The Platelet Glycoprotein Ib-von Willebrand Factor Interaction Activates the Collagen Receptor α₂β₁ and the Two Adhesive Receptors then Collaborate to Fully Activate Platelets to Form Thrombi.

Integrin α₂β₁ (GP Ia/IIa) is a major platelet receptor for collagen, containing its collagen binding site within the α₂ I domain. α₂β₁ changes conformation upon platelet activation, increasing its affinity for collagen. The conformational changes are reflected in the markedly different crystal structures obtained for the α₂ I domain depending on whether it is free or bound to a collagen peptide. However, it is not known whether in flowing blood α₂β₁ on platelets is activated before binding to collagen. To address this issue, we identified an antibody that has higher affinity for the activated α₂β₁. We found that two antibodies that bind within the α₂ I domain, 12F1 and 6F1, bound preferentially to ADP-activated platelets, with 12F1 displaying the most marked increase in binding with activation. We corroborated this result for 12F1 by showing that it binds with higher affinity to a gain-of-function I domain mutant than to either the wild-type I domain or to a loss-of-function mutant. In addition, when whole blood was perfused over a surface coated with 12F1, the antibody did not support the adhesion of unstimulated platelets. Because thrombus formation on collagen at a high shear stress is initiated by the binding of the platelet glycoprotein Ib-IX-V complex (GP Ib) to von Willebrand factor (VWF), we tested whether this interaction can activate α₂β₁, using 12F1 as a probe for integrin activation. We perfused blood over a surface coated with a mixture of VWF A1 domain (a GP Ib ligand) and 12F1, or VWF A1 and mouse IgG. Platelets rolled and did not attach stably on the A1/IgG surface, but they firmly bound and covered the A1/12F1 surface. The fact that 12F1 alone failed to capture resting platelets under flow but supported firm platelet adhesion if GP Ib interacted with VWF A1 strongly suggests that GP Ib ligation by VWF induces signals that activate α₂β₁ and increase its affinity for collagen. The two receptors (GP Ib and α₂β₁) then cooperate in platelet adhesion to collagen, which was demonstrated by perfusing, at a high shear stress, reconstituted blood lacking VWF and fibrinogen over surfaces coated with collagen or A1/collagen. The A1/collagen surface contained more firmly adherent platelets than the collagen surface; firm adhesion was blocked by 6F1. We then tested whether the signals from GP Ib and α₂β₁ cooperate to fully activate platelets and allow thrombus growth. For this, we perfused whole blood over a mixed matrix of A1 and the α₂β₁-specific type I collagen-derived triple-helical peptide, CP10. We observed that platelets not only firmly adhered to this surface, they also formed thrombi, similar to those seen on collagen surfaces.

Thrombus formation was inhibited by either the α_IIbβ₃ antibodies or blocking the A1/CP10 surface with the recombinant α₂ I domain. Together, our data indicate that platelets adhere to collagen in a stepwise fashion, beginning with the interaction of GP Ib with VWF, which rapidly activates α₂β₁ to engage collagen. The combination of adhesive ligand-receptor interactions induces the activation of integrin α_IIbβ₃, which enables thrombus formation.