Functional Analysis of a Recombinant Glycoprotein Ia/IIa (Integrin α2β1) I Domain That Inhibits Platelet Adhesion to Collagen and Endothelial Matrix under Flow Conditions*

The interaction of platelets with collagen plays an important role in primary hemostasis. Glycoprotein Ia/IIa (GPIa/IIa, integrin α2β1) is a major platelet receptor for collagen. The binding site for collagen has been mapped to the I domain within the α2 subunit (GPIa). In order to assess the role of the α2-I domain structure in GPIa/IIa binding to collagen, a recombinant I domain (amino acids 126–337) was expressed in Escherichia coli. The α2-I protein bound human types I and III collagen in a saturable and divalent cation-dependent manner and was blocked by the α2β1 function blocking antibody 6F1. The α2-I protein inhibited collagen-induced platelet aggregation (IC50 = 600 nm). Unexpectedly, 6F1, an antibody that fails to inhibit platelet aggregation in platelet-rich plasma, blocked the inhibitory effect of the α2-I protein. The α2-I protein was able to prevent platelet adhesion to a collagen surface exposed to flowing blood under low shear stress. Interestingly, it inhibited platelet adhesion to extracellular matrix at high shear stress. These results, taken together, provide firm evidence that GPIa/IIa directly mediates the first contact of platelets with collagen under both stirring and flow conditions.

Platelet adhesion to subendothelium at sites of vascular injury is a critical initial step in hemostasis. Platelets adhere to collagen by two mechanisms: directly by the interaction of platelet membrane proteins with collagen and indirectly via bridging molecules such as von Willebrand factor (vWF) 1 that bind to both platelet glycoprotein (GP) Ib␣ and collagen. This latter interaction is necessary to confer resistance to detachment under high flow and shear conditions. It also stabilizes platelet adhesion via the collagen receptor (1).
GPIa/IIa (integrin ␣ 2 ␤ 1 , VLA2, CD49b/29) is member of the integrin family of heterodimeric molecules that mediate both cell-cell adhesion and adhesion between cells and the extracellular matrix (ECM) (2). The integrin ␣ 2 ␤ 1 is found on several different cell types, and its function may vary depending on the particular cell on which it is expressed. While it is a collagen receptor in platelets and fibroblastic cells, it functions as both a collagen and a laminin receptor on endothelial and epithelial cells (3,4). It also acts as receptor for the human pathogen echovirus-1 (5) and is involved in the migration of tumor cells within collagenous matrices (6). Integrin ␣ 2 ␤ 1 is a major collagen receptor in platelets (7). Although ␣ 2 ␤ 1 -mediated adhesion appears to be an essential primary step in collagen-platelet interactions, it is still not known whether collagen-␣ 2 ␤ 1 binding alone is sufficient to support platelet adhesion and to induce collagen-dependent platelet activation (8 -10). Platelets from patients described as having mild bleeding disorders due to deficient expression of either the ␣ 2 -integrin (11)(12)(13)(14) or GPVI (15) have demonstrated impaired aggregation in vitro in response to collagen. Antibodies against the collagen receptor GPIV (CD36) partly inhibit platelet adhesion to fibrillar collagen under both static and flow conditions (15 -17). However, patients who constitutively lack CD36 have shown normal collagen-induced platelet aggregation (18 -20). Recently, an additional collagen receptor has been cloned from human platelets (the 65-kDa protein) and expressed as a recombinant protein (21). This receptor protein binds specifically to type I collagen but not to type III collagen.
The integrin ␣ 2 ␤ 1 is composed of a 150-kDa ␣ 2 and a 130-kDa ␤ 1 subunit (5). Within the ␣ 2 subunit, the 200-amino acid I domain shares homology with the A domains of vWF, complement proteins, cartilage matrix protein, and certain other integrins. There is increasing evidence that I (A) domains play an important role in cell-adhesion protein and cell-matrix interactions. Like other I domains, the ␣ 2 -I domain contains a cation binding site described as a metal ion-dependent adhesion site (MIDAS) motif (22,23), which may explain why collagen-induced platelet adhesion and activation are dependent on divalent cations.
