anti-GST (Ig-peroxidase conjugate), for ₂ I-domain detection. The wells were washed four times, and bound ligand was detected using a 3,3',5,5'-tetramethylbenzidine-peroxidase substrate system (Pierce and Warriner). Absorbance at 450 nm was measured using a Maxline Emax plate reader. Results were corrected for background as above and are scaled to an absorbance reading of 1 for collagen.

Calcium Concentration Calculations-- These were performed using the program WinMAXC v2.05 (36) from Dr. Chris Patton at Stanford University.

Replication and Presentation of Data-- Data were obtained from Glanzmann's platelets for two identical experiments using blood from different donors. All other experiments were performed on at least three separate occasions. Mean values ± S.D. from single representative experiments are shown throughout, with each condition tested in triplicate. Where error bars are absent, they were too small to reproduce.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Platelet Adhesion via Integrin ₂₁ Is Calcium-dependent-- We first demonstrate the linearity of the assay used to measure platelet adhesion (Fig. 1) and are in agreement with Bellavite and coworkers (35) that the relationship between absorbance at 405 nm and platelet number is linear, in our conditions up to A₄₀₅ = 3.0. Platelet adhesion to monomeric bovine type I collagen and to the peptide GFOGER-GPP requires the presence of Mg²⁺ (14, 24, 25), although other divalent cations such as Co²⁺ and Mn²⁺, but not Ca²⁺, can replace Mg²⁺ in ₂₁ binding (24) and ₂ I-domain binding.³ Previous work (5, 24, 25) suggests that Ca²⁺ inhibits Mg²⁺-dependent adhesion. However, we found that micromolar concentrations of EGTA, a Ca²⁺ chelator, blocked platelet adhesion to collagen and to GFOGER-GPP in the presence of 2 mM Mg²⁺ (Fig. 2). Platelet adhesion to collagen-related peptide (CRP), which is mediated by the receptor glycoprotein VI in a cation-independent manner (37), was unaffected by EGTA (data not shown).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Platelet number is directly proportional to A405 nm. Platelets were loaded into 96-well plates and lysed for 1 h, and the absorbance of the p-nitrophenol product was read at 405 nm, as described under "Experimental Procedures." Data are from a single experiment, representative of five identical experiments using platelets from different donors and expressed as the mean of triplicate readings ± S.D. Where error bars are absent, they were too small to be reproduced.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   EGTA inhibits platelet adhesion to collagen and to GFOGER-GPP. Platelets were preincubated with EGTA for 20 min in the presence of 2 mM MgCl2 and allowed to adhere to wells coated with monomeric bovine type I collagen or GFOGER-GPP as described under "Experimental Procedures." Data are from a single experiment, representative of three identical experiments using platelets from different donors and expressed as the mean of triplicate readings ± S.D., scaled to an A405 = 1 for adhesion to collagen in the presence of 2 mM Mg2+ alone. Where error bars are absent, they were too small to be reproduced.

When platelets from normal donors were preincubated with an RGD mimetic, GR144053F, at the previously established maximal level for blockade of _IIb3,⁴ adhesion to either monomeric collagen I or to GFOGER-GPP was inhibited (Fig. 3). One explanation for the involvement of _IIb3 might be that platelet microaggregates form on the initial layer of adherent platelets. Alternatively, the adhesion might involve indirect binding of platelets to substrate in an _IIb3-dependent manner, as has been proposed previously (26, 28). We and others (14, 26, 28) have shown that this component of adhesion is secondary to the initial ₂₁-dependent adhesive process, being entirely inhibited by 6F1 (Fig. 3).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   EGTA inhibition of platelet adhesion to collagen and to GFOGER-GPP is independent of integrin IIb3. Normal platelets were preincubated for 20 min with 25 µM EGTA and 2 µM GR144053F individually or in combination, or with anti-(human 2 integrin) monoclonal antibody 6F1 at 2 µg/ml. Platelets from type I Glanzmann's patients, lacking the IIb3 receptor, were preincubated for 20 min with 25 µM EGTA or 2 µM GR144053F. All adhesions were performed in the presence of 2 mM MgCl2. Data for normal platelets are from a single experiment, representative of four identical experiments using platelets from different donors and expressed as the mean of triplicate readings ± S.D. Data for Glanzmann platelets are from a single experiment, representative of two identical experiments from two donors and expressed as the mean of triplicate readings ± S.D. Adhesion of both the normal and Glanzmann platelets are scaled to an A405 = 1 for adhesion of normal platelets to collagen in the presence of 2 mM Mg2+ alone. nd denotes "not done," and where error bars are absent, they were too small to be reproduced.

