TREM-like Transcript-1 Research Articles

Emerging Roles for Platelets in Inflammation and Disease

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.

TLT-1s, Alternative Transcripts of Triggering Receptor Expressed on Myeloid Cell-like Transcript-1 (TLT-1), Inhibits the Triggering Receptor Expressed on Myeloid Cell-2 (TREM-2)-mediated Signaling Pathway during Osteoclastogenesis

Triggering receptor expressed on myeloid cells (TREM)-like transcript-1 (TLT-1) is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-baring TREM family protein. In this study, we identified an alternative transcript form of TLT-1, namely TLT-1s, which has very short extracellular immunoglobulin domain consisting of only 202 amino acids. TLT-1s was mainly expressed in macrophages and osteoclast precursor cells. Upon receptor activator of nuclear factor-κB ligand stimulation, TLT-1s mRNA and protein levels were gradually decreased in BMMs. We also showed the TLT-1s is localized to the cytoplasmic membrane in osteoclast precursor cells. TLT-1s silencing strongly enhanced the formation and resorption activity of osteoclast. In addition, forced expression of TLT-1s showed reduced formation of osteoclast. Because ITIM-baring proteins inhibit immunoreceptor tyrosine-based activation motif (ITAM)-mediated receptor signaling, we tested whether TLT-1s physically interacted with TREM-2, the ITAM-associated co-stimulatory receptor essential for osteoclast differentiation. We showed that TLT-1s is associated with TREM-2 in osteoclast precursor cells. TLT-1s is also associated with tyrosine Src homology 2 domain-containing phosphatase-1 and SH2 domain-containing inositol phosphatase-1 and recruited them to the TREM2-ITAM signaling complex. In addition, knockdown of TLT-1s markedly elevated the intracellular calcium concentration and oscillation in osteoclast precursor cells. In addition, calcium-mediated induction of nuclear factor of activated T cells was also increased by TLT-1s silencing. Furthermore, TREM-2-mediated Akt activation and proliferation of osteoclast precursor cells were also enhanced in TLT-1s silenced cells. In this paper, we found the noble ITIM-baring inhibitory membrane protein; TLT-1s, which regulates ITAM-mediated signaling on osteoclastogenesis.

Soluble Trem-like Transcript-1 Regulates Leukocyte Activation and Controls Microbial Sepsis

The triggering receptor expressed on myeloid cells (TREM)-1 plays a crucial role during the onset of sepsis by amplifying the host immune response. The TREM-like transcript-1 (TLT-1) belongs to the TREM family, is selectively expressed on activated platelets, and is known to facilitate platelet aggregation through binding to fibrinogen. In this study, we show that a soluble form of TLT-1 is implicated in the regulation of inflammation during sepsis by dampening leukocyte activation and modulating platelet-neutrophil crosstalk. A 17-aa sequence of the TLT-1 extracellular domain (LR17) is responsible for this activity through competition with the TREM-1 ligand. Whereas early or late LR17 treatment of septic mice improves survival, treml-1(-/-) animals are highly susceptible to polymicrobial infection. The present findings identify platelet-derived soluble TLT-1 as a potent endogenous regulator of sepsis-associated inflammation and open new therapeutic perspectives. We anticipate soluble TLT-1 to be important in regulating leukocyte activation during other noninfectious inflammatory disorders.

TREM-like transcript-1: at the frontier between haemostasis and inflammation

TLT-1 ( TREM-like transcript-1) est un nouveau recepteur plaquettaire recemment caracterise et jouant un role important dans la biologie de la plaquette. TLT-1 appartient a la famille TREM ( triggering receptors expressed on myeloid cells), composee d’immunorecepteurs exprimes par les cellules de l’immunite innee. TLT-1 est exclusivement exprime par les megacaryocytes et les plaquettes, inclus dans les granules α et rapidement adresse a la membrane plasmique lors de l’activation plaquettaire. De par ses capacites a lier le fibrinogene et a moduler les flux calciques intraplaquettaires, mais egalement a moduler la reponse inflammatoire, TLT-1 pourrait jouer un role important dans le maintien de l’homeostasie vasculaire en regulant l’agregation et l’inflammation localement en cas de dommages tissulaires ou d’infection. Une meilleure comprehension du role et de la regulation de TLT-1 et surtout la capacite de moduler son activite permettront tres certainement le developpement de nouvelles approches therapeutiques.

