Protease-activated receptors 1-3 (PAR1, PAR2, and PAR3) are members of a unique G protein-coupled receptor family. They are characterized by a tethered peptide ligand at the extracellular amino terminus that is generated by minor proteolysis. A partial cDNA sequence of a fourth member of this family (PAR4) was identified in an expressed sequence tag database, and the full-length cDNA clone has been isolated from a lymphoma Daudi cell cDNA library. The ORF codes for a seven transmembrane domain protein of 385 amino acids with 33% amino acid sequence identity with PAR1, PAR2, and PAR3. A putative protease cleavage site (Arg-47͞Gly-48) was identified within the extracellular amino terminus. COS cells transiently transfected with PAR4 resulted in the formation of intracellular inositol triphosphate when treated with either thrombin or trypsin. A PAR4 mutant in which the Arg-47 was replaced with Ala did not respond to thrombin or trypsin. A hexapeptide (GYPGQV) representing the newly exposed tethered ligand from the amino terminus of PAR4 after proteolysis by thrombin activated COS cells transfected with either wild-type or the mutant PAR4. Northern blot showed that PAR4 mRNA was expressed in a number of human tissues, with high levels being present in lung, pancreas, thyroid, testis, and small intestine. By f luorescence in situ hybridization, the human PAR4 gene was mapped to chromosome 19p12.

The activation of human platelets by thrombin is mediated primarily by protease-activated receptors (PARs). PAR1 and PAR4 are present on human platelets and are activated by the hexapeptides SFLLRN and GYPGQV, respectively. To further characterize the involvement of PAR1 and PAR4 in platelet activation, the ability of SFLLRN or GYPGQV to generate annexin V binding to newly exposed phospholipids on the platelet surface and generate procoagulant activity has been examined. Exposure of phosphatidylserine and phosphatidylethanolamine on platelets, as determined by an increase in annexin V binding, was strongly stimulated by SFLLRN, thrombin, and collagen, but only to a minor extent by GYPGQV. In a clotting assay initiated with factor VIIa, soluble tissue factor, and calcium, the clotting time in the absence of platelets was >5 min. In the presence of unstimulated platelets, the clotting time was 200 ؎ 20 sec. In the presence of platelets activated with SFLLRN or collagen, the clotting time decreased to 100 ؎ 10 sec. This shortening of the clotting time is equivalent to about a 5-fold increase in coagulant activity when stimulated platelets are compared with unstimulated platelets and activated platelets are used as a reference. These results indicate that thrombin initiates a very strong response in platelets through PAR1, leading to exposure of anionic phospholipids that support blood clotting. The response mediated by PAR4, however, was limited to platelet aggregation and similar to that triggered in platelets by weaker agonists such as ADP or epinephrine.

This communication describes an assay for the relative translation efficiency of individual codons which makes use of the pyrE attenuator to probe the coupling between transcription and translation at the end of an artificial leader peptide. By cloning of short synthetic DNA fragments the codons to be tested were placed in the middle of the leader peptide and the downstream transcription of a pyrE"lacZ gene was monitored by measuring beta-galactosidase activity. The substitution, one by one, of three AGG codons for arginine with three CGT codons for the same amino acid residue was found to cause a two fold increase per codon of transcription over the pyrE attenuator, such that an eight fold higher frequency of pyrE expression was seen when all three AGG codons were replaced by CGT codons. No such effect of codon composition was observed, when the cells were grown with a low UTP pool which causes a reduction of the mRNA chain growth rate.

Microglia are the resident immune cells of the CNS. Brain injury triggers microglial activation, leading to proliferation, changes in antigenic profile, NO production and cytokine release. It is widely believed that serum factors inundating the injured tissue can prompt this activation, leading to long-term phenotypic changes. We and others have recently reported that commercial-grade preparations of thrombin, a serine protease known for its central function in blood coagulation, activate microglial cells. Recent findings, however, have called into question the involvement of thrombin itself in the induction of microglial cytokine release and led us to systematically re-investigate the ability of the protease to induce a broad spectrum of microglial activation parameters. We used a pharmaceutical-grade recombinant human thrombin (rh-thr) and compared it with a commercial-grade plasma-derived bovine thrombin (pb-thr) preparation that has been used extensively in the literature, including in our own earlier report. We investigated the effect of these two thrombin preparations on proliferation, NO production, interleukin-6 and tumour necrosis factor-a release, intracellular calcium signaling and cell surface expression of CD95 (Fas) and CD40. Pb-thr induced robust responses in all variables tested. In contrast, rh-thr triggered calcium signals and induced small but significant changes in the expression of cell surface antigens, but had no effect on proliferation, NO production or cytokine release. Control studies assured equivalent thrombin potencies and excluded both species-specific effects and endotoxin (lipopolysaccharide) contamination as possible causes of the disparity. Our results indicate a substantially more restricted role for thrombin itself in microglial activation than previously appreciated, but point to several potentially important co-stimulatory effects. In addition, these results suggest that previous studies examining thrombin's activation of microglia should be cautiously re-interpreted.

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