. The human EPCR cDNA was PCR amplified and subcloned into pEGFP-N1 (Clontech) introducing a linker of two Gly residues between EPCR and the C-terminal EGFP protein. A mutant EPCR construct that lacks PC/APC binding activity was generated by replacing Tyr154 with Ala (
) using oligonucleotide directed mutagenesis. A palmitoylation resistant mutant of EPCR was generated by replacing the single cytoplasmic Cys221 with Ser. The coding sequences of all constructs were confirmed by DNA sequencing. Thrombin, APC, hirudin, the PAR1 agonist peptide TFLLRNPNDK, the PAR2 agonist peptide SLIGRL, and monoclonal anti PAR1 antibodies ATAP2 (used at 10
). APC was active site modified with 0.5 mM H-D-Phe-L-Phe-Arg chloromethylketone (Bachem Bioscience Inc.) and the absence of residual APC activity and free chloromethyl ketone was confirmed following extensive dialysis. The murine PAR deficient M6-11 cell line was co-transfected with the relevant cDNAs. Overnight serum starved cells were incubated with 20 nM APC, 10
M PAR2 agonist SLIGRL, or 5 nM thrombin (IIa) for 5 h. All experiments involving APC contained 100 nM hirudin to exclude thrombin signaling. Cleavage blocking antibodies against PAR1 were added 10 min prior to the agonists where indicated.
. Primary human umbilical vein endothelial cells (HUVEC) were incubated for 5 hours with fresh M199 containing 2% serum prior to agonist stimulation as above. Erk1/2 phosphorylation was analyzed by Western blotting after 6 min of stimulation. To block APC binding to EPCR, 100 nM chloromethylketone modified APC (APC-CK) was added. To block PAR1 cleavage, anti PAR1 antibodies were added 10 minutes prior to agonist stimulation. MAP kinase phosphorylation was quantified by laser densitometry of blots and statistical significance was determined by t-test.
. Confluent human umbilical vein endothelial cells HUV-EC-C (ATCC CRL 1730) were serum deprived for 5 h in M199 medium, 2 mM CaCl
M SLIGRL, or 10 nM APC/100 nM hirudin, for 90 min at 37°C. Total RNA was isolated followed by reverse transcription. Purified double stranded cDNA was
transcribed in the presence of biotinylated dUTP and dCTP, fragmented cRNA was hybridized to the HG-U95Av2 array (Affymetrix, Santa Clara, CA), followed by staining with streptavidin-phycoerythrin. Arrays were scanned and analyzed using GeneChip3.1 software. Three independent experiments analyzing gene induction by PAR1, PAR2, or APC yielded 12 data sets from high density microarrays with 9 comparisons between the agonists and control. An average fold-induction for the three independent experiments was obtained by calculating the means of the logarithm of the individual fold-increases for each agonist. To eliminate non-expressed and highly variable genes, the following criteria were set for genes shown: (1) The gene's expression level had to be scored "Present" by the GeneChip software algorithm in more than two of the 12 data sets. (2) GeneChip3.1 comparison analysis of stimulated versus control had to score "Increased" in more than one out of the 9 comparisons. (3) The ratio of the average fold induction for all agonists and the mean of the standard deviations for the inductions by each agonist had to be larger than 1 for inclusion in Fig. 2 or larger than 0.5 for the evaluation of PAR1 and PAR2 selective responses in the larger gene set.
. For TaqMan (Applied Biosystems) real-time RT-PCR 4
g total cellular RNA from stimulated primary HUVEC was reverse transcribed followed by RNAse treatment using the Superscript Kit (Invitrogen). TaqMan probes were custom designed for genes of interests. All samples were normalized with human GAPDH probes and primers obtained from Applied Biosystems.
| Supplemental Table 1. Gene induction profiles determined by microarray analysis of endothelial cells stimulated with APC, PAR1, or PAR2 agonists. The first set shows genes that were induced with high reproducibility by the three agonists. Selected genes from a larger gene set with higher variability between repeat experiments is shown below, grouped according to PAR1 and PAR2 selective expression. Certain genes were not induced by APC, but PAR agonists.