Proteomic analysis of palmitoylated platelet proteins

AbstractProtein palmitoylation is a dynamic process that regulates membrane targeting of proteins and protein-protein interactions. We have previously demonstrated a critical role for protein palmitoylation in platelet activation and have identified palmitoylation machinery in platelets. Using a novel proteomic approach, Palmitoyl Protein Identification and Site Characterization, we have begun to characterize the human platelet palmitoylome. Palmitoylated proteins were enriched from membranes isolated from resting platelets using acyl-biotinyl exchange chemistry, followed by identification using liquid chromatography-tandem mass spectrometry. This global analysis identified > 1300 proteins, of which 215 met criteria for significance and represent the platelet palmitoylome. This collection includes 51 known palmitoylated proteins, 61 putative palmitoylated proteins identified in other palmitoylation-specific proteomic studies, and 103 new putative palmitoylated proteins. Of these candidates, we chose to validate the palmitoylation of triggering receptors expressed on myeloid cell (TREM)–like transcript-1 (TLT-1) as its expression is restricted to platelets and megakaryocytes. We determined that TLT-1 is a palmitoylated protein using metabolic labeling with [3H]palmitate and identified the site of TLT-1 palmitoylation as cysteine 196. The discovery of new platelet palmitoyl protein candidates will provide a resource for subsequent investigations to validate the palmitoylation of these proteins and to determine the role palmitoylation plays in their function.

Proteomic Analysis of Palmitoylated Platelet Proteins

AbstractAbstract 2017Protein palmitoylation is a dynamic process that regulates membrane targeting of proteins and protein-protein interactions. It is unique among the fatty acid modifications as it is reversible, and its reversibility suggests that it can participate in the regulation of cell signaling. We have previously demonstrated a critical role for protein palmitoylation in platelet activation and have begun to characterize the palmitoylation machinery in platelets. We have now employed a novel proteomic approach termed Palmitoyl Protein Identification and Site Characterization (PalmPISC) to define the platelet “palmitoylome.” Using acyl biotin exchange (ABE) chemistry, we have purified palmitoylated proteins from membranes of resting platelets and identified them using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Spectral counting analysis identified 131 putative palmitoylated proteins including 58 novel palmitoylated proteins. Components of the G protein signal transduction pathways (15% of palmitoylated proteins) and membrane fusion proteins (10% of palmitoylated proteins) were highly represented. Platelets undergo a dramatic phenotypic change upon activation and platelet proteins are known to undergo activation-dependent palmitoylation. Changes in the palmitoylation state of proteins during platelet signaling may be reflective of the activation process. We have compared changes in protein palmitoylation in resting and thrombin-activated platelets to identify proteins that undergo activation-dependent palmitoylation or depalmitoylation. To quantify these changes by mass spectrometry, we employed iTRAQ labeling and identified 32 proteins that increase or decrease their palmitoylation upon activation. We have focused our initial efforts on one of these proteins, Triggering Receptor Expressed on Myloid cells (TREM)-like transcript-1 (TLT-1), an immunoglobulin domain-containing receptor expressed exclusively in platelets and megakaryocytes. We have validated that platelet TLT-1 is palmitoylated using [3H]palmitate labeling and have identified the site of TLT-1 palmitoylation as juxtamembrane Cys196, which is adjacent to an ITIM domain. Our iTRAQ results reveal that TLT-1 exhibits a 2-fold decrease in palmitoylation upon activation. A decrease in TLT-1 palmitoylation upon Par1-mediated activation was confirmed using an ABE strategy, which detects total protein palmitoylation. In contrast, there is a 2.5-fold increase in [3H]palmitate labeling of TLT-1 upon activation of platelets, indicating increased turnover of palmitate with activation. These observations suggest that activation-dependent depalmitoylation of TLT-1 occurs more rapidly than activation-dependent palmitoylation and underscores the importance of measuring both total palmitoylation and palmitate turnover in assessing activation-dependent palmitoylation. This global analysis of platelet protein palmitoylation provides a platform to inform future investigations identifying the role of palmitoylation in the function of specific platelet proteins. Identification of proteins that undergo activation-dependent palmitoylation or depalmitoylation will enable studies of the platelet protein palmitoylation machinery. Disclosures:No relevant conflicts of interest to declare.

