New insights in platelet signaling by Low Density Lipoprotein

Abstract

Contact between LDL and human blood platelets enhances their responsiveness to various aggregation-inducing agents. Although the sensitization and upstream signaling has been well characterized, the identity of the platelet surface receptor for LDL-particles has remained obscure. We report that the initiation of platelet signaling towards p38MAPK is mediated by the LDL-receptor binding domain of apolipoprotein B100 (apoB100), the B-site. Here, we describe that the activation of p38MAPK is inhibited by receptor-associated protein. This inhibitor blocks ligand binding to members of the LDL-receptor family, suggesting a role for a family member in LDL-induced platelet sensitization. This was further investigated by confocal fluorescence microscopy, which revealed a high degree of colocalisation of apoB100 and the LDL-receptor homologue Apolipoprotein E Receptor 2 (ApoER2) at the platelet surface. In addition, association of LDL to platelets was associated with tyrosine phosphorylation of ApoER2. Tyrosine phosphorylation of ApoER2 appeared to be independent of integrin aIIbb3, but required the presence of cell surface proteoglycans. Furthermore, LDL-dependent phosphorylation of ApoER2 was inhibited in the presence of PP1, an inhibitor of Src-like tyrosine kinases. Moreover, phosphorylated ApoER2 coprecipitated with the Src-family member Fgr in immunoprecipitation experiments. This suggests that exposure of platelets to LDL induces association of ApoER2 to Fgr, a kinase that is able to activate p38MAPK. In conclusion, our data indicate that ApoER2 serves a critical role in LDL-dependent sensitization of platelets. LDL sensitizes platelets by inducing a transient activation of p38MAPK, a Ser/Thr kinase which is activated by the simultaneous phosphorylation of Thr180 and Tyr182 and which is an upstream regulator of cytosolic phospholipase A2 (cPLA2). A similar, transient phosphorylation of p38MAPK is induced by a peptide mimicking amino acids 3359-3369 in apoB100 called the B-site. Here we report that the transient nature of the p38MAPK activation is caused by PECAM-1, a receptor with an immunoreceptor tyrosine-based inhibitory motif. PECAM-1 activation by cross-linking induces tyrosine phosphorylation of PECAM-1 and a fall in phosphorylated p38MAPK and cPLA2. Interestingly, also LDL and B-site peptide induce tyrosine phosphorylation of PECAM-1 and studies with immuno-precipitates indicate the involvement of c-Src. Inhibition of Ser/Thr-phosphatases PP1/PP2A (okadaic acid) makes the transient p38MAPK activation by LDL and B-site peptide persistent. Inhibition of Tyr-phosphatases (vanadate) increases Tyr-phosphorylated PECAM-1 and blocks the activation of p38MAPK. Together these findings suggest that following a first phase in which LDL, through its B-site, phosphorylates and thereby activates p38MAPK, a second phase is initiated in which LDL activates PECAM-1 and induces dephosphorylation of p38MAPK via activation of the Ser/Thr phosphatases PP1/PP2A