Platelets and their interaction with cells of the immune system contribute through a variety of molecular mechanisms to support hemostasis and inflammation. These simple yet essential cells exert their effects in lymphocytes, monocytes, and neutrophils, both recruiting and modulating their function after activation. Emerging evidence is starting to define the mechanisms that allow platelets to also play pivotal roles in host defense. For example, platelet cell-surface expression of toll-like receptors allows platelets to direct neutrophil activation toward extracellular trap formation and facilitate the elimination of blood pathogens. In addition to these well-known receptors, two of the most recently discovered platelet receptors, C-type lectin receptor 2 (CLEC-2), and TREM-like transcript-1 (TLT-1), have been shown to modulate hemostatic and inflammation-related roles in platelets. This review will discuss the evolution of our understanding of platelet functions from hemostasis to inflammation, and highlight novel mechanisms that platelets use to mediate hemostasis under inflammatory pressure.
Platelet activation at sites of inflammation triggers the secretion of molecules that induce the transition of atherosclerosis from fatty streak to an acute disease, featuring an increased vulnerability of the atherosclerotic lesion that results in plaque rupture and thrombosis. TLT-1 (Triggering Receptor Expressed in Myeloid cells (TREM)-like transcript-1) is a molecule exclusively found in the α-granules of megakarocytes and platelets and has a demonstrated effect in inflammatory responses. Upon platelet activation, TLT-1 is moved to the platelet surface, while its soluble form, s-TLT-1, is secreted and detected in serum. Studies using the C57Bl/6 treml1 - /- mouse demonstrated a predisposition to hemorrhage after an acute inflammatory challenge suggesting that TLT-1 may be a key regulatory molecule in the interface between hemostatic and inflammatory mechanisms. Because we have found that sTLT-1 levels are significantly elevated in apoE mice when compared to wild type, we hypothesized that TLT-1 may be playing an important role in the progression of atherosclerosis. To address this possibility, we generated apoE - /- / treml1 - /- double knockout mice [DN]. Assessment of lesions after 4 weeks high-fat diet (HFD) demonstrated that at early stages, TLT-1 deficiency accelerates fatty streak formation. After 20 weeks on HFD, lesions in both apoE - /- and [DN] mice progressed to an advance fibrous plaque stage. Although their lesion sizes were not substantially different, lesion compositions were. The mechanistic basis of these differences appears to be that the [DN] mice have significantly higher cholesterol levels when compared to apoE - /- mice. The increased cholesterol levels extend to the treml1 -/- mouse when compared to wild type mice at 4 weeks on HFD, this difference, however, gradually subsides as wild type mice cholesterol levels increase over 20 weeks. Interestingly, cholesterol levels in 50 week old mice on chow diet revealed minimal differences between test and control mice suggesting the higher cholesterol levels are related to increased dietary intake. Our work defines a surprising role for TLT-1 in the regulation of serum cholesterol levels during atherogenesis.
While it is known that high blood cholesterol levels are associated with increased risk and poor outcomes during cardiovascular disease (CVD), all of the mechanisms that regulate cholesterol levels remain unclear. Recent studies on the Triggering Receptor Expressed in Myeloid (TREM) like transcript (TLT)-1 null mice demonstrate changes in cholesterol regulation. TLT-1 was identified as a platelet specific immunoreceptor prompting our studies on TLT-1’s potential involvement in CVD. We crossed our treml1-/- mice on to the apoe-/- background and placed them on high fat diet (HFD). To our surprise, the treml1-/- /apoe-/- (DKO) mice had total cholesterol levels 1.5 fold higher compared to apoE controls. These changes can be seen in treml1-/- mice that are not on the apoE background as well. There are no cholesterol differences in mice not on HFD, demonstrating that these changes are diet-induced. These differences led us to investigate, “why the treml1-/- mouse has higher cholesterol?” Serum chemistry panels reveal high triglycerides, increased glucose levels, and potential liver damage in the treml1-/- mice. Histological evaluation of liver sections revealed large lipid-filled vacuoles and ballooning of DKO and treml1-/- hepatocytes suggesting direct liver involvement in regulating the aforementioned cholesterol levels. What brings these findings together is that we have identified an alternate transcript of TLT-1, TLT-1s, associated with mitochondrial fractions of hepatocytes. Immunohistochemistry of liver sections demonstrate a unique TLT-1s expression in the perivascular hepatocytes of the central vein. Preliminary metabolic analysis of mice livers confirm the hyperlipidemia and demonstrate significant changes in protein metabolism as well as Krebs cycle intermediates. Interestingly, treml1-/- mice have smaller lesions in the aortic sinus which may be explained by less reactive treml1-/- platelets (Gonzalez et al.). Our data suggests that treml1 gene products may affect energy metabolism and mutations in TLT-1 may affect energy use in platelets. This uncanny hypothesis potentially explains how we could have high cholesterol levels, with smaller atherosclerotic lesions and less reactive platelets in the face of hyperlipidemia.