Soluble TLT-1 modulates platelet–endothelial cell interactions and actin polymerization

Triggering receptor expressed on myeloid cells (TREM) like transcript-1 (TLT-1) is a membrane protein receptor found in alpha-granules of platelets and megakaryocytes. Upon platelet activation TLT-1 is rapidly brought to the surface of platelets. Recently, we demonstrated that activated platelets release a soluble form of TLT-1 (sTLT-1) that is found in serum but not in the plasma of healthy individuals and can enhance platelet aggregation in vitro. Furthermore, evaluation of patients diagnosed with inflammatory diseases, such as sepsis, show that these patients have significantly elevated levels of sTLT-1 in their blood. Accordingly, mice deficient in TLT-1 are predisposed to bleeding in response to an inflammatory challenge; however, the mechanism of TLT-1 function remains unknown. In this investigation, we demonstrate an increase in the amount of platelets that adhere to endothelial cell monolayers in the presence of recombinant sTLT-1 (rsTLT-1). Additionally, we present evidence that rsTLT-1 increases platelet adherence to glass slides by stimulating actin polymerization in platelets, as determined by increased staining of rodamine phalloidin. These results suggest that during inflammation, sTLT-1 may mediate hemostasis by enhancing actin polymerization, resulting in increased platelet aggregation and adherence to the endothelium.

Soluble TLT-1 is a naturally occurring TREM-1 inhibitor and protects mice from hyperresponsiveness and death during sepsis

Triggering receptor expressed on myeloid cells-1 (TREM-1) and TREM-like transcript 1 (TLT-1) belong to the TREM family. TREM-1 is expressed on neutrophils and monocytes/macrophages, and plays a crucial role during the onset of sepsis by cooperating with pattern recognition receptors in a synergistic way, thus amplifying the host immune response. TLT-1 is selectively expressed on activated platelets and is known to facilitate platelet aggregation through binding to fibrinogen. Interestingly, TLT-1 null mice displayed higher plasma cytokines concentrations and death rates than WT mice during experimental sepsis. We identified a 17 amino acid peptide derived from the extracellular part of TLT-1, named LR17, which is responsible for TLT-1 anti-inflammatory properties.

TREM-like transcript-1 protects against inflammation-associated hemorrhage by facilitating platelet aggregation in mice and humans

Triggering receptor expressed on myeloid cells-like (TREM-like) transcript-1 (TLT-1), a type 1 single Ig domain orphan receptor specific to platelet and megakaryocyte alpha-granules, relocates to the platelet surface upon platelet stimulation. We found here that patients diagnosed with sepsis, in contrast to healthy individuals, had substantial levels of soluble TLT-1 (sTLT-1) in their plasma that correlated with the presence of disseminated intravascular coagulation. sTLT-1 bound to fibrinogen and augmented platelet aggregation in vitro. Furthermore, the cytoplasmic domain of TLT-1 could also bind ezrin/radixin/moesin family proteins, suggesting its ability to link fibrinogen to the platelet cytoskeleton. Accordingly, platelets of Treml1-/- mice failed to aggregate efficiently, extending tail-bleeding times. Lipopolysaccharide-treated Treml1-/- mice developed higher plasma levels of TNF and D-dimers than wild-type mice and were more likely to succumb during challenge. Finally, Treml1-/- mice were predisposed to hemorrhage associated with localized inflammatory lesions. Taken together, our findings suggest that TLT-1 plays a protective role during inflammation by dampening the inflammatory response and facilitating platelet aggregation at sites of vascular injury. Therefore, therapeutic modulation of TLT-1-mediated effects may provide clinical benefit to patients with hypercoagulatory conditions, including those associated with inflammation.

TLT-1 (TREM-Like transcript-1) Protects against Hemorrhage Associated with Inflammation by Facilitating Platelet Aggregation.