Several studies have localized the binding site for collagen to the I domain (amino acids 140 -349) of the ␣ 2 subunit. First, monoclonal antibodies that block ␣ 2 ␤ 1 interaction with collagen recognize epitopes located between amino acids 173 and 259 within the I domain (24). Second, a polyclonal antiserum prepared against recombinant ␣ 2 -I domain inhibited endothelial cell attachment to collagen and laminin substrates (25). Finally, some groups have expressed the ␣ 2 -I domain in bacteria as a glutathione S-transferase (GST) or a maltose binding protein fusion product (26 -29). Although some conflicting results have been reported, all functional studies of these expressed proteins confirmed that this region contains the binding site for collagen.
To learn more about the collagen-GPIa/IIa-I domain interac-tion, we have cloned and expressed in bacteria the ␣ 2 -I domain spanning residues 126 -337. The ␣ 2 -I protein was purified to homogeneity and assessed for its biological and biochemical properties. The ␣ 2 -I protein bound to various types of collagen and, when incubated with platelet-rich plasma (PRP), inhibited collagen-induced platelet aggregation. Furthermore, although this protein bound to collagen by a mechanism distinct from that of vWF, it inhibited platelet adhesion to both collagen and endothelial matrix under flow conditions.

EXPERIMENTAL PROCEDURES
Monoclonal Antibodies and Collagen-Antibody 6F1 (30) was obtained from B. Coller (Mt. Sinai Medical Center, New York, NY), antibody 12F1 (31) from V. Woods (University of California, San Diego, CA). Acid-soluble calfskin type I collagen was purchased from Worthington (Lakewood, NJ). Acid-soluble human placenta type I and type III collagen were purchased from Sigma. ␣ 2 -I Domain Protein-Complementary DNA encoding the human ␣ 2 integrin subunit I domain (amino acids 126 -337) was generated by polymerase chain reaction (PCR) using full-length human ␣ 2 cDNA (provided by M. E. Hemler, Dana-Farber Cancer Institute, Boston, MA) as template. The PCR end primers were designed to introduce a BamHI restriction site at the 5Ј end (forward primer, 5Ј-AAGGCCGGATC-CGATTTTCAGCTCTCA -3Ј) and HindIII restriction site at the 3Ј end (reverse primer, 5Ј-AAGGCCAAGCTTACCTTCAATGCTGAA-3Ј). The PCR product was digested with BamHI and HindIII restriction enzymes and inserted into pQE9 (Qiagen, Chatsworth, CA) for expression in Escherichia coli. The recombinant ␣ 2 -I domain protein was expressed as a Histag fusion protein containing 12 residues at the N terminus from the expression vector (MRGSHHHHHHGS).
Purification of Recombinant Proteins-Recombinant ␣ 2 -I protein was expressed in E. coli M15(pREP4) (Qiagen, Chatsworth, CA). Bacteria containing the pQE9-␣ 2 -I fragment were cultured overnight at 37°C in 30 ml of 25 g/liter tryptone, 15 g/liter yeast extract, 5 g/liter NaCl, pH 7.3, containing 100 g/ml ampicillin and 25 g/ml kanamycin. The overnight culture was diluted 1:30 and grown to an A 595 of 0.8. The culture was adjusted to 1.5 mM IPTG and incubated for 3.5 h at 37°C. The cells were then harvested and resuspended in 25 ml of lysis buffer (50 mM Tris-Cl, 0.1 M NaCl, 1 mM EDTA, pH 8.0) containing a final concentration of 250 g/ml lysozyme and allowed to stand for 15 min at 4°C. The bacterial cells were lysed in the presence of 1.25 mg/ml deoxycholic acid and 70 g/ml RNase/DNase. The lysate was centrifuged at 45,000 ϫ g for 25 min. The resulting supernatant was collected and passed over a Ni 2ϩ -chelated Sepharose (Amersham Pharmacia Biotech) column equilibrated with 50 mM Tris-HCl, 500 mM NaCl, pH 7.4 buffer. The ␣ 2 -I protein was eluted from the column with 150 mM imidazole. The highly purified protein was dialyzed against 35 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.5 (TBS-T), overnight at 4°C.
Recombinant vWF-A3 protein was prepared as described previously (32). Briefly, E. coli M15(pREP4) cells containing pQE9-vWF-A3 were cultured overnight at 37°C as described above for ␣ 2 -I protein. The culture was adjusted to 1.5 mM IPTG and incubated for 4 h at 37°C. The bacterial cells were lysed as described above. The lysate was centrifuged at 12,000 ϫ g for 15 min, and the resulting pellet was washed with lysis buffer containing 0.5% Triton X-100 and 10 mM EDTA, followed by recentrifugation.