|
| Average Induction |
| Gene | PAR1 | PAR2 | APC |
| Genes represented in Fig.2 A-C |
| L13740 /FEATURE= /DEFINITION=HUMTR3A Human TR3 orphan receptor mRNA | 4.8 | 5.4 | 3.3 |
| L13740 /FEATURE= /DEFINITION=HUMTR3A Human TR3 orphan receptor mRNA | 4.7 | 5.4 | 3.2 |
| Cluster Incl M60278:Human heparin-binding EGF-like growth factor mRNA | 3.6 | 3.9 | 3.5 |
| U12767 /FEATURE= /DEFINITION=HSU12767 Human mitogen induced nuclear orphan receptor (MINOR) | 4.9 | 3.3 | 2.6 |
| M29039 /FEATURE=cds /DEFINITION=HUMJUNCAA Human transactivator (jun-B) gene | 2.8 | 2.8 | 4.9 |
| M60721 /FEATURE=mRNA /DEFINITION=HUMHB24 Human homeobox gene | 2.8 | 3.7 | 2.3 |
| U15932 /FEATURE= /DEFINITION=HSU15932 Human dual-specificity protein phosphatase | 2.9 | 3.3 | 1.8 |
| Cluster Incl J02931:Human placental tissue factor (two forms) mRNA | 3.0 | 2.7 | 2.1 |
| M22489 /FEATURE= /DEFINITION=HUMBMP2A Human bone morphogenetic protein 2A (BMP-2A) | 2.3 | 3.2 | 2.4 |
| Cluster Incl X75918:H.sapiens mRNA for NOT | 2.3 | 3.3 | 2.2 |
| Cluster Incl U75697:Human transcription regulator RPD3-2B mRNA | 2.7 | 2.6 | 2.3 |
| Cluster Incl U12767:Human mitogen induced nuclear orphan receptor (MINOR) | 2.4 | 3.4 | 1.7 |
| Cluster Incl M92843:H.sapiens zinc finger transcriptional regulator mRNA, | 2.0 | 3.4 | 1.8 |
| Cluster Incl M30474:Human kidney gamma-glutamyl transpeptidase type II mRNA | 2.7 | 2.1 | 2.3 |
| L19871 /FEATURE= /DEFINITION=HUMATF3X Human activating transcription factor 3 (ATF3) | 2.5 | 2.6 | 1.8 |
| Cluster Incl M22489:Human bone morphogenetic protein 2A (BMP-2A) | 2.4 | 2.9 | 1.6 |
| Cluster Incl X63741:H.sapiens pilot mRNA | 1.9 | 3.0 | 1.9 |
| X68277 /FEATURE=cds /DEFINITION=HSCL100 H.sapiens CL 100 mRNA for protein tyrosine phosphatase | 2.1 | 2.8 | 1.9 |
| Cluster Incl M28211:Homo sapiens GTP-binding protein (RAB4) mRNA | 2.0 | 2.7 | 1.8 |
| Cluster Incl AI288757:qm11h01.x1 Homo sapiens cDNA, 3' end /clone=IMAGE-1881553 | 1.8 | 2.3 | 2.3 |
| Cluster Incl M36820:Human cytokine (GRO-beta) mRNA | 2.1 | 1.6 | 2.6 |
| Cluster Incl AL031432:Human DNA sequence from clone 465N24 on chromosome 1p35.1-36.13. | 1.9 | 2.6 | 1.7 |
| Cluster Incl Z48054:H.sapiens mRNA for peroxisomal targeting signal 1 (SKL type) receptor | 2.0 | 1.9 | 2.2 |
| Cluster Incl D15050:Human mRNA for transcription factor AREB6 | 2.1 | 1.7 | 2.2 |
| Cluster Incl X89066:H.sapiens mRNA for TRPC1 protein | 2.6 | 2.2 | 0.9 |
| L05072 /FEATURE=expaned_cds /DEFINITION=HUMIFNRF1A Homo sapiens interferon regulatory factor 1 gene | 1.9 | 2.0 | 1.7 |
| Cluster Incl X51345:Human jun-B mRNA for JUN-B protein | 1.8 | 1.8 | 2.1 |
| Cluster Incl D78579:Homo sapiens mRNA for neuron derived orphan receptor | 2.6 | 1.4 | 1.5 |
| Cluster Incl J04076:Human early growth response 2 protein (EGR2) mRNA | 2.0 | 2.0 | 1.5 |