TREM-like transcript-1 (TLT-1) is a novel, platelet-specific, membrane protein that is stored in a-granules and brought to the platelet surface upon activation. Little is known about its function. Here we build upon our recent findings that a soluble fragment of TLT-1 (sTLT) is released upon platelet activation and can be detected in the serum, but not the plasma, of healthy individuals. Evaluation of patients admitted to the University Hospital, Ramon Ruiz Arnau, in Bayamon Puerto Rico with the diagnosis of sepsis for the presence of plasma sTLT demonstrated significant levels of sTLT in septic individuals as compared to controls, indicating that TLT-1 plays a role in the early stages of sepsis. To directly assess this possibility we generated TLT-1 knockout mice and characterized their hemostatic function. Compared to wild type mice, TLT-1−/− platelets displayed decreased aggregation when treated with thrombin, collagen, ADP, or U46619 in vitro. The reduced platelet aggregation of TLT-1−/− mice translates to a 30% increase in bleeding time in tail snip assays compared to controls, supporting TLT-1's role in vascular homeostasis. To evaluate TLT-1's role in sepsis we challenged mice with lipopolysaccharide (LPS). LPS challenged TLT-1 null mice die faster and have a significantly lower rate of survival than wild type mice. We used the localized Shwartzman reaction to model the pathology associated with sepsis in a controlled lesion. Although neutrophil infiltration, thrombus formation, and the formation of microclots was not significantly higher in TLT-1−/− mice, the hemorrhage associated with Shwartzman lesions of these mice was nearly double and lesion size was greater than three times that of wild type mice. Moreover, we demonstrate a recombinant sTLT augments platelet aggregation in vitro suggesting a mechanistic explanation for the increased hemorrhage in TLT-1−/− mice. Thus these studies reveal an important role for TLT-1 in the maintenance of vascular hemostasis by facilitating platelet aggregation through incorporation of sTLT into the forming clot. This work suggests that therapeutic intervention using tools such as anti-TLT-1 single chain antibodies that inhibit platelet aggregation may provide a novel approach to the control of the thrombotic response.

Phenotypic heterogeneity in the gray platelet syndrome extends to the expression of TREM family member, TLT-1

The Gray platelet syndrome (GPS) is a rare inherited disorder linked to undefined molecular abnormalities that prevent the formation and maturation of alpha-granules. Here, we report studies on two patients from unrelated families that confirm phenotypic heterogeneity in the disease. First we used immunoelectron microscopy (I-EM) to confirm that TREM-like transcript-1 (TLT-1) is mostly localized to alpha-granule membranes of normal platelets. Then we performed Western blotting (WB) and flow cytometry with permeabilized platelets to show that TLT-1 is selectively reduced in the platelets of patient 1, previously noted to be deficient in glycoprotein (GP)VI (Nurden et al., Blood 2004; 104: 107-114). Yet both TLT-1 and GPVI were normally expressed in platelets of patient 2. Usual levels of JAM-C and claudin-5, also members of the immunoglobulin receptor family, were detected in platelets of both patients. In contrast, P-selectin was markedly decreased for patient 1 but not patient 2. Two metalloproteases, MMP-2 and MMP-9 were normally present. As predicted, platelets of patient 1 showed little labelling for TLT-1 in I-EM, whereas residual Fg was seen in small vesicular structures and P-selectin lining vacuoles or channels of what may be elements of the surface-connected canalicular system. Our results identify TLT-1 as a glycoprotein potentially targeted in platelets of GPS patients, while decreases in at least three membrane glycoproteins suggest that an unidentified proteolytic activity may contribute to the phenotype in some patients with this rare disease.

Inhibition of thrombin-induced platelet aggregation using human single-chain Fv antibodies specific for TREM-like transcript-1

TREM-like transcript-1 (TLT-1) is a novel platelet membrane receptor, which has been recently characterized in mice. TLT-1 is expressed exclusively in platelets and megakaryocytes, and its expression is dramatically upregulated upon platelet activation, suggesting that it plays a unique role in hemostasis and/or thrombosis. In this study we identified and characterized highly specific human monoclonal antibodies that bind to TLT-1 by screening a naïve library of single chain Fv fragments (scFvs) displayed on filamentous phage (Thomlinson I library). These scFvs detected plate-bound TLT-1, captured soluble TLT-1, and readily reacted with cell-bound TLT-1 on transfectants and primary human platelets. Most importantly, anti-TLT-1 scFvs inhibited thrombin-mediated human platelet aggregation. This inhibition was specific for thrombin-induced aggregation and was reversible with higher doses of agonist. These data are the first to demonstrate a biological role for TLT-1 and its potential as a therapeutic target. The human scFvs isolated in this study may represent novel anti-platelet therapeutic agents.