If the observed effects of EGTA were solely due to inhibition of _IIb3, the degree of inhibition of adhesion induced by GR144053F would be the same as for EGTA alone and no additional inhibition of adhesion would occur in the presence of both of these substances. However, the left-hand side of Fig. 3 demonstrates that this is not the case; with normal platelets, 2 µM GR144053F resulted in ~50% inhibition of adhesion to collagen I and ~70% inhibition of adhesion to GFOGER-GPP, whereas 25 µM EGTA reduced adhesion to about a quarter of these values. The effect of EGTA was greater than that of GR144053F for adhesion to both collagen and to GFOGER-GPP (p < 0.001, analysis of variance). Therefore, blockade of normal platelet adhesion by EGTA cannot be attributed solely to inhibition of _IIb3 but must include inhibition of binding through ₂₁. We do not understand why GR144053F differentially inhibits platelet adhesion to collagen I and GFOGER-GPP.

Preincubation with a monoclonal antibody specific for the integrin ₂ subunit, 6F1, completely abrogated platelet adhesion to either collagen or to GFOGER-GPP (Fig. 3), thus demonstrating the absolute requirement of this receptor for adhesion to these ligands. In control experiments (Fig. 3), 6F1 did not block adhesion of platelets to CRP (37). These observations suggest that the inhibition of normal platelet adhesion by EGTA in excess of that caused by GR144053F is due to direct action on ₂₁.

To confirm the independence of the effect of EGTA from _IIb3, we examined adhesion using platelets lacking the _IIb3 receptor,⁵ from two Type I Glanzmann's patients (Fig. 3, right-hand side). Adhesion of these platelets to collagen I and to the ₂₁-specific GFOGER-GPP was 80% inhibited by micromolar EGTA (Fig. 3) but was completely insensitive to GR144053F, validating the specificity of GR144053F for _IIb3. The data in Fig. 3 show that the EGTA-induced inhibition of platelet adhesion occurs in the absence of _IIb3, supporting the concept that the inhibition occurs at the level of ₂₁.

Although EGTA chelates Ca²⁺, its inhibitory effect could be due to the removal of other ions such as Zn²⁺ or Co²⁺, possibly present at trace levels in the medium, which might be essential for ligand binding. However, addition of micromolar Ca²⁺ to EGTA-inhibited platelets restored adhesive function, confirming that Ca²⁺ is sufficient to support adhesion in the presence of Mg²⁺ (Fig. 4). However, platelet adhesion to collagen I and to GFOGER-GPP was inhibited in the presence of millimolar Ca²⁺, in agreement with the work of others (24, 25). Over several different experiments, rescue of platelet adhesion occurred in the estimated free Ca²⁺ concentration ranges (36): 58-323 nM (collagen I) and 20-323 nM (GFOGER-GPP), where 50% maximal adhesion occurred at a free Ca²⁺ concentration of 88 ± 28 or 110 ± 57 nM for collagen I and GFOGER-GPP, respectively (the latter values are given as mean ± S.E. of five determinations).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Ca2+ restores EGTA-inhibited platelet adhesion to collagen and to GFOGER-GPP. Platelets were preincubated with 25 µM EGTA and the concentrations of CaCl2 shown, in the presence of 2 mM MgCl2, before allowing adhesion to ligand-coated wells. Each point is scaled to an absorbance reading of 1 for adhesion to collagen in the presence of 2 mM MgCl2 alone, and under these conditions, adhesion to GFOGER-GPP gave an absorbance of 0.9. Note: the x axis is non-linear. Data are from a single experiment, representative of three identical experiments using platelets from different donors and expressed as the mean of triplicate readings ± S.D. Where error bars are absent, they were too small to be reproduced.