For purification of vWF-A3 protein, the washed pellet was solubilized by the addition of 7.5 M urea in 25 mM Tris-HCl, pH 8.8, and the solubilized protein was dialyzed against 25 mM Tris-HCl, pH 8.2. The solubilized protein was passed over a Q-Sepharose column (Amersham Pharmacia Biotech) and eluted from the column with sodium chloride. The purified vWF-A3 protein was dialyzed against TBS.
Radiolabeling of ␣ 2 -I Protein-E. coli were grown overnight and diluted in the medium described above. When grown to A 595 ϭ 0.8, the cells (800 ml) were pelleted by centrifugation, washed with M9 minimal medium (12.8 g/liter Na 2 HPO 4 , 3 g/liter KH 2 PO 4 , 0.5 g/liter NaCl, 1 g/liter NH 4 Cl, 1 mM MgCl 2, 0.1 mM CaCl 2 , 0.5% glucose, 2 mg/liter thiamine), and pelleted again. The pellet was resuspended in 500 ml of M9 minimal medium supplemented with 150 mg/liter amounts of each of the following amino acids: threonine, phenylalanine, leucine, isoleucine, lysine, valine, and proline. Bacteria were grown for 60 min, IPTG was added, and the medium was supplemented with 2 Ci/ml [ 35 S]Na 2 SO 4 (NEN Life Science Products) overnight at 37°C. Labeled ␣ 2 -I protein was purified as described above. The specific activity of the purified 35 S-␣ 2 -I protein was determined to 2.4 ϫ 10 4 cpm/g.
Collagen Binding Assay-A final concentration of either 500 or 10 g/ml collagen or a 1% solution of bovine serum albumin (BSA) was added to microtiter wells in 65 mM sodium phosphate buffer, pH 7.2, for 90 min at 37°C. After washing twice with phosphate-buffered saline, pH 7.4, to remove unadsorbed collagen, residual binding sites were blocked by the addition of 3% BSA in TBS-T for 60 min at 37°C. Varying concentrations of the ␣ 2 -I protein were added to the wells and incubated for 60 min at 37°C. Wells were then washed with TBS-T and the remaining bound ␣ 2 -I detected by enzyme-linked immunosorbent assay (ELISA). Wells were incubated with a 1:20,000 dilution of peroxidase-conjugated monoclonal anti-polyhistidine antibody (Sigma) for 60 min at 37°C. The wells were again washed and the substrate (ophenylenediamine, Sigma) was added. After 30 min of substrate conversion, reactions were stopped with 0.025 ml of 2 N H 2 SO 4 , and the plates were read at 490 nm. Net specific binding was determined by subtracting optical density values from wells coated only with BSA from the total binding values obtained as described above.
Collagen-binding assays in the presence of EDTA or divalent cations were carried out with 35 S-labeled ␣ 2 -I protein (500 nM) and EDTA, MgCl 2 , or CaCl 2 (2 mM). After a 60-min incubation, the wells were washed and separated for scintillation counting. For the antibodyblocking assay, 35 S-labeled ␣ 2 -I protein (500 nM) was preincubated with anti-human ␣ 2 antibodies 6F1 or 12F1 (1.5 M) for 30 min and added to the wells. After 60 min, the wells were washed and removed for scintillation counting.
For competition assays, a constant concentration of plasma (diluted 1:40 in TBS) was added to the wells with increasing concentrations of ␣ 2 -I protein or vWF-A3 protein. Wells were washed with TBS-T and anti-vWF-horseradish peroxidase conjugate (1:500; Dako, Carpinteria, CA) was added and incubated for 60 min at 37°C. The bound antibody was detected as described above.
Collagen-induced Platelet Aggregation Assay-Blood was collected from a healthy donor into a tube containing 3.8% sodium citrate. PRP was prepared by centrifuging the blood at 250 ϫ g for 10 min. Platelet aggregation was carried out in siliconized glass cuvettes at 37°C with constant stirring at 1200 rpm in a four-channel aggregometer (Bio/Data Corp., Horsham, PA). PRP was diluted to a platelet concentration of 1.5 or 2 ϫ 10 8 /ml in TBS and incubated with varying concentrations of ␣ 2 -I protein at 37°C for 5 min. Aggregation was initiated by the addition of 5 g/ml soluble equine tendon collagen (Helena Laboratories, Beaumont, TX).