Inhibition of Thrombin-Induced Platelet Aggregation Using Human Single Chain Fv Antibodies Specific for TREM-Like Transcript-1.

Platelets play a duel role in our cardiovascular system. They are important in maintaining our vascular integrity. They also facilitate the progression of cardiovascular diseases (CVD) such as arteriosclerosis. In more severe cases, displaced platelet aggregates can lead to myocardial infarction or stroke. Because of their prominent role, anti-platelet therapies are used to reduce mortality associated with CVD. However, the use of these therapies is sometimes associated with bleeding complications, thrombocytopenia or non-responsiveness and the search for new therapeutic targets continues. Some of the recent treatment strategies include molecules that target platelet membrane receptors. The success shown by this class of therapeutics has prompted us to evaluate the recently characterized murine platelet receptor, TREM like transcript 1 (TLT-1), as a therapeutic target. Murine studies suggest that TLT-1 is a platelet specific protein found in α-granules of platelets and megakaryocytes. Here we asked if human TLT-1 was also linage specific and if platelet aggregation could be modulated through interaction with TLT-1 specific antibodies. We evaluated human TLT-1 (hTLT-1) for pattern of expression in the peripheral blood and we found that hTLT-1, like its mouse counterpart, is platelet specific. To address TLT-1 function we panned a naïve human library of single chain fragments (scFvs) with a TLT-1 extracellular domain/human fc fusion protein to produce monoclonal antibodies specific for hTLT-1. We isolated six scFv clones using ELISA to detect reactivity to the fusion protein and confirmed their specificity by flow cytometry on cells transfected with TLT-1 and purified human platelets. These scFvs were then tested to see if they would disrupt or enhance platelet aggregation. Here we demonstrate, for the first time, inhibition of thrombin induced platelet activation using scFv that are specific for TLT-1. Taken together these results suggest TLT-1 is a potential target for therapeutic intervention of cardiovascular disease.

Heterogeneity in the Gray Platelet Syndrome: Variable Expression of the TREM Family Member, TLT-1, in Platelets of Patients of Two Unrelated Families.

The Gray platelet syndrome is a rare inherited disorder of platelets linked to undefined molecular abnormalities that prevent the normal formation and maturation of alpha-granules. This leads to the spontaneous release of endogenously synthesized alpha-granule proteins from megakaryocytes into the marrow, a factor favouring the development of fibrosis. The platelet functional response varies greatly in patients in the GPS. Here we examine patients from two unrelated families, patient 1 has platelets that have a much reduced aggregation response to collagen, and patient 2 has a normal response. Both patients possess platelets severely deficient in alpha-granules. Features of their internal stucture include numerous vacuoles, thin channels and a reduced surface-connected canalicular system (SCCS) and small vesicular structures (SVs) some of which are open to the surface and rapidly accessed by abciximab used as a probe for ligand trafficking in immunoelectron microscopy (I-EM). Residual amounts of fibrinogen, and VWF are often associated with the SVs, P-selectin delimits some elements of the SCCS and vacuoles while most internal and surface membranes are labelled for seven transmembrane receptors such as PAR-1 and TPalpha, Immunoblotting showed that unlike platelets of patient 1 where GPVI was severely decreased (Nurden P et al, Blood 2004; 104:107–114), those of patient 2 normally expressed this collagen receptor. TREM-like transcript-1 (TLT-1), a membrane glycoprotein, has recently been predominantly localized to the alpha-granules of human platelets on the basis of its codistribution with P-selectin. Using a rabbit antibody to a cytoplasmic domain sequence we have confirmed this distribution in normal platelets. Significantly, we then further showed by Western blotting (WB) that TLT-1, like GPVI, is severely reduced in the platelets of patient 1 but normally expressed in those of patient 2. Flow cytometry using permeabilized platelets showed that this decrease was selective compared to normal levels of JAM-3 and claudin-5, also members of the immunoglobulin family. I-EM showed residual TLT-1 associated with both internal and external membranes. One possibility is that a pathology-related metalloprotease activity (MMP) leads to spontaneous cleavage of the extracellular domains of GPVI and TLT-1 in the platelets of some patients. We therefore examined the platelets of patient 1 to see if MMP-2 and MMP-9, two proteases expressed in normal platelets, were present. I-EM first showed that while for normal platelets both proteases were in their majority localized to alpha-granules, other pools were present in the cytoplasmic and surface-linked membrane systems. The latter were retained in the platelets of patient 1 but flow cytometry with permeabilized platelets failed to show over-expression. Our results identify TLT-1 as a new glycoprotein potentially cleaved in GPS platelets and further show that so-far undefined protease activity contributes to phenotypic heterogeneity in this rare disease.

