To date, immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been shown to mediate inhibitory properties. We report a novel triggering receptor expressed on myeloid cells (TREM) family member, TREM-like transcript-1 (TLT1), which differs from the activating members because its cytoplasmic tail contains two ITIMs at Y245 and Y281. A TLT1 splice variant (TLT1sp) encodes a different cytoplasmic tail lacking ITIMs. Both isoforms are expressed in resting platelet α-granules, which are up-regulated to the cell surface following activation. TLT1 recruited Src homology 2 domain-containing tyrosine phosphatase (SHP)-2 to the “classical” ITIM (Y281) but not the “nonclassical” ITIM (Y245). In contrast to previously characterized ITIM receptors, TLT1 enhanced, rather than inhibited, FcεRI-mediated calcium signaling in rat basophilic leukemia cells, a property dependent on the SHP-2 recruiting classical Y281 ITIM. Therefore, TLT1 represents a new costimulatory ITIM immunoreceptor and is the second ITIM-bearing receptor to be identified in platelets after platelet endothelial cell adhesion molecule-1.

SummaryLow Density Lipoprotein (LDL) is known to sensitize platelets for physiological agonists. To clarify the basis of this sensitization, we investigated the involvement of p38MAP Kinase (p38MAPK). As dual phosphorylation on Thr180 and Tyr182 of p38MAPK is the trigger for activation of the kinase, p38MAPK-activity was measured with an antibody that recognizes the dual-phosphorylated sequence. LDL induced a rapid and dose dependent activation of p38MAPK. The activation was not inhibited by a wide variety of inhibitors of platelet signalling, including TxA2-formation, Phospholipase C-activation, Ca2+-mobilization and ERK 1/2-activation. Only a slight reduction in p38MAPK-activation was observed when protein kinase C was inhibited. Activation of p38MAPK was strongly inhibited by a rise in cAMP. Thus, p38MAPK-activation was upstream of most signalling pathways and close to the LDL-receptor. A number of platelet receptors was screened with the use of antibodies. Integrins αIIbβ3 and α2β1, as well as the FcγRII-receptor, CD36 (platelet glycoprotein IV), CD68 (gp110) and Low Density Lipoprotein-receptor related protein (LRP) were not implicated in LDL-induced p38MAPK-activation. Inhibition of LDL binding by modification of apo B100 lysines reduced p38MAPK-activation by 80 %. Activation of p38MAPK resulted in an increase in release of arachidonic acid, the precursor for thromboxane A2 synthesis. In conclusion, activation of p38MAPK might be the first step in platelet sensitization by LDL, leading to formation of arachidonate metabolites and increased aggregation and secretion responses to physiological agonists.

Events leading to hyperactivity of human blood platelets are accompanied by an enhanced risk of atherosclerosis and arterial thrombosis. Lipoprotein disorders affect platelet functions, and hypersensitive platelets are observed in various stages of hyperlipidemia. Low-density lipoprotein (LDL), a circulating complex of lipids and proteins that is increased in hypercholesterolemia, enhances platelet function and increases sensitivity of platelets to several naturally occurring agonists. LDL sensitizes platelets via binding of apoB-100 to a receptor on the platelet membrane and via transfer of lipids to the platelet membrane. The receptor that mediates binding of LDL to the platelet and initiates subsequent intracellular signaling cascades has not yet been identified. Modification of native LDL generates a platelet-activating particle, and this interaction might contribute to the development of the atherosclerotic plaque. Lysophosphatidic acid is formed upon mild oxidation of LDL and is responsible for subsequent platelet activation induced by the modified LDL particle. Thus, LDL changes the functions of platelets via a broad spectrum of interactions.

Binding of low density lipoprotein (LDL) to platelets enhances platelet responsiveness to various aggregation-inducing agents. However, the identity of the platelet surface receptor for LDL is unknown. We have previously reported that binding of the LDL component apolipoprotein B100 to platelets induces rapid phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK ). Here, we show that LDL-dependent activation of this kinase is inhibited by receptor-associated protein (RAP), an inhibitor of members of the LDL receptor family. Confocal microscopy revealed a high degree of co-localization of LDL and a splice variant of the LDL receptor family member apolipoprotein E receptor-2 (apoER2) at the platelet surface, suggesting that apoER2 may contribute to LDL-induced platelet signaling. Indeed, LDL was unable to induce p38 MAPK activation in platelets of apoER2-deficient mice. Furthermore, LDL bound efficiently to soluble apoER2, and the transient LDL-induced activation of p38 MAPK was mimicked by an anti-apoER2 antibody. Association of LDL to platelets resulted in tyrosine phosphorylation of apoER2, a process that was inhibited in the presence of PP1, an inhibitor of Src-like tyrosine kinases. Moreover, phosphorylated but not native apoER2 co-precipitated with the Src family member Fgr. This suggests that exposure of platelets to LDL induces association of apoER2 to Fgr, a kinase that is able to activate p38 MAPK . In conclusion, our data indicate that apoER2 contributes to LDL-dependent sensitization of platelets. Platelets and low density lipoproteins (LDL)1 are key elements in the development of atherothrombotic complications.The interplay between both elements is apparent from the notion that LDL particles markedly enhance the responsiveness of platelets to various aggregation-inducing agents (1-4). These agonists mediate the release of growth factors, vasoactive substances, and chemotactic agents that are known to stimulate atherosclerotic plaque formation. Sensitization of platelets by LDL involves the major LDL constituent apolipoprotein B100 (apoB100) (5), a 4563 amino acid protein that is wrapped around the LDL particle (6). LDL particles are recognized by the classical hepatic LDL receptor (LDL-R) through the apoB100 moiety, and in particular through a region within the apoB100 protein that is enriched in positively charged amino acids, the so-called B-site (7). Like LDL, a synthetic peptide mimicking this B-site associates to the platelet surface (5). Moreover, this peptide interferes with binding of LDL to platelets (5), suggesting that both elements share similar binding sites. This possibility is supported by the observation that binding of either LDL or the B-site peptide to the platelet results in a near immediate activation of the intracellular enzyme p38 mitogen-activated protein kinase (p38 MAPK ) (5, 8). Activation of this Ser/Thr kinase is associated with downstream phosphorylation and activation of cytosolic phospholipase A 2 , which leads to the formation of thromboxane A 2 (9, 10). Fi...