Mer receptor tyrosine kinase signaling participates in platelet function.

OBJECTIVE: Recently, mice made deficient in growth arrest-specific gene 6 product (Gas6) or in which Gas6 gene expression was inhibited were shown to have platelet dysfunction and to be less susceptible to thrombosis. The aim of this study was to define and characterize the relevant Gas6 receptor or receptors involved in platelet function. METHODS AND RESULTS: Using RT-PCR and Western blot analysis we found that mer was the predominantly expressed subtype in mouse and human platelets, whereas axl and rse were not detected. We generated mer-deficient mice by targeted disruption of the mer receptor gene. Platelets derived from mer-deficient mice had decreased platelet aggregation in responses to low concentrations of collagen, U46619, and PAR4 thrombin receptor agonist peptide in vitro. However, the response to ADP was not different from wild-type platelets. Knockout of the mer gene protected mice from collagen/epinephrine-induced pulmonary thromoembolism and inhibited ferric chloride-induced thrombosis in vivo. Tail bleeding times, coagulation parameters, and peripheral blood cell counts in mer-deficient mice were similar to wild-type mice. CONCLUSIONS: Our data provide the first evidence that mer, presumably through activation by its ligand Gas6, participates in regulation of platelet function in vitro and platelet-dependent thrombosis in vivo.

Virus-based expression systems facilitate rapid target in vivo functionality validation and high-throughput screening.

Target validation is one of rate-limiting steps in the modern drug discovery. The authors developed a strategy of combining adenovirus-mediated gene transfer for efficient target functionality validation, both in vivo and in vitro, with baculovirus expression to produce sufficient quantities of protein for high-throughput screening (HTS). The incorporation of green fluorescent protein (GFP) in the adenovirus vectors accelerates recombinant adenovirus plaque purification, whereas the use of epitope and affinity tags facilitates the identification and purification of recombinant protein. In this generalized scheme, the flexible modular design of viral vectors facilitates the transition between target validation and HTS. In the example presented, functional target validation in vivo was achieved by overexpressing the target gene in cell-based models and in the mouse cortex following adenovirus-mediated gene delivery. In this context, target overexpression resulted in the accumulation of a disease-related biomarker both in vitro and in vivo. A baculovirus-based expressional system was then generated to produce enough target protein for HTS. Thus, the use of these viral expression systems represents a generalized method for rapid target functionality validation and HTS assay development, which could be applied to numerous target candidates being elucidated in gene discovery programs.

A novel serine protease predominately expressed in macrophages.

We have identified a novel serine protease designated EOS by sequence identity searches. The deduced protein contains 284 amino acids with an active form containing 248 amino acids starting from an Ile-Val-Gly-Gly motif. The active form comprises a catalytic triad of conserved amino acids: His77, Asp126 and Ser231. It shares 44% identity with beta-tryptase and belongs to the S1 trypsin-like serine-protease family. Interestingly, this gene also maps to human chromosome 16p13.3. The purified protease showed amidolytic activity, cleaving its substrates before arginine residues. Tissue distribution by immunohistochemistry analysis demonstrated that EOS is highly expressed in spleen and moderately expressed in intestine, colon, lung and brain. We confirmed this expression pattern at the mRNA level by performing in situ hybridization. The results from both immunohistochemistry and in situ hybridization indicate that EOS is associated with macrophages. We corroborated this observation by double immunofluorescence using the anti-EOS antibody and an anti-CD68 antibody, a macrophage specific marker. Furthermore, we have detected a dramatic increase in immune staining of EOS in cultured U937 cells treated with PMA, which represent activated macrophages. This up-regulation is also reflected by elevated EOS mRNA in the PMA-treated U937 cells detected by Northern blotting. Since macrophages have important roles in various pathological conditions, such as wound healing, atherosclerosis and numerous inflammatory diseases, the localization of this novel serine protease to active macrophages may help to further the elucidation of the roles of this gene product in modulating these disorders.

Mechanisms of protease-activated receptor-4 actions in cardiomyocytes. Role of Src tyrosine kinase.

Protease-activated receptor (PAR)-4 is a low affinity thrombin receptor with slow activation and desensitization kinetics relative to PAR-1. This study provides novel evidence that cardiomyocytes express functional PAR-4 whose signaling phenotype is distinct from PAR-1 in cardiomyocytes. AYPGKF, a modified PAR-4 agonist with increased potency at PAR-4, activates p38-mitogen-activated protein kinase but is a weak activator of phospholipase C, extracellular signal-regulated kinase, and cardiomyocyte hypertrophy; AYPGKF and thrombin, but not the PAR-1 agonist SFLLRN, activate Src. The observation that AYPGKF and thrombin activate Src in cardiomyocytes cultured from PAR-1(-/-) mice establishes that Src activation is via PAR-4 (and not PAR-1) in cardiomyocytes. Further studies implicate Src and epidermal growth factor receptor (EGFR) kinase activity in the PAR-4-dependent p38-mitogen-activated protein kinase signaling pathway. Thrombin phosphorylates EGFRs and ErbB2 via a PP1-sensitive pathway in PAR-1(-/-) cells that stably overexpress PAR-4; the Src-mediated pathway for EGFR/ErbB2 transactivation underlies the protracted phases of thrombin-dependent extracellular signal-regulated kinase activation in PAR-1(-/-) cells that overexpress PAR-4 and in cardiomyocytes. These studies identify a unique signaling phenotype for PAR-4 (relative to other cardiomyocyte G protein-coupled receptors) that is predicted to contribute to cardiac remodeling and influence the functional outcome at sites of cardiac inflammation.

The nucleotide-binding site of human sphingosine kinase 1.

Sphingosine kinase catalyzes the formation of sphingosine 1-phosphate, a lipid second messenger that has been implicated in a number of agonist-driven cellular responses including mitogenesis, anti-apoptosis, and expression of inflammatory molecules. Despite the importance of sphingosine kinase, very little is known regarding its structure or mechanism of catalysis. Moreover, sphingosine kinase does not contain recognizable catalytic or substrate-binding sites, based on sequence motifs found in other kinases. Here we have elucidated the nucleotide-binding site of human sphingosine kinase 1 (hSK1) through a combination of site-directed mutagenesis and affinity labeling with the ATP analogue, FSBA. We have shown that Gly(82) of hSK1 is involved in ATP binding since mutation of this residue to alanine resulted in an enzyme with an approximately 45-fold higher K(m)((ATP)). We have also shown that Lys(103) is important in catalysis since an alanine substitution of this residue ablates catalytic activity. Furthermore, we have shown that this residue is covalently modified by FSBA. Our data, combined with amino acid sequence comparison, suggest a motif of SGDGX(17-21)K is involved in nucleotide binding in the sphingosine kinases. This motif differs in primary sequence from all previously identified nucleotide-binding sites. It does, however, share some sequence and likely structural similarity with the highly conserved glycine-rich loop, which is known to be involved in anchoring and positioning the nucleotide in the catalytic site of many protein kinases.