Preparation of Collagen-coated Dishes-Equine tendon collagen was diluted to 5 g/ml with 20 mM MES, 150 mM NaCl, pH 5.0, and added to 35-mm culture dishes. After 1 h incubation at 37°C, coated dishes were subsequently rinsed and incubated with TBS containing 1% BSA for 30 min at 37°C to block nonspecific interactions.
Preparation of Extracellular Matrix-coated Dishes-Human umbilical vein endothelial cells were cultured in growth medium 199 (Bio-Whittaker, Walkersville, MD) containing 20% fetal bovine serum (Summit Biotech, Ft. Collins, CO), 2 mM L-glutamine, 60 units/ml penicillin, 60 mg/ml streptomycin (Life Technologies, Inc.), 50 mg/ml endothelial mitogen (Biomedical Technologies Inc., Stoughton, MA), and 100 mg/ml tissue culture grade heparin (Sigma). The cells were grown on sterile 35-mm dishes (Corning, Corning, NY) for 4 days to reach confluence in a humidified 5% CO 2 incubator at 37°C. The cells were detached by incubation for 20 min at room temperature with 5 mM EDTA in Ca 2ϩand Mg 2ϩ -free Hanks' balanced salt solution (Life Technologies, Inc.). The extracellular matrix deposited on the dishes was washed repeatedly with phosphate-buffered saline. For functional inhibition of the platelet collagen receptor GPIa/IIa, 4 M ␣ 2 -I protein was added to the coated dishes and incubated for 1 h at 37°C and then used immediately in blood perfusion studies as described below.
Flow Assays-A parallel plate flow chamber was assembled as described by the manufacturer (Glycotech, Rockville, MD). Briefly, the collagen-coated dish formed the lower surface of the chamber and a silicone rubber gasket determined the flow path height of 254 m. The flow chamber was assembled and filled with TBS. A syringe pump (Harvard Apparatus Inc., Holliston, MA) was used to aspirate blood through the flow chamber. Using different gasket thicknesses, flow rates of 0.48 and 0.6 ml/min produced a wall shear rate of 150 s Ϫ1 and 1500 s Ϫ1 , respectively. Blood was collected from healthy adult donors into syringes containing 3.8% sodium citrate as anticoagulant. Blood was then perfused for 3 min, and the coated dish was washed with TBS. Attached platelets were observed with phase contrast objectives and recorded by videomicroscopy. For the inhibition experiments, the collagen or subendothelial matrix surface were incubated with ␣ 2 -I protein (4 M) for 60 min at 37°C. Blood was incubated with ␣ 2 -I protein (4 M) for 5 min at 37°C. All experiments were performed in duplicate on different days.

RESULTS
After induction with IPTG, bacteria transformed with the plasmid pQE9-␣ 2 -I and expressing ␣ 2 -I cDNA were lysed. The resultant bacterial lysate was then passed over a Ni 2ϩ -column and the bound protein eluted with a step imidazole gradient. The final yield of purified protein was 10 mg/liter of bacterial culture. The calculated molecular mass for the sequence between Asp-126 and Gly-337 is 23,207 Da. The 12 additional amino acids from the vector sequence add another 1613 Da, bringing the total estimated molecular mass to 24,820 Da. This is in good agreement with the estimated molecular mass of the purified material of 27,000 Da (Fig. 1, inset) as assessed by SDS-polyacrylamide gel electrophoresis. Purified ␣ 2 -I protein was evaluated for its ability to bind directly to collagen. As shown in Fig. 1, the ␣ 2 -I protein bound to calf skin and human type I and III collagen in a concentration-dependent and saturable manner. The half-maximal binding to the three types of collagen occurred at 650 nM (Fig. 1).
It has been reported that the binding of integrin ␣ 2 ␤ 1 to collagen is a Mg 2ϩ -dependent process. To confirm the dependence on divalent cation in ␣ 2 -I domain/collagen interactions, we examined the extent of collagen binding in the presence of Mg 2ϩ , Ca 2ϩ , and EDTA. Since EDTA impaired the interaction of the anti-polyhistidine-HRP monoclonal antibody with the His tag motif, we expressed and purified metabolically labeled 35 S-␣ 2 -I protein. Consistent with previous reports, 35 S-␣ 2 -I protein binding to collagen type I was greatly reduced by the addition of 2 mM EDTA (Fig. 2). More detailed studies of the divalent cation dependence of the 35 S-␣ 2 -I protein/collagen interaction were then undertaken. It was observed that the addition of Mg 2ϩ (2 mM) supported binding while Ca 2ϩ (2 mM) only supported low levels of interaction. These results confirmed the integrity of the MIDAS motif in the 35 S-␣ 2 -I protein.
