Miguel A. Cruz

Crystal Structure of the von Willebrand Factor A1 Domain and Implications for the Binding of Platelet Glycoprotein Ib

von Willebrand Factor (vWF) is a multimeric protein that mediates platelet adhesion to exposed subendothelium at sites of vascular injury under conditions of high flow/shear. The A1 domain of vWF (vWF-A1) forms the principal binding site for platelet glycoprotein Ib (GpIb), an interaction that is tightly regulated. We report here the crystal structure of the vWF-A1 domain at 2.3-Å resolution. As expected, the overall fold is similar to that of the vWF-A3 and integrin I domains. However, the structure also contains N- and C-terminal arms that wrap across the lower surface of the domain. Unlike the integrin I domains, vWF-A1 does not contain a metal ion-dependent adhesion site motif. Analysis of the available mutagenesis data suggests that the activator botrocetin binds to the right-hand face of the domain containing helices α5 and α6. Possible binding sites for GpIb are the front and upper surfaces of the domain. Natural mutations that lead to constitutive GpIb binding (von Willebrand type IIb disease) cluster in a different site, at the interface between the lower surface and the terminal arms, suggesting that they disrupt a regulatory region rather than forming part of the primary GpIb binding site. A possible pathway for propagating structural changes from the regulatory region to the ligand-binding surface is discussed.

Mapping the Collagen-binding Site in the I Domain of the Glycoprotein Ia/IIa (Integrin α2β1)

The I domain present within the α2 chain of the integrin α2β1 (GPIa/IIa) contains the principal collagen-binding site. Based on the crystal structure of the α2-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 βA-α1 turn, N190D in the βB-βC turn, D219R in the α3-α4 turn, and E256V and H258V in the βD-α5 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.

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 aggregation by membrane-expressed A1 domains of von Willebrand Factor is dependent on residues Asp 560 and Gly 561☆

We have previously demonstrated that the isolated von Willebrand Factor (vWF)-A1-domain can activate platelets in a GPIb-dependent manner. Here we evaluated the functional impact of targeted point mutations Gly561His (G1324(561)H), an analog of a previously described 2M von Willebrand disease variant, and Asp560Ser (D1323(560)S) in the model of the membrane expressed A1-domain. Platelet aggregation in response to COS-7-cells stably transfected with wild type A1-domain was abrogated by both substitutions. Ristocetin did not increase the aggregatory potential of mutant vWF-A1, in contrast to native forms. Botrocetin boosted the aggregatory responses of all A1-domains tested. These data suggest that G1324(561) and D1323(560) comprise part of the GPIb binding motif essential for subsequent platelet aggregation. Botrocetin seems to alter the potential of vWF for GPIb interaction independently of that motif. The experimental system tested here provides a rapid and reproducible approach for the functional analysis of isolated A1-domain interactions with platelet-GPIb.