Inflammatory Bowel Disease (IBD) is a chronic disease of the gut that currently afflicts 7 million people worldwide and currently has no cure. A hallmark of IBD is hyperactive platelets, leading to increased venous thromboembolism, or deep vein clots. Previous research has shown that these hyperactive platelets also lose the ability to properly metabolize hyaluronan (HA). Our lab sought to determine how this loss of platelet-mediate HA degradation is linked to IBD platelet hyperactivity.

We found layilin to be the primary HA receptor for platelets via HA binding assays rather than the well-studied HA receptor, CD44. While some research has shown that layilin functions to alter integrin activation in T-cells and modulates cell structure in endothelial cells, the role of layilin in platelets is unknown. We began our research into layilin by asking, “what does it’s ligand, HA, do to platelet function?” Through platelet aggregation and flow cytometry assays, we discovered that HA led to inhibiting platelet clotting and activation. We repeated these experiments with an antibody that bound specifically to layilin and found the same results. Therefore, HA-layilin binding inhibits platelet function.

The next question, of course, was “how?” We found that layilin and integrin aiibB3 (a key receptor for platelet binding) are close together on the platelet surface using pulldown, staining, and proximity ligation assays. Perhaps layilin’s proximity to integrin was allowing it to mediate integrin activation. Using layilin knock-out (KO) platelets from mice, we found that our KO platelets showed significant hyperactivation of integrin when we activated their PAR receptor pathway with thrombin.  These KO platelets also showed increased binding onto fibrinogen, integrin’s ligand, under slow venous flow when compared to the wild-type platelets.

However, our layilin KO platelets did show a reduction in clotting, which we believe is proof that completely deleting layilin leads to unstable clots (despite their hyperactivation). We measured the internal levels of platelet signaling molecules with western blot and found that the RAC1 GTPase pathway was dysregulated in resting platelets.  Layilin loss made platelets “primed to activate.” When we turned to IBD patient platelets, we not only found that these hyperactive platelets showed reduced layilin expression, but they also had increased active RAC1. However, these platelets were also highly sensitive to RAC1 inhibition, which suggests that drugs targeting RAC1 could be effective in treating IBD symptoms.

Our work not only examines the crucial role of an HA receptor in the context of platelet regulation but also suggests a possible treatment route for IBD.