Inhibition of platelet GPIa/IIa binding to collagen by ␣ 2 -I protein was assessed in two ways. As shown in Fig. 3, we studied the effect of ␣ 2 -I protein on collagen-induced platelet aggregation (CIPA). ␣ 2 -I protein inhibited platelet aggregation in a dose-dependent manner with an IC 50 of 600 nM. At a sufficiently high concentration of ␣ 2 -I protein (2 M), CIPA was completely inhibited. We also examined the effect of the ␣ 2 -I protein on platelet interactions with collagen or subendothelial matrix under shear stress. ␣ 2 -I protein (4 M) prevented platelet adhesion to collagen fibrils exposed to whole blood at shear rate of 150 s Ϫ1 (Fig. 4A). At higher shear rates (1500 s Ϫ1 ), the protein failed to inhibit this interaction (data not shown). However, the ␣ 2 -I protein (4 M) inhibited platelet adhesion to subendothelial matrix at a shear rate of 1500 s Ϫ1 (Fig. 4B). These data confirmed the important role of ␣ 2 ␤ 1 in platelet activation (aggregation) and platelet adhesion under flow conditions.
Since vWF binds to collagen via the vWF-A3 domain, which The purified protein was then tested for its ability to bind collagen. Increasing concentrations of the ␣ 2 -I protein were incubated with immobilized collagen: calf skin type I (q), human type I (OE), and human type III (ࡗ). Bound protein was determined by ELISA as described under "Experimental Procedures." Error bars represent S.E. of six similar experiments. possesses a similar three-dimensional structure to ␣ 2 -I domain, we next examined the ability of the ␣ 2 -I protein to block the binding of multimeric vWF to collagen. Inhibition of the binding of multimeric vWF to immobilized collagen by ␣ 2 -I and vWF-A3 proteins is shown in Fig. 5. Both I (A) domain proteins compete with vWF for binding to collagen, but with varying affinities. The IC 50 for vWF-A3 protein was 1.0 M while that for the ␣ 2 -I protein was 10.2 M.
The anti-human ␣ 2 integrin monoclonal antibodies 6F1 and 12F1 were tested for their effects on the collagen binding activity of ␣ 2 -I protein in two ways. Antibody 6F1, which inhibits the binding of intact ␣ 2 ␤ 1 integrin to collagen (30), also effectively blocked the binding of the ␣ 2 -I protein to type I collagen (Fig. 6A). Surprisingly, 12F1, an antibody that does not inhibit the binding of the intact integrin ␣ 2 ␤ 1 to collagen (31), inhibited 50% of the binding of the ␣ 2 -I protein to collagen. We also studied the effect of the monoclonal antibodies on the ␣ 2 -I protein in the CIPA assay. As expected, neither 6F1 nor 12F1 antibody inhibited platelet aggregation in PRP. However, they were able to block the inhibitory effect of the ␣ 2 -I protein in the CIPA assay (Fig. 6B). DISCUSSION Among the platelet membrane proteins proposed as mediators of platelet adhesion to collagen, GPIa/IIa (integrin ␣ 2 ␤ 1 ) is considered the major collagen receptor. The I domain present in the ␣ 2 chain (GPIa) contains the collagen binding site (24 -29). To further our knowledge of ␣ 2 subunit structure and function, we have expressed a protein encompassing the ␣ 2 -I domain (amino acids126 -337) in E. coli. This recombinant protein 1) can be readily purified in a soluble form from bacteria, 2) binds specifically to collagen, 3) inhibits collagen-induced platelet aggregation, 4) blocks platelet interaction with collagen or ECM under flow conditions, and 5) inhibits the binding of the multimeric vWF to collagen with a lower affinity than the vWF-A3 protein. Furthermore, its collagen binding ability is blocked by the anti-human ␣ 2 -integrin monoclonal antibodies 6F1 and 12F1.
