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 α-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.

The three-dimensional crystal structure of an arginine kinase transition-state analogue complex has been re®ned at 1.2 A Ê resolution, with an overall R factor of 12.3%. The current model provides a unique opportunity to analyze the structure of a bimolecular (phosphagen kinase) enzyme in its transition state. This atomic resolution structure con®rms in-line transfer of the phosphoryl group and the catalytic importance of the precise alignment of the substrates. The structure is consistent with a concerted proton transfer that has been proposed for an unrelated kinase. Re®nement of anisotropic temperature factors and translation±libration±screw (TLS) analyses led to the identi®cation of four rigid groups and their prevalent modes of motion in the transition state. The relative magnitudes of the mobility of rigid groups are consistent with their proposed roles in catalysis.

No abstract

Arginine kinase is a member of the phosphagen kinase family that includes creatine kinase and likely shares a common reaction mechanism in catalyzing the buffering of cellular ATP energy levels. Abstraction of a proton from the substrate guanidinium by a catalytic base has long been thought to be an early mechanistic step. The structure of arginine kinase as a transition state analog complex (Zhou, G., Somasundaram, T., Blanc, E., Parthasarathy, G., Ellington, W. R., and Chapman, M. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8449 -8454) showed that Glu-225 and Glu-314 were the only potential catalytic residues contacting the phosphorylated nitrogen. In the present study, these residues were changed to Asp, Gln, and Val or Ala in several single and multisite mutant enzymes. These mutations had little impact on the substrate binding constants. The effect upon activity varied with reductions in k cat between 3000-fold and less than 2-fold. The retention of significant activity in some mutants contrasts with published studies of homologues and suggests that acid-base catalysis by these residues may enhance the rate but is not absolutely essential. Crystal structures of mutant enzymes E314D at 1.9 Å and E225Q at 2.8 Å resolution showed that the precise alignment of substrates is subtly distorted. Thus, pre-

Triggering receptor expressed on myeloid cells like transcript-1 (TLT-1) is an abundant platelet-specific, type I transmembrane receptor. The extracellular fragment of TLT-1 consists of a single, immunoglobulin-like domain connected to the platelet cell membrane by a linker region called the stalk. Here we present evidence that a soluble fragment of the TLT-1 extracellular domain is found in serum of humans and mice and that an isoform of similar mass is released from platelets following activation with thrombin. We also report the crystal structure of the immunoglobulin domain of TLT-1 determined at the resolution of 1.19 Å . The structure of TLT-1 is similar to other immunoglobulin-like variable domains, particularly those of triggering receptor expressed on myeloid cells-1 (TREM-1), the natural killer cell-activating receptor NKp44, and the polymeric immunoglobulin receptor. Particularly interesting is a 17-amino acid segment of TLT-1, homologous to a fragment of murine TREM-1, which, in turn, showed activity in blocking the TREM-1-mediated inflammatory responses in mice. Structural similarity to TREM-1 and polymeric immunoglobulin receptor, and evidence for a naturally occurring soluble fragment of the TLT-1 extracellular domain, suggest that this immunoglobulin-like domain autonomously plays an as yet unidentified, functional role.Triggering receptor expressed on myeloid cells like transcript-1 (TLT-1) 2 is a membrane-bound protein, abundant in the ␣-granules of resting platelets and on the surface of activated platelets (1). The gene that codes for TLT-1 is located in the TREM gene cluster on human chromosome 6, locus 6p21.1, and murine chromosome 17. Related genes in the human TREM gene cluster include TREMs 1 and 2, TLTs 1 through 5, and the natural killer cell-activating receptor NKp44 (2). In the murine genome, chromosome 17 includes genes encoding TREMs 1 through 5, and TLTs 1 and 2 (2). Gene products from the TREM gene cluster are expressed in specific populations of cells of the myeloid lineage. To date, only TREM-1, TREM-2, NKp44, and TLT-1 have been characterized.The immunoglobulin-like domain of human TLT-1 (hTLT-1) consists of 105 residues and is attached to the membrane by a 37-amino acid stalk. The putative transmembrane segment of hTLT-1 is 20 amino acids long and lacks the positively charged amino acid that other TREM family members use to interact with a negatively charged side chain of the adaptor molecule DAP12 (3). Inside the membrane-spanning region, DNAX activation protein 12 contains a signaling motif known as an immunoreceptor tyrosine-based activation motif (3). Two cDNA sequences, which code for proteins with identical extracellular and membrane-spanning regions but different cytoplasmic domains (2), have been reported for hTLT-1. The shorter transcript has a cytoplasmic domain of 18 amino acids with no recognizable signaling motifs, and the cytoplasmic domain of the longer hTLT-1 transcript consists of 127 amino acids, including two tyrosine residues. Both cytoplasmic tyrosines ar...