Anti-Inflammatory Effect of Blocking the Interaction between Platelet Glycoprotein Ibα and the Leukocyte Integrin α_Mβ₂.

The interaction between the leukocyte integrin Mac-1 (α _M β ₂) and platelet glycoprotein (GP) Ibα allows leukocytes to firmly adhere to platelet thrombi, enabling their subsequent transmigration into the vessel wall. This interaction is blocked by the GP Ibα monoclonal antibody AP1, which we have previously mapped to the GP Ibα sequence Arg218 to Tyr228. In the current investigation we examined the effectiveness of a GP Ibα-based peptide containing the AP1 epitope in blocking platelet-leukocyte interactions under flow and in an inflammation model based on corneal wound healing. The peptide bound the α M I-domain specifically and saturably and was able to inhibit several GP Ibα-dependent functions under flow:

• the adhesion of GP Ib-IX complex-expressing CHO cells to immobilized α M I-domain,

• the adhesion of THP-1 cells - a monocytic cell line expressing α _Mβ ₂ - to immobilized glycocalicin (soluble extracellular region of GP Ibα ),

• adhesion of activated THP-1 cells to surface-adherent GP Ib-IX complex-expressing CHO cells, and

• the binding of neutrophils to immobilized glycocalicin or a platelet monolayer.

We therefore tested whether the peptide could inhibit inflammatory processes that may be dependent on the interactions between platelets and neutrophils. Neutrophils play an important role in corneal wound healing, but because the cornea is avascular, they must migrate to the wound from the limbal vessels at the periphery of the cornea. Therefore we chose this model of mouse corneal wounding as we have previously demonstrated that antibody-induced platelet depletion markedly reduces neutrophil localization and emigration from the limbal vessels and therefore postulated that platelets may directly facilitate neutrophil emigration. In our model, a 2 mm wound was made at the center of the cornea by removing the epithelium without injuring the stroma. Neutrophil infiltration across the cornea, from periphery to center, was assessed at 12 and 30 hr after the injury. Mice were injected with either AP1 peptide or control peptide (scrambled AP1 peptide) three times at 4 hour intervals beginning immediately after corneal wounding. Mice treated with AP1 peptide showed markedly fewer neutrophils infiltrating all regions of the cornea. These results indicate that the interaction between GP Ibα and Mac-1 is required for efficient leukocyte extravasation. They also suggest that drugs targeting the GP Ibα-α _Mβ ₂ axis could have anti-inflammatory properties without compromising hemostasis, as the peptide had minimal effects on the interaction of GP Ibα with von Willebrand factor.