Other investigators have used different expression systems to produce ␣ 2 -I domain fragments in E. coli. In their methods, they have used sonication (26,27,29) or denaturing agents (28) to solubilize their recombinant proteins. In contrast, our ␣ 2 -I protein was purified from the cytoplasmic soluble fraction of enzymatically disrupted bacterial membranes by single-step Ni 2ϩ -affinity chromatography. Also of note are the further manipulations to which some groups subjected their recombinant proteins, such as biotinylation or iodination, in order to permit subsequent detection. However, in this study, we expressed and purified metabolically labeled ␣ 2 -I protein. We believe that the solubility of our recombinant protein is related to the short fusion protein (12 amino acids) coded for in the expression vector and that minimal processing of the purified protein results in a superior starting material for biochemical analysis.
Among the four recombinant ␣ 2 -I domain expressed and characterized to date, distinctions exist among them with respect to the particular sequence selected for expression, purity of the final product, biophysical properties, and the methods used to functionally analyze the recombinant proteins. However, our results agree, in general, with the previously published observations. The half-maximal binding values reported by three of the previous studies has been calculated using ELISA for protein quantitation. Using similar methods, our value of 650 nM for the three types of collagen used is in good agreement with the range of half-maximal binding concentrations reported previously (400 nM to 1.3 M) (27)(28)(29). In addi- Collagen-coated dishes were then incubated with ␣ 2 -I protein (4 M). Whole blood containing the same concentration of the ␣ 2 -I protein was perfused through the chamber at 150 s Ϫ1 . As seen in the lower panel (ϩ), few platelets attached to collagen fibrils. B, upper panel (Ϫ), platelet attachment after whole blood was perfused over subendothelial matrix shear rate of 1500 s Ϫ1 . Lower panel (ϩ), platelet attachment was reduced after perfusion with whole blood containing 4 M ␣ 2 -I protein over a surface of subendothelial matrix previously incubated with ␣ 2 -I protein (4 M). tion, we are the first group to analyze the binding of the isolated ␣ 2 -I domain to type III collagen. Our results are in agreement with the work of Saelman et al. (33), who demonstrated the interaction of platelets (via GPIa/IIa) with type III collagen. In one of the four previous studies, Kamata et al. (26) reported the binding of 125 I-GST-␣ 2 -I protein to collagen with a half-maximal binding of 500 nM. In our study, utilizing purified metabolically labeled 35 S-␣ 2 -I protein, we obtained a half-max-imal binding of 200 nM (data not shown).
Our ␣ 2 -I protein bound to collagen in a similar manner to the intact integrin ␣ 2 ␤ 1 . In this respect, the present study extends the observations of three previously published reports that Mg 2ϩ sustained, while EDTA or Ca 2ϩ inhibited the binding of ␣ 2 -I protein to collagen. On the other hand, our findings contrast with those of Kamata et al. (26), who reported that recombinant 125 I-GST-␣ 2 -I domain bound to collagen in a metalindependent manner. It is possible that this discrepancy is an artifactual consequence of the iodination procedure since we could demonstrate the metal dependence using intrinsically labeled 35 S-␣ 2 -I protein. The results of our binding studies support the conclusion that the MIDAS motif in the I domain plays an important role in the collagen binding activity of the ␣ 2 -I domain.
It has been reported that collagen failed to induce platelet aggregation on either integrin ␣ 2 ␤ 1 -deficient platelets or in the presence of anti-integrin ␣ 2 ␤ 1 antibody. In this study, the ␣ 2 -I protein effectively inhibited platelet aggregation in a concentration-dependent fashion. Although the IC 50 calculated from our data agrees with a report by Depraetere et al. (29), our study is the first to examine the inhibitory effect of the ␣ 2 -I protein using PRP. This approach has provided an interesting new insight in that the 6F1 antibody blocked the inhibitory capacity of the ␣ 2 -I protein. As shown in this study, and in agreement with Coller et al. (30) and Kunicki et al. (34), while 6F1 inhibits CIPA with gel-filtered platelets, 6F1 does not prevent platelet aggregation in PRP. It is of interest that washed platelets were used to produce this antibody. Thus, the discordant 6F1 results might be explained by a continued exposure of the collagen-binding site while a conformational FIG. 6. Antibody blocking of ␣ 2 -I protein binding to collagen. A, monoclonal antibodies 6F1 and 12F1 were tested for their ability to inhibit the binding of ␣ 2 -I protein to type I collagen. Metabolically labeled 35 S-␣ 2 -I protein (500 nM) was preincubated with either antibody (1.5 M) for 30 min and then tested in the collagen-binding assay. Total bound was normalized to 100%. B, neither monoclonal antibody 6F1 nor 12F1 inhibited platelet aggregation at concentrations up to 100 g/ml. However, when a suspension of PRP (1:2) was incubated with antibody 6F1 or 12F1 and ␣ 2 -I protein (2 M), the inhibitory effect of the ␣ 2 -I protein was abolished by over 95%. change occurs in the ␣ 2 -I domain during platelet preparation. Another possibility is that the epitope recognized by the antibody could be blocked by a plasma component that is removed by gel filtration.
