Monomeric C-reactive protein is prothrombotic and dissociates from circulating pentameric C-reactive protein on adhered activated platelets under flow

Abstract

We previously reported that C-reactive protein bioactivity on thrombogenesis was based on loss of its pentameric symmetry, resulting in formation of monomeric C-reactive protein. Our purpose was to provide mechanistic information on the direct effects of C-reactive protein isoforms on platelet activation and provide a C-reactive protein dissociation mechanism in circulating blood. C-reactive protein-induced platelet activation was evaluated by flow cytometry. Platelet aggregation, clot properties, and coagulation were also measured. Washed platelets were incubated with C-reactive protein isoforms and vasodilator-stimulated phosphoprotein (VASP) phosphorylation was analysed by western blot and immunofluorescence. C-reactive protein dissociation under flow was evaluated by confocal microscopy on the surface of adhered platelets after perfusing human blood containing pentameric C-reactive protein at different shear rates. Dissociated monomeric C-reactive protein thrombogenicity was measured in flow experiments. Platelet aggregation and flow cytometry analysis revealed that monomeric C-reactive protein significantly induced platelet aggregation, surface P-selectin and CD63 exposure, and glycoprotein IIb-IIIa activation, whereas pentameric C-reactive protein was unable to produce any effect. p38 mitogen-activated protein kinase (MAPK) and Jun N-terminal kinase (JNK) inhibitors, as well as CD36 blocking antibody partially inhibited monomeric C-reactive protein-induced platelet activation and aggregation. Additionally, monomeric C-reactive protein significantly induced VASP dephosphorylation at serine 239. We found that pentameric C-reactive protein dissociated into monomeric C-reactive protein on the surface of activated adhered platelets under flow conditions and that this generated monomeric C-reactive protein promoted further platelet recruitment. These data indicate that whereas serum pentameric C-reactive protein may not affect platelet activation, monomeric C-reactive protein, which dissociates from pentameric C-reactive protein on the surface of activated platelets, could contribute to atherothrombotic complications by promoting thrombosis.