PB0890 - The heparan sulfate proteoglycan perlecan induces conflicting patterns of thrombus formation upon activation of GPVI: a novel role in thrombus stability?

Background: Proteoglycans are a heterogeneous family of proteins with covalently bound sulfated glycosaminoglycans. Perlecan, a matrix heparan sulfate proteoglycan secreted by endothelial cells and smooth muscle cells, has been proved to bind to the platelet and megakaryocyte-specific ITIM receptor G6bB, regulating glycoprotein VI (GPVI) signaling. Contrastingly its C-term fragment can enhance collagen induced platelet adhesion via the integrin α2β1.

Aims: We investigated the potential dual role of perlecan to induce platelet adhesion and to regulate GPVI mediated thrombus formation.

Methods: Platelet degranulation and integrin αIIbβ3 activation was measured by flow cytometry following pre-incubation with proteoglycans and dose-dependent activation with suboptimal concentrations of collagen-related peptide (CRP-XL). In spreading assays, platelets were allowed to interact with collagen and perlecan for 45 minutes. Whole blood thrombus formation under flow was measured on CRP-XL surfaces with(out) perlecan. After perfusion the formed thrombi were stained for P-Selectin.

Results: Perlecan enhanced CRP-XL-induced platelet degranulation and integrin αIIbβ3 activation in a time and concentration-dependent manner. Immobilised perlecan supported platelet adhesion and spreading. However, in combination with collagen, perlecan reduced the platelet spreading. In whole blood perfusion over a combination of CRP-XL and von Willebrand factor (VWF), co-immobilised perlecan reduced the buildup of thrombi, but simultaneously changed the thrombus structure. Morphological analyses indicated smaller, denser and more regularly shaped thrombi, with increased P-selectin exposure when perlecan was present. Perfusion onto perlecan alone resulted in a monolayer of platelets without multilayered thrombus formation.

Conclusion(s): Perlecan enhanced adhesion and GPVI-mediated platelet activation. However, when co-immobilised, it suppressed GPVI-mediated spreading and thrombus formation, while still enhancing local platelet activation. Whether this is due to a slower kinetic of platelet deposition or to an enhanced thrombus contractility remains unclear. These findings reflect the intrinsic structural features of perlecan, acting as a ligand for platelets and simultaneously competing with GPVI ligands.