Our study is also one of the first to analyze simultaneously the interaction between GPIa and collagen under flow conditions using a soluble, high affinity receptor fragment as a ligand. It is known that, at high shear stress, binding of plasma vWF to collagen or ECM and subsequent platelet GPIb binding to bound vWF is required for initial platelet adhesion (1,35). However, we observed that integrin ␣ 2 ␤ 1 was critical for platelet attachment to subendothelial matrix at high shear rates. This observation agreed with a recent study that used a monoclonal antibody against integrin ␣ 2 ␤ 1 and reported a relatively modest inhibition of platelet attachment to ECM (1). Deposited ECM contains vWF, collagen, and other components that may participate in matrix-platelet interactions (36). However, it was surprising to find that endogenous subendothelial vWF and plasma vWF were not sufficient to support platelet adhesion to ECM. Competition between vWF and the ␣ 2 -I domain for collagen binding sites is unlikely since in this study we demonstrated that they bind to collagen by different mechanisms. One explanation could be that these cells synthesized a small amount of either endogenous vWF or collagen. Therefore, the consequence would be a limited availability of binding sites for the platelet GPIb/IX/V complex or for plasma vWF, respectively. On the other hand, our data support previous reports that platelet adhesion to collagen fibrils at low shear stress is integrin ␣ 2 ␤ 1 -dependent (33,38). Compared with endogenous subendothelial collagen, immobilized isolated collagen fibrils are assembled by raising the pH of a solution and the lack of other subendothelial components may contribute to the formation of an alternative collagen structure. Then, at high shear stress, this conformational change may affect the basic properties of the triple-helical structure resulting in a low affinity by the ␣ 2 -I domain for the binding sites in collagen.
Further interesting results were obtained with the non-function blocking antibody, 12F1 (31). In contrast to previous reports by Kamata et al. (26) and Dickeson et al. (28), we found that 12F1 blocked the ␣ 2 -I protein binding to collagen in CIPA. As discussed above, the fact that 6F1 does not bind to GPIa in PRP, but binds to the isolated I domain, suggests that the epitope recognized by the antibody in the I domain of the GPIa/IIa complex may have a different conformation or may be cryptic in the intact receptor. Therefore, one possibility is that, although the collagen binding remains normal, the recombinant I domain acquired a different conformation. It may be also possible that binding of antibody 12F1 to our ␣ 2 -I protein destroys the integrity of the MIDAS motif (which is essential for collagen binding) rather than simply blocking sterically.
Integrin ␣ 2 ␤ 1 , GPIV (CD36), and GPVI have all been implicated in platelet-collagen adhesive interactions. It has been suggested that the collagen receptor GPIV is an important mediator of platelet adhesion to collagen (16,37) while the receptor GPVI seems to play an important role in collagenmediated signaling events (8,10). Our findings strongly suggest that among these collagen receptors, the integrin ␣ 2 ␤ 1 is a necessary complement to the vWF-GPIb interaction for the initial tethering of platelets to collagen or ECM. Our results may also explain the occurrence of bleeding in patients with genetic defects on ␣ 2 ␤ 1 (11).
Thus, while there is little doubt that the ␣ 2 -I domain is essential for the binding of platelet integrin ␣ 2 ␤ 1 (GPIa/IIa) to collagen, it is possible that other regions of ␣ 2 subunit may mediate collagen binding (28). The techniques described here for the expression, purification, and the biochemical analysis of recombinant ␣ 2 -I domain can be employed in future studies to analyze the different ligand specificities of the integrin ␣ 2 ␤ 1 expressed in other cell types. The biological properties of this soluble receptor fragment should provide an interesting model system to design agents that can selectively inhibit shear-dependent platelet adhesion to vascular subendothelium. These results strongly suggest that platelet GPIa/IIa is the major collagen receptor and that the isolated ␣ 2 -I protein is a promising inhibitor of the platelet-collagen interaction.