The impact of low density lipoprotein oxidation on platelet responsiveness

Contact between native low-density lipoprotein (nLDL) and platelets enhances platelet responsiveness to various aggregation-inducing agents. Confocal microscopy reveals a high degree of co-localization of nLDL and a splice variant of the LDL-receptor family member apolipoprotein E Receptor-2 (apoER2’) at the platelet surface. nLDL induces tyrosine phosphorylation of apoER2’. The activation of apoER2’ is transient reaching its maximum after 1 minute. These findings suggest that apoER2’ may contribute to nLDL-induced platelet signaling. Indeed, (i) nLDL binds efficiently to soluble apoER2’, (ii) nLDL is unable to induce activation of the enzyme p38MAPK in apoER2’-deficient platelets, and (iii) nLDL-induced platelet activation is blocked by RAP, an inhibitor of LDL receptor family members. Activated apoER2’ initiates the transient phosphorylation of p38MAPK. The deactivation of p38MAPK is caused by PECAM-1, which is also activated by LDL, albeit later. For this action, PECAM-1 recruits the Ser/Thr phosphatase PP2A. A second mechanism for down-regulation of p38MAPK involves the Tyr phosphatases SHP-1 and SHP-2, which are activated by LDL in a PECAM-1-dependent manner, and co-precipitate with phosphorylated p38MAPK. Following a first stimulation with LDL, a second dose added 30 min later again activates apoER2’, but p38MAPK activation is absent. P38MAPK blockade is not caused by PECAM-1 or PP2A, which also remain in an inactive state. In contrast, SHP-1 and SHP-2 are persistently active thereby preventing the activation of PECAM-1 and PP2A by LDL, and the activation of p38MAPK by the activated apoER2’ receptor. These data reveal two mechanisms for down-regulation of LDL-induced p38MAPK phosphorylation: (i) the activation of PECAM-1 and the subsequent recruitment and activation of the Ser/Thr phosphatase PP2A, (ii) the activation of the Tyr phosphatases SHP- and SHP-2. These mechanisms reflect an important negative feedback inhibition of LDL-induced platelet sensitization, which prevents extensive platelet activation in the circulation by LDL. Mild oxidation of nLDL is associated with the generation of lysophosphatidic acid (LPA), which induces Ca2+ mobilization and platelet aggregation. Hence, the platelet-activating properties of nLDL might be caused by LPA, generated during nLDL isolation. However, nLDL enhances TRAP-induced fibrinogen binding to αIIbβ3 in an LPA-independent manner. To investigate how oxidation changes the platelet-sensitizing property of nLDL, nLDL was oxidized by FeSO4 to obtain different oxLDL preparations ranging from 1 to >60 % oxidation. Like nLDL, oxLDL signals via apoER2’ but in addition initiates a second pathway that further activates p38MAPK. In contrast with signaling via apoER2’, the second pathway does not involve an LDL receptor family since it is insensitive to RAP and is not dependent on proteoglycans. The separate addition of antibodies against the scavenger receptors CD36 and SR-A failed to block p38MAPK activation by oxLDL. In contrast, combined blockade of CD36 and SR-A resulted in >95% inhibition. Platelets from mice deficient in either CD36 or SR-A showed normal oxLDL-induced signaling to p38MAPK. However, a deficiency in SR-A in combination with an antibody against CD36, and vice versa, disrupted signaling to p38MAPK by oxLDL. These findings reveal a novel platelet signal transduction pathway initiated by oxLDL, which involves both CD36 and SR-A. An increase in LDL oxidation enhances platelet activation via two independent pathways, one signaling via p38MAPK phosphorylation and one via Ca2+ mobilization. Below 15% oxidation, the p38MAPK-route enhances fibrinogen binding induced by TRAP and signaling via CA2+ is absent. Above 30% oxidation, p38MAPK-signaling increases further and is accompanied by Ca2+ mobilization and platelet aggregation in the absence of a second agonist. Despite the increase in p38MAPK-signaling, synergism with TRAP disappears and oxLDL becomes an inhibitor of fibrinogen binding. Inhibition is due to binding of oxLDL to the scavenger receptor CD36, which is associated with the fibrinogen receptor, αIIbβ3 thereby inhibiting fibrinogen binding to αIIbβ3 and aggregation. An antibody that blocks oxLDL binding to CD36 rescues aggregation. Thus, above 30% oxidation, LDL interferes with ligand binding to integrin αIIbβ3 thereby attenuating platelet functions