Trem-Like Transcript 2 Is Expressed on Cells of the Myeloid/Granuloid and B Lymphoid Lineage and Is Up-Regulated in Response to Inflammation

The triggering receptor expressed on myeloid cells (TREM) gene cluster encodes a group of transmembrane proteins that are emerging as important components in innate and adaptive immunity. In both mice and humans, the TREM gene cluster encodes eight receptors; only four of these, however, are direct homologs: TREM-1, TREM-2, TREM-like transcript 1 (TLT1), and TLT2. Of the transmembrane receptors encoded by the four conserved genes within this cluster, TLT2 has not been studied previously. Data presented in this study demonstrate that TLT2 is expressed early in B cell development in conjunction with B220 and is detected on all developing mouse B cell populations as well as B cells in the periphery. TLT2 expression on B cells in the periphery exhibits a distinct hierarchy with the highest detectable levels observed on B1 B cells in the peritoneum. The overall gradation of TLT2 expression on B cells is: B1 > marginal zone/transitional 2 > transitional 1 > follicular. Additionally, TLT2 expression was observed on mouse neutrophils throughout the body. Although monocytes were not observed to express TLT2, resident peritoneal and lung macrophages do express TLT2, suggesting that it is up-regulated in association with terminal differentiation of monocytes. Finally, both neutrophils and macrophages were observed to up-regulate TLT2 expression in vivo in response to inflammatory stimuli, whereas TLT2 expression on B cells remained unchanged. In conclusion, the data suggest that TLT2 may be involved in the innate immune response based on its expression profile and the fact that it is up-regulated in response to inflammation.

Preparation and Characterization of Monoclonal Antibody Against Protein TREM-Like Transcript-1 (TLT-1)

TREM-like transcript-1 (TLT-1) was initially characterized as the putative inhibitory receptor within the TREM locus on chromosome 6 and was suggested to play a critical role in modulating TREM-associated immunity. Recently, TLT-1 was found not to function as an inhibiting member of the TREM family; however, it may play a significant role in maintaining vascular hemostasis, coagulation, and inflammation at sites of injury through its location in platelets. In this study, we have successfully generated a monoclonal antibody that can be applied to assays, including enzyme-linked immunosorbent assay (ELISA), Western immunoblotting, immunofluorescence, and Fluorescence-Activated Cell Sorter (Becton-Dickinson) (FACS), for the study of TLT-1 functions in physiological and pathological conditions.

A TREM family member, TLT-1, is found exclusively in the α-granules of megakaryocytes and platelets

AbstractThe triggering receptors expressed on myeloid cells (TREMs) have drawn considerable attention due to their ability to activate multiple cell types within the innate immune system, including neutrophils, monocyte/macrophages, and dendritic cells, via their association with DAP12. TLT-1 (TREM-like transcript-1) lies within the TREM gene cluster and contains the characteristic single V-set immunoglobulin (Ig) domain of the family, but its longer cytoplasmic tail is composed of both a proline-rich region and an immune receptor tyrosine-based inhibitory motif, the latter known to be used for interactions with protein tyrosine phosphatases. Here we report that TLT-1 is expressed exclusively in platelets and megakaryocytes (MKs) and that TLT-1 expression is up-regulated dramatically upon platelet activation. Consistent with this observation, confocal microscopy demonstrates that TLT-1 is prepackaged, along with CD62P, into both MK and platelet α-granules. Differences in thrombin-induced redistribution of CD62P and TLT-1 indicate that TLT-1 is not simply cargo of α-granules but may instead regulate granule construction or dispersal. Together these data show that that TLT-1 does not function to inhibit members of the TREM family but instead may play a role in maintaining vascular hemostasis and regulating coagulation and inflammation at sites of injury.

Cutting edge: TREM-like transcript-1, a platelet immunoreceptor tyrosine-based inhibition motif encoding costimulatory immunoreceptor that enhances, rather than inhibits, calcium signaling via SHP-2.

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 alpha-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, FcepsilonRI-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.