Schoolmeester and colleagues report the development of a monoclonal antibody that binds specifically to the active conformation of the I-domain of the collagen-binding integrin α2β1, solving the dilemma of specifying the activation state of this integrin when it mediates platelet adhesion to collagen.
The ability of integrins to interact with ligands can be regulated by intracellular signaling. In the most frequently cited example, the platelet fibrinogen receptor, αIIbβ3, only binds soluble ligands on stimulated platelets. Thus, it is not possible to measure an affinity between fibrinogen and αIIbβ3 on resting platelets, but a dissociation constant for fibrinogen of approximately 100 nM is readily measured when platelets are stimulated by adenosine diphosphate (ADP). This change in the affinity of αIIbβ3 for ligands can also be detected using the monoclonal antibody PAC-1, which only binds to the activated conformation of αIIbβ3.1 Similarly, a derivative of PAC-1 named WOW-1 binds exclusively to the active conformation of αvβ3.2
Platelet adhesion to collagen is a complex process involving 2 receptors. One receptor, glycoprotein VI (GPVI), is a member of the immunoglobulin gene superfamily that mediates the initial adhesion of platelets to collagen and, via the associated Fc receptor γ chain, is responsible for initiating collagen-stimulated platelet signaling. In turn, GPVI-stimulated signaling enables the I-domain of the second receptor, α2β1, to bind firmly to collagen, stabilizing platelet adhesion to this substrate.3 Subsequentα2β1-initiated signaling serves to amplify the GPVI-initiated signals.4 Heretofore, it has been difficult to directly demonstrate an agonist-induced change in the affinity of α2β1 for soluble collagen and impossible to demonstrate a change in α2β1 affinity when platelets adhere to collagen-coated surfaces.
Interaction of IAC-1 to platelets deposited on collagen under flow. See the complete figure in the article beginning on page .
Interaction of IAC-1 to platelets deposited on collagen under flow. See the complete figure in the article beginning on page .
To remedy these problems, Schoolmeester and colleagues (page ) produced monoclonal antibodies against a chimeric protein composed of the α2 I-domain and maltose-binding protein. After selecting for antibodies that bound to the I-domain, they identified one, integrin activated conformation-1 (IAC-1), that only bound to platelets stimulated by the GPVI agonist convulxin. But they also found that IAC-1 bound to platelets stimulated by ADP, U46619, and thrombin. These observations confirm that GPVI-initiated signals shift the α2 I-domain from its inactive to its active conformation and show that the effect of platelet agonists on integrins is not confined to the β3 subfamily.
The crystal structure of a 21-residue collagen peptide bound to the α2 I-domain revealed that 3 loops on the upper surface of the I-domain coordinate a metal ion and bind to collagen.5 However, IAC-1 binds to an epitope on the opposite side of the I-domain. This means that platelet stimulation induces a global change in the I-domain conformation. More important from the standpoint of future experimentation, it means that collagen and IAC-1 can bind to the I-domain simultaneously, enabling the antibody to detect activated α2βl when platelets adhere to a collagen-coated surface.FIG1
Platelet adhesion to collagen is an essential facet of platelet function in vivo. IAC-1 has already provided new insights into this process, and it promises to be an exquisite tool for future studies.
