The I domain present within the ␣2 chain of the integrin ␣ 2  1 (GPIa/IIa) contains the principal collagenbinding site. Based on the crystal structure of the ␣2-I domain, a hypothetical model was proposed in which collagen binds to a groove on the upper surface of the I domain (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517). We have introduced point mutations into 13 residues on the upper surface of the domain. Recombinant mutant proteins were assayed for binding to monoclonal antibodies 6F1 and 12F1, to collagen under static conditions, and for the ability to retain adhesive activity under flow conditions. The mutations to residues surrounding the metal ion-dependent adhesion site that caused the greatest loss of collagen binding under both static and flow conditions are N154S in the A-␣1 turn, N190D in the B-C turn, D219R in the ␣3-␣4 turn, and E256V and H258V in the D-␣5 turn. Mutation in one of the residues that coordinate the metal binding, S155A, completely lost the adhesive activity under flow but bound normally under static conditions, whereas the mutation Y285F had the converse effect. We conclude that the upper surface of the domain, including the metal ion-dependent adhesion site motif, defines the collagen recognition site. The glycoprotein (GP)1 Ia/IIa (integrin ␣ 2  1 ) is a major platelet adhesion receptor for collagen. Integrins are a family of heterodimeric cell adhesion molecules that mediate cell-cell and cell-matrix adhesion (1). The integrin ␣ 2  1 is expressed on several different cell types. Although it is a collagen receptor in platelet and fibroblastic cells, it functions both as a collagen and laminin receptor on endothelial and epithelial cells (2, 3). It also acts as the receptor for the human pathogen echovirus-1 (4).␣ 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 von Willebrand factor, 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 interaction.Within the ␣ 2  1 integrin, the I domain (amino acids 140 -349) provides the principal binding site for collagen. Thus, antibodies that block ␣ 2  1 interaction with collagen recognize epitopes within the I domain (6 -8). Three groups have shown that recombinant ␣2-I domain fusion proteins bind specifically to collagen in a divalent cation-dependent manner (9 -11), and a fourth group has presented conflicting data suggesting that collagen binding is independent of divalent cation (12).Like other I domains, the ␣2-I domain contains a cationbinding site called the metal ion-dependent adhesion site (MI-DAS) motif (13). In the crystal structure (14), the side chains of residues Ser-153, Ser-155, and Asp-254 directly coordinate a Mg 2ϩ ion, and Asp-151 and Thr-221 provide water-mediated bonds. One study reported that mutation of residues A...
The interaction of platelets with collagen plays an important role in primary hemostasis. Glycoprotein Ia/ IIa (GPIa/IIa, integrin ␣ 2  1 ) is a major platelet receptor for collagen. The binding site for collagen has been mapped to the I domain within the ␣ 2 subunit (GPIa). In order to assess the role of the ␣ 2 -I domain structure in GPIa/IIa binding to collagen, a recombinant I domain (amino acids 126 -337) was expressed in Escherichia coli. The ␣ 2 -I protein bound human types I and III collagen in a saturable and divalent cation-dependent manner and was blocked by the ␣ 2  1 function blocking antibody 6F1. The ␣ 2 -I protein inhibited collagen-induced platelet aggregation (IC 50 ؍ 600 nM). Unexpectedly, 6F1, an antibody that fails to inhibit platelet aggregation in plateletrich plasma, blocked the inhibitory effect of the ␣ 2 -I protein. The ␣ 2 -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 (11-14) or GPVI (15) have demonstrated impaired aggregation in vitro in response to collagen. Antibodies against the collagen receptor GPI...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.