It is not surprising that the amount of Ca²⁺ needed to restore EGTA-inhibited platelet adhesion varied between experiments. The platelets obtained from individual donors are likely to vary both in expression of ₂₁ and in sensitivity to activation, so that they may secrete their granule load of Ca²⁺ to different extents. However, it is important to note that Ca²⁺ concentrations of around 100-200 nM can restore EGTA-inhibited, Mg²⁺-dependent platelet adhesion via the ₂₁ receptor. This suggests that Ca²⁺ binds at high affinity site(s) on the platelet surface. The location of these sites may be either on ₂₁ itself or on other surface proteins that interact with ₂₁ and modify its binding affinity.

Ligand Binding to Isolated ₂₁ Shows Partial Ca²⁺ Dependence-- The purity of the ₂₁ preparation used in these assays was judged to be ~90% by densitometric analysis of the polyacrylamide gel shown in Fig. 5. As with platelets, binding of purified ₂₁ to collagen and to GFOGER-containing peptides is completely abolished by 6F1 (14, 30) and is also inhibited by increasing concentration of EGTA (Fig. 6A). At 2 mM EGTA, there is very little further inhibition of adhesion (data not shown). Eleven repeat experiments found that in the presence of 50 µM EGTA, ₂₁ binding to GFOGER-GPP is reduced to 30 ± 3% but adhesion to collagen is only reduced to 66 ± 4% (mean ± S.E.), perhaps because ₂₁ binds to collagen sequences other than GFOGER in a Ca²⁺-independent manner. It is possible that, when removed from its proper membrane context, the unconstrained integrin displays novel collagen binding activity in regions other than its I-domain. It has also been speculated that integrin  subunits may contain I-domain-like elements (7, 38), and it is possible that ₁ adheres to sites other than GFOGER within collagen. However, use of monoclonal antibodies directed against the ₁ subunit did not result in significant blockade of binding (data not shown). Others have found that recombinant constructs, including ₂ sequence up to the end of the first EF-hand as well as the I-domain show enhanced capacity to bind collagen (4), although the mechanism is unclear. These regions of ₂ may either increase the affinity of the MIDAS or bind to collagen at a site distinct from GFOGER. However, it is clear that adhesion of isolated ₂₁ to the ₂ I-domain-specific peptide, GFOGER-GPP, is highly sensitive to EGTA, suggesting that Ca²⁺ has a role in affinity regulation of the I-domain.

View larger version (91K): [in this window] [in a new window]   Fig. 5.   21 preparation is at least 90% pure. 9 µg of 21 preparation was separated by SDS-polyacrylamide gel electrophoresis on a 6% gel.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   EGTA inhibits 21 adhesion to collagen and to GFOGER-GPP, but adhesion of 2 I-domain to these ligands is insensitive to EGTA or Ca2+. A, biotinylated 21 was preincubated with EGTA for 15 min, before measuring adhesion. B, recombinant GST-2 I-domain fusion protein was preincubated with EGTA or CaCl2 for 15 min before measuring adhesion. All adhesions were performed in the presence of 2 mM MgCl2. Data are from a single experiment, representative of three identical experiments and expressed as the mean of triplicate readings ± S.D., scaled to an A450 = 1 for adhesion to collagen in the presence of 2 mM Mg2+ alone. Where error bars are absent, they were too small to be reproduced.