| Cluster Incl AB014569:Homo sapiens mRNA for KIAA0669 protein | 1.3 | 2.0 | 2.1 |
| Cluster Incl AJ005821:Homo sapiens mRNA for X-like 1 protein | 2.4 | 1.6 | 1.4 |
| Cluster Incl Y00630:Human mRNA for Arg-Serpin (plasminogen activator-inhibitor 2, PAI-2) | 2.2 | 1.7 | 1.5 |
| Cluster Incl M31516:Human decay-accelerating factor mRNA | 1.7 | 1.9 | 1.5 |
| X94216 /FEATURE=cds /DEFINITION=HSVEGFC H.sapiens mRNA for VEGF-C protein | 1.4 | 2.1 | 1.7 |
| Cluster Incl AL023584:Human DNA sequence from clone 67K17 on chromosome 6q24.1-24.3. | 1.8 | 2.0 | 1.3 |
| Cluster Incl M28225:Human JE gene encoding a monocyte secretory protein | 2.1 | 1.1 | 2.0 |
| Cluster Incl D86181:Homo sapiens DNA for galactocerebrosidase | 1.9 | 1.5 | 1.6 |
| Cluster Incl AF017786:Homo sapiens phosphatidic acid phosphohydrolase homolog (Dri42) | 1.6 | 1.6 | 1.8 |
| U04636 /FEATURE=mRNA /DEFINITION=HSU04636 Human cyclooxygenase-2 (hCox-2) gene | 1.3 | 1.3 | 2.3 |
| Guanine Nucleotide Exchange Factor 2 | 1.4 | 1.4 | 2.1 |
| Cluster Incl AF039843:Homo sapiens Sprouty 2 (SPRY2) mRNA | 1.6 | 1.8 | 1.5 |
| Cluster Incl U08015:Human NF-ATc mRNA | 1.4 | 2.0 | 1.4 |
| M59465 /FEATURE= /DEFINITION=HUMA20 Human tumor necrosis factor alpha inducible protein A20 mRNA | 1.4 | 1.3 | 2.1 |
| U43142 /FEATURE= /DEFINITION=HSU43142 Human vascular endothelial growth factor related protein VRP | 1.4 | 1.9 | 1.4 |
| Cluster Incl U79259:Human clone 23945 mRNA | 1.5 | 1.7 | 1.5 |
| Cluster Incl AF068197:Homo sapiens BCE-1 mRNA | 1.5 | 1.8 | 1.4 |
| Cluster Incl AF054182:Homo sapiens mitochondrial processing peptidase beta-subunit mRNA | 1.8 | 1.3 | 1.5 |
| Cluster Incl AL050078:Homo sapiens mRNA; cDNA DKFZp566G0746 | 1.6 | 1.6 | 1.3 |
| Cluster Incl J04111:Human c-jun proto oncogene (JUN) | 1.7 | 1.5 | 1.4 |
| M69043 /FEATURE= /DEFINITION=HUMMAD3A Homo sapiens MAD-3 mRNA encoding IkB-like activity | 1.4 | 1.4 | 1.8 |
| Cluster Incl AF050110:Homo sapiens TGFb inducible early protein and early growth response protein alpha genes | 1.7 | 1.4 | 1.4 |
| S81439 /FEATURE= /DEFINITION=S81439 EGR alpha=early growth response gene alpha | 1.8 | 1.4 | 1.3 |
| Cluster Incl AI670788:tz10c02.x1 Homo sapiens cDNA, 3' end /clone=IMAGE-2288162 | 1.5 | 1.4 | 1.6 |
| Cluster Incl AB022718:Homo sapiens mRNA for DEPP (decidual protein induced by progesterone) | 1.3 | 2.0 | 0.9 |
| Cluster Incl U25997:Homo sapiens stanniocalcin precursor (STC) mRNA | 1.6 | 1.4 | 1.2 |
| Cluster Incl AB004066:Homo sapiens mRNA for DEC1 | 1.5 | 1.3 | 1.5 |
| Cluster Incl U07802:Human Tis11d gene | 1.5 | 1.6 | 1.2 |
| Cluster Incl AL096858:Novel human gene mapping to chomosome 1 | 1.3 | 1.5 | 1.4 |
| M26683 /FEATURE= /DEFINITION=HUMIFNIND Human interferon gamma treatment inducible mRNA | 1.6 | 1.0 | 1.6 |
