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.

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

The 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 alpha-granules. Differences in thrombin-induced redistribution of CD62P and TLT-1 indicate that TLT-1 is not simply cargo of alpha-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.

Anthrax toxin: structures, functions and tumour targeting.

Anthrax toxin, the major virulence factor of Bacillus anthracis, consists of three polypeptides: protective antigen (PrAg), lethal factor (LF) and oedema factor (EF). To intoxicate mammalian cells, PrAg binds to its cellular receptors and is subsequently activated via proteolysis, yielding a carboxyl-terminal fragment which coordinately assembles to form heptamers that bind and translocate LF and EF into the cytosol to exert their cytotoxic effects. Substantial progress has been made in recent years towards the characterisation of the structure and function of anthrax toxin, and this has greatly facilitated rational drug design of antianthrax agents. There is also emerging evidence that toxins can be manipulated for cancer therapy. LF can efficiently inactivate several mitogen-activated protein kinase kinases (MAPKKs) via cleavage of their amino-terminal sequences. Consequently, antitumour effects of wild type lethal toxin were observed after treatment of mitogen-activated protein kinase (MAPK)-dependent tumours such as human melanomas. Modification of the toxin's proteolytic activation site limits its cytotoxicity to certain cell types and creates a versatile method of treatment. One approach that has successfully achieved specific tumour targeting is the alteration of the furin cleavage of PrAg so that it is not activated by furin, but, alternatively, by proteases that are highly expressed by tumour tissues, including matrix metalloproteases and urokinase.

Specificity of cognate ligand-receptor interactions: fusion proteins of human chorionic gonadotropin and the heptahelical receptors for human luteinizing hormone, thyroid-stimulating hormone, and follicle-stimulating hormone.

The family of glycoprotein hormones and their homologous heptahelical receptors represent an excellent system for comparative structure-function studies. We have engineered single chain molecules of human chorionic gonadotropin (hCG) fused to its cognate receptor, LH receptor (LHR), and to the noncognate receptors, TSH receptor (TSHR) and FSH receptor (FSHR; N-beta-alpha-receptor-C), to create the yoked (Y) complexes YCG/LHR, YCG/TSHR, and YCG/FSHR. The expression and bioactivity of these fusion proteins were examined in transiently transfected HEK 293 cells. Western blot analysis and antibody binding assays demonstrated that each of the proteins was expressed. In the case of YCG/LHR, minimal binding of exogenous hormone was observed due to the continued occupation of receptor by the fused ligand. The presence of hCG in the YCG/TSHR and YCG/FSHR, however, did not prevent binding of exogenous cognate ligand, presumably due to the lower affinity of hCG. The basal cAMP levels in cells expressing the YCG/LHR complex was approximately 20-fold higher than that in cells expressing LHR. Increases in basal cAMP production were also observed with YCG/TSHR and YCG/FSHR, e.g. 13- and 4-fold increases, respectively. Whereas the affinity and specificity of hCG for LHR are extraordinarily high, the hormone is capable of binding to and activating both TSHR and FSHR under these conditions that mimic high ligand concentrations. These findings were confirmed by adding high concentrations of hCG to cells expressing TSHR and FSHR. Although the functional interaction of hCG and TSHR has been recognized in gestational hyperthyroidism, there are no reports linking hCG to FSHR activation. This study, however, suggests that such a functional interaction is capable of occurring under conditions of high circulating levels of hCG, e.g. the first trimester of pregnancy and in patients with hCG-secreting tumors.