Binding of Recombinant ₂ I-domain Is Ca²⁺-independent-- By marked contrast, binding of recombinant GST-₂ I-domain fusion protein to collagen and to GFOGER-GPP was completely unaffected by EGTA, even up to 2 mM levels (Fig. 6B). In addition, up to 10 mM Ca²⁺ had no significant effect on adhesion of the I-domain to these ligands. The insensitivity to EGTA suggests that the activatory Ca²⁺ binding site is not found within the I-domain and must lie elsewhere within ₂₁. Also, the well-documented inhibitory effect of Ca²⁺ on ₂₁-mediated adhesion (24, 25) cannot be due to direct competition for the Mg²⁺ ion bound at the MIDAS site within the I-domain, which is in agreement with others (4).

Ca²⁺Restores EGTA-inhibited ₂₁ Binding-- The EGTA-induced inhibition of isolated ₂₁ adhesion can be restored by the addition of Ca²⁺ (Fig. 7). A 60% reduction in ₂₁ binding to GFOGER-GPP was observed in the presence of 50 µM EGTA, which could subsequently be restored by addition of Ca²⁺. A similar pattern was observed for binding to collagen. Recovery of adhesion corresponds to estimated free Ca²⁺ concentrations in the range of 2-50 µM (36). The rescue of adhesion observed here is similar to that observed with whole platelets, but a striking difference is that 2 mM Ca²⁺ does not significantly inhibit adhesion of integrin ₂₁ to its ligands. In fact, the presence of 10 mM Ca²⁺ results in only partial reduction of adhesion to either collagen I or to GFOGER-GPP (Fig. 7), suggesting that the inhibitory Ca²⁺-binding site is either not present or non-functional in the isolated integrin. Alternatively, millimolar Ca²⁺ might bind other cell surface proteins, which then regulate ₂₁ ligand-binding affinity.

View larger version (32K): [in this window] [in a new window]   Fig. 7.   The EGTA-induced inhibition of 21 adhesion to collagen or to GFOGER-GPP can be restored by addition of Ca2+. Biotinylated 21 was preincubated for 15 min with the concentrations of CaCl2 shown and EGTA in the presence of 2 mM MgCl2, before application to the ligand-coated wells. Data are from a single experiment, representative of three identical experiments and expressed as the mean of triplicate readings ± S.D., scaled to an A450 = 1 for adhesion to collagen in the presence of 2 mM Mg2+ alone. Where error bars are absent, they were too small to be reproduced.

In conclusion, we have demonstrated an essential requirement for micromolar concentrations of Ca²⁺ in Mg²⁺-dependent ₂₁-mediated platelet adhesion. Adhesion of purified ₂₁ to GFOGER-GPP was significantly Ca²⁺-dependent, whereas, adhesion to collagen had a large Ca²⁺-independent component. The stimulatory Ca²⁺ binding site(s) is not situated within the I-domain and, in agreement with others, Ca²⁺-mediated inhibition of ₂₁ adhesion does not act by competition with the Mg²⁺ ion at the MIDAS (4). These findings contribute to the understanding of the role of cations in the regulation of ₂₁ function.

	FOOTNOTES

^* This work was supported in part by the Medical Research Council of the United Kingdom.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Supported by a British Heart Foundation of the United Kingdom PhD Studentship. To whom correspondence should be addressed: Tel.: 44-1223-333643; Fax: 44-1223-333345; E-mail: djo21@hermes.cam. ac.uk.

Published, JBC Papers in Press, May 25, 2000, DOI 10.1074/jbc.M004111200

² Protected by International Patent Application PCT/GB99/00992.

³ L. F. Morton (this laboratory), unpublished observations.

⁴ E. M. Wijnen (this laboratory), unpublished observations.

⁵ Determined by flow cytometry (M. Makris, unpublished observation).