Platelets have a well documented role in atherosclerotic lesion formation. They play a role from the initiation of the lesion to final occlusion of an atherosclerotic vessel. While it is known that regulating aggregation is important to control the thrombus that leads to occlusion, the platelet’s role in promoting lesion formation is less defined. Molecules stored in the platelet a-granules such as p-selectin and cytokines, which promote the inflammatory side of platelet function, have been shown to affect lesion progression. These molecules, however, only provide an outline of the platelet function during this process. and thus our understanding of how platelets participate in the progression of atherosclerosis remains a gap in our knowledge. The Triggering Receptor Expressed Myeloid cells (TREM)-like transcript (TLT)-1 is a receptor found in the α-granules of both megakaryocytes and platelets. Antibodies to TLT-1 have been shown to inhibit platelet aggregation and accordingly studies using the treml1 -/- mouse have demonstrated that TLT-1 null mice have reduced platelet aggregation and extended tail-bleeding times. Subsequent studies using the treml1 -/- mouse demonstrated a predisposition to hemorrhage derived from an inflammatory challenge suggesting that TLT-1 may be a key regulatory molecule in the interface between hemostatic and inflammatory mechanisms. Additionally, apoE null mice have significantly higher levels of the soluble form of TLT-1 (sTLT-1) in their plasma compared to wild type mice suggesting that sTLT-1 may play a role in the progression of atherosclerotic lesions. Therefore, we hypothesized that removal of TLT-1 receptor will delay the formation of atherosclerotic lesions in an apoE deficient genetic background. To address our hypothesis, we have created an apoE/treml1 double null mouse and investigated the development of lesions in the aortic sinus. Our preliminary data show an unexpected trend where the lesions in apoE/treml1 double null mice have exacerbated compared to apoE or treml1 -/- mice suggesting that TLT-1 may actually have a anti-inflammatory effect during the progression of atherosclerosis. The status of the current research will be presented here.
Atherosclerosis, the hardening and narrowing of the arteries, is a chronic inflammatory disease driven by a crosstalk of signaling molecules between circulating immune cells and the vessel wall. In the classic view of this cardiovascular disease, hyperlipidemia initiates an inflammatory cascade that promotes macrophage infiltration into the vessel wall leading to a feedback mechanism which exacerbates lesion growth and ultimately, atherothrombosis. Our laboratory has characterized a novel atherosclerotic mice model, in which deletion of the platelet receptor Triggering Receptor Expressed on Myeloid Cells (TREM)-Like Transcript-1 (TLT-1) on a apolipoprotein E ( apoe -/- /treml1 -/- ) background dampens early macrophage infiltration into the vessel wall. This leads to decreased lesion size, instability, and calcification of atherosclerotic lesions. We have demonstrated that one of the mechanisms for dampened lesion progression is decreased platelet activation and we hypothesize that the macrophage infiltration differences may also be due to fundamental differences in macrophage phenotypic functions. In order to further define the mechanism by which TLT-1 modulates macrophage infiltration, we analyzed macrophage function in vitro . Analysis of bone marrow-derived macrophages (BMDM) from apoe -/- /treml1 -/- and apoe -/- /treml1 -/+ mice revealed no significant differences in the phagocytic function of long chain fatty acids. However, analysis of basal levels of monocyte chemoattractant protein-1 (MCP1), a key chemokine regulating monocyte infiltration, were significantly decreased in the apoe -/- /treml1 -/- BMDM supernatants. Accordingly, levels of Interleukin 6 (IL6) and Interleukin 10 (IL10) were significantly decreased in the apoe -/- /treml1 -/- BMDM supernatants after two hours of lipopolysaccharide (LPS) stimulation (p<0.05; n=5-8). These findings define an underlying difference in the BMDM secretory phenotype of the apoe -/- /treml1 -/- mice. However, it also opens the possibility of treml1 expression in macrophages, which may account for these differences. We will now investigate if an alternate isoform of treml1 is expressed in macrophages or at some period of their differentiation.
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