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Activated protein C-cleaved protease activated receptor-1 is retained on the endothelial cell surface even in the presence of thrombin.

ABSTRACT: Activated protein C (APC) signals in endothelial cells ex vivo through protease activated receptor-1 (PAR1). However, it is controversial whether PAR1 can mediate APC's protective effects in sepsis because the inflammatory response results in thrombin generation and thrombin proteolytically activates PAR1 much more efficiently than APC. Here we show that APC can induce powerful barrier protective responses in an endothelial cell monolayer in the presence of thrombin. Using cell surface immunoassays with conformation sensitive monoclonal anti-PAR1 antibodies we analyzed cleavage of endogenous PAR1 on the endothelial cell surface by APC in the absence and presence of thrombin. Incubation with APC caused efficient PAR1 cleavage and upon coincubation with thrombin APC supported additional PAR1 cleavage. Thrombin-cleaved PAR1 rapidly disappeared from the cell surface whereas, unexpectedly, the APC-cleaved PAR1 remained and could be detected on the cell surface, even when thrombin at concentrations of up to 1 nM was also present. Our findings demonstrate for the first time directly that APC can generate a distinct PAR1 population on endothelial cells in the presence of thrombin. The data suggest that different trafficking of activated PAR1 might explain how PAR1 signaling by APC can be relevant when thrombin is present.

SUBMITTER: Schuepbach RA

PROVIDER: S-EPMC2254534 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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Activated protein C-cleaved protease activated receptor-1 is retained on the endothelial cell surface even in the presence of thrombin.

Schuepbach Reto A RA Feistritzer Clemens C Brass Lawrence F LF Riewald Matthias M

Blood 20071218 5

Activated protein C (APC) signals in endothelial cells ex vivo through protease activated receptor-1 (PAR1). However, it is controversial whether PAR1 can mediate APC's protective effects in sepsis because the inflammatory response results in thrombin generation and thrombin proteolytically activates PAR1 much more efficiently than APC. Here we show that APC can induce powerful barrier protective responses in an endothelial cell monolayer in the presence of thrombin. Using cell surface immunoass ...[more]

PMID: 18089851

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Project description:Thrombin is a serine protease that elicits a variety of cellular responses. Molecular cloning of a thrombin receptor revealed a G protein-coupled receptor that is activated by a novel proteolytic mechanism. Recently, a second protease-activated receptor was discovered and dubbed PAR2. PAR2 is highly related to the thrombin receptor by sequence and, like the thrombin receptor, is activated by cleavage of its amino terminal exodomain. Also like the thrombin receptor, PAR2 can be activated by the hexapeptide corresponding to its tethered ligand sequence independent of receptor cleavage. Thus, functionally, the thrombin receptor and PAR2 constitute a fledgling receptor family that shares a novel proteolytic activation mechanism. To further explore the relatedness of the two known protease-activated receptors and to examine the possibility that a protease-activated gene cluster might exist, we have compared the structure and chromosomal locations of the thrombin receptor and PAR2 genes.The genomic structures of the two protease-activated receptor genes were determined by analysis of lambda phage, P1 bacteriophage, and bacterial artificial chromosome (BAC) genomic clones. Chromosomal location was determined with fluorescent in situ hybridization (FISH) on metaphase chromosomes, and the relative distance separating the two genes was evaluated both by means of two-color FISH and analysis of YACs and BACs containing both genes.Analysis of genomic clones revealed that the two protease-activated receptor genes share a two-exon genomic structure in which the first exon encodes 5'-untranslated sequence and signal peptide, and the second exon encodes the mature receptor protein and 3'-untranslated sequence. The two receptor genes also share a common locus with the two human genes located at 5q13 and the two mouse genes at 13D2, a syntenic region of the mouse genome. These techniques also suggest that the physical distance separating these two genes is less than 100 kb.The fact that the thrombin receptor and PAR2 genes share an identical structure and are located within approximately 100 kb of each other in the genome demonstrates that these genes arose from a gene duplication event. These results define a new protease-activated receptor gene cluster in which new family members may be found.

Project description:Essentials The rs773902 SNP results in differences in platelet protease-activated receptor (PAR4) function. The functional consequences of rs773902 were analyzed in human platelets and stroke patients. rs773902 affects thrombin-induced platelet function, PAR4 desensitization, stroke association. Enhanced PAR4 Thr120 effects on platelet function are blocked by ticagrelor. SUMMARY: Background F2RL3 encodes protease-activated receptor (PAR) 4 and harbors an A/G single-nucleotide polymorphism (SNP) (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue 120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP) AYPGKF. Objectives To determine the functional effects of rs773902 on stimulation by a physiological agonist, thrombin, and on antiplatelet antagonist activity. Methods Healthy human donors were screened and genotyped for rs773902. Platelet function in response to thrombin was assessed without and with antiplatelet antagonists. The association of rs773902 alleles with stroke was assessed in the Stroke Genetics Network study. Results As compared with rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation in response to subnanomolar concentrations of thrombin, increased granule secretion, and decreased sensitivity to PAR4 desensitization. In the presence of PAR1 blockade, this genotype effect was abolished by higher concentrations of or longer exposure to thrombin. We were unable to detect a genotype effect on thrombin-induced PAR4 cleavage, dimerization, and lipid raft localization; however, rs773902 AA platelets required a three-fold higher level of PAR4-AP for receptor desensitization. Ticagrelor, but not vorapaxar, abolished the PAR4 variant effect on thrombin-induced platelet aggregation. A significant association of modest effect was detected between the rs773902 A allele and stroke. Conclusion The F2RL3 rs773902 SNP alters platelet reactivity to thrombin; the allelic effect requires P2Y12 , and is not affected by gender. Ticagrelor blocks the enhanced reactivity of rs773902 A platelets. PAR4 encoded by the rs773902 A allele is relatively resistant to desensitization and may contribute to stroke risk.

Dataset Information

Activated protein C-cleaved protease activated receptor-1 is retained on the endothelial cell surface even in the presence of thrombin.

Publications

Activated protein C-cleaved protease activated receptor-1 is retained on the endothelial cell surface even in the presence of thrombin.

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