	ABBREVIATIONS

The abbreviations used are: MIDAS, metal ion-dependent adhesion site; BSA, bovine serum albumin; CRP, collagen-related peptide; GST, glutathione S-transferase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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				J. O. Humtsoe, J. K. Kim, Y. Xu, D. R. Keene, M. Hook, S. Lukomski, and K. K. Wary A Streptococcal Collagen-like Protein Interacts with the {alpha}2{beta}1 Integrin and Induces Intracellular Signaling J. Biol. Chem., April 8, 2005; 280(14): 13848 - 13857. [Abstract] [Full Text] [PDF]

				P. R.-M. Siljander, S. Hamaia, A. R. Peachey, D. A. Slatter, P. A. Smethurst, W. H. Ouwehand, C. G. Knight, and R. W. Farndale Integrin Activation State Determines Selectivity for Novel Recognition Sites in Fibrillar Collagens J. Biol. Chem., November 12, 2004; 279(46): 47763 - 47772. [Abstract] [Full Text] [PDF]

				K. Ajroud, T. Sugimori, W. H. Goldmann, D. M. Fathallah, J.-P. Xiong, and M. A. Arnaout Binding Affinity of Metal Ions to the CD11b A-domain Is Regulated by Integrin Activation and Ligands J. Biol. Chem., June 11, 2004; 279(24): 25483 - 25488. [Abstract] [Full Text] [PDF]

				P. R.-M. Siljander, I. C. A. Munnix, P. A. Smethurst, H. Deckmyn, T. Lindhout, W. H. Ouwehand, R. W. Farndale, and J. W. M. Heemskerk Platelet receptor interplay regulates collagen-induced thrombus formation in flowing human blood Blood, February 15, 2004; 103(4): 1333 - 1341. [Abstract] [Full Text] [PDF]

				J. Lahav, E. M. Wijnen, O. Hess, S. W. Hamaia, D. Griffiths, M. Makris, C. G. Knight, D. W. Essex, and R. W. Farndale Enzymatically catalyzed disulfide exchange is required for platelet adhesion to collagen via integrin {alpha}2{beta}1 Blood, September 15, 2003; 102(6): 2085 - 2092. [Abstract] [Full Text] [PDF]

				S. Perret, J. A. Eble, P. R.-M. Siljander, C. Merle, R. W. Farndale, M. Theisen, and F. Ruggiero Prolyl Hydroxylation of Collagen Type I Is Required for Efficient Binding to Integrin {alpha}1{beta}1 and Platelet Glycoprotein VI but Not to {alpha}2{beta}1 J. Biol. Chem., August 8, 2003; 278(32): 29873 - 29879. [Abstract] [Full Text] [PDF]

				W.-M. Zhang, J. Kapyla, J. S. Puranen, C. G. Knight, C.-F. Tiger, O. T. Pentikainen, M. S. Johnson, R. W. Farndale, J. Heino, and D. Gullberg alpha 11beta 1 Integrin Recognizes the GFOGER Sequence in Interstitial Collagens J. Biol. Chem., February 21, 2003; 278(9): 7270 - 7277. [Abstract] [Full Text] [PDF]

				T. Kamata, K. K. Tieu, T. Tarui, W. Puzon-McLaughlin, N. Hogg, and Y. Takada The Role of the CPNKEKEC Sequence in the {beta}2 Subunit I Domain in Regulation of Integrin {alpha}L{beta}2 (LFA-1) J. Immunol., March 1, 2002; 168(5): 2296 - 2301. [Abstract] [Full Text] [PDF]

				B. Nieswandt, V. Schulte, W. Bergmeier, R. Mokhtari-Nejad, K. Rackebrandt, J.-P. Cazenave, P. Ohlmann, C. Gachet, and H. Zirngibl Long-term Antithrombotic Protection by In Vivo Depletion of Platelet Glycoprotein VI in Mice J. Exp. Med., February 12, 2001; 193(4): 459 - 470. [Abstract] [Full Text] [PDF]

				M. Achison, C. M. Elton, P. G. Hargreaves, C. G. Knight, M. J. Barnes, and R. W. Farndale Integrin-independent Tyrosine Phosphorylation of p125fak in Human Platelets Stimulated by Collagen J. Biol. Chem., January 26, 2001; 276(5): 3167 - 3174. [Abstract] [Full Text] [PDF]

P. D. Arora, L. Silvestri,