| Cluster Incl AF022375:Homo sapiens vascular endothelial growth factor mRNA | 1.5 | 1.3 | 1.4 |
| Cluster Incl U81607:Homo sapiens gravin mRNA | 1.4 | 1.3 | 1.5 |
| Cluster Incl L43821:Homo sapiens enhancer of filamentation (HEF1) mRNA | 1.4 | 1.2 | 1.6 |
| Cluster Incl AF045451:Homo sapiens transcriptional regulatory protein p54 | 1.5 | 1.4 | 1.2 |
| Cluster Incl AF078077:Homo sapiens growth arrest and DNA-damage-inducible protein GADD45beta | 1.3 | 1.3 | 1.5 |
| Cluster Incl D50917:Human mRNA for KIAA0127 gene | 1.4 | 1.3 | 1.2 |
| Cluster Incl AI985272:ws06b05.x1 Homo sapiens cDNA, 3' end /clone=IMAGE-2496369 | 1.3 | 1.4 | 1.1 |
| S81914 /FEATURE= /DEFINITION=S81914 IEX-1=radiation-inducible immediate-early gene | 1.3 | 1.2 | 1.3 |
| Cluster Incl Z25821:H.sapiens gene for mitochondrial dodecenoyl-CoA delta-isomerase | 1.1 | 1.7 | 1.0 |
| Cluster Incl W27545:32c4 Homo sapiens cDNA | 1.2 | 1.2 | 1.2 |
| Cluster Incl U59863:Human TRAF-interacting protein I-TRAF mRNA | 1.3 | 1.1 | 1.2 |
| | | |
| Genes with higher variability (not represented in Fig.2) |
| PAR1 enhanced (<1.3 fold with PAR2 agonist) |
| Cluster Incl AI186701:qe82d12.x1 Homo sapiens cDNA, 3' end /clone=IMAGE-1745495 | 1.9 | 1.1 | 1.6 |
| Cluster Incl U35735:Human RACH1 (RACH1) mRNA | 1.8 | 1.1 | 1.2 |
| Cluster Incl AL035297:H.sapiens gene from PAC 747L4 | 1.7 | 1.0 | 1.4 |
| Cluster Incl M22299:Human T-plastin polypeptide mRNA | 1.6 | 1.2 | 2.0 |
| Cluster Incl U87460:Human putative endothelin receptor type B-like protein mRNA | 1.4 | 0.9 | 1.8 |
| AF024710 /FEATURE= /DEFINITION=AF024710 Homo sapiens vascular endothelial growth factor (VEGF) | 1.4 | 1.1 | 1.1 |
| Cluster Incl AI953789:wx69d10.x1 Homo sapiens cDNA, 3' end /clone=IMAGE-2548915 | 1.3 | 0.9 | 1.4 |
| PAR2 enhanced (<1.3 fold with PAR1 agonist) |
| Cluster Incl AI038821:ox96d03.x1 Homo sapiens cDNA, 3' end /clone=IMAGE-1664165 | 1.2 | 2.0 | 1.3 |
| Cluster Incl U65093:Human msg1-related gene 1 (mrg1) mRNA | 1.3 | 1.7 | 1.0 |
| Cluster Incl Y13115:Homo sapiens mRNA for serine/threonine protein kinase SAK | 1.2 | 1.6 | 1.0 |
| Cluster Incl W26851:17b12 Homo sapiens cDNA | 1.2 | 1.6 | 1.1 |
| Cluster Incl U53831:Homo sapiens interferon regulatory factor 7B | 1.1 | 1.5 | 1.3 |
| Cluster Incl U27655:Human RGP3 mRNA | 1.2 | 1.5 | 1.2 |
| Cluster Incl U58334:Human Bcl2, p53 binding protein Bbp/53BP2 (BBP/53BP2) mRNA | 1.1 | 1.5 | 1.1 |
| Cluster Incl AC004475:Homo sapiens chromosome 19, cosmid F23858 | 1.2 | 1.4 | 1.2 |
| Cluster Incl L38696:Homo sapiens autoantigen p542 mRNA | 1.0 | 1.3 | 1.1 |
| Cluster Incl AL049354:Homo sapiens mRNA; cDNA DKFZp566E183 | 1.1 | 1.3 | 0.9 |
| Cluster Incl Z25821:H.sapiens gene for mitochondrial dodecenoyl-CoA delta-isomerase | 1.1 | 1.7 | 1.0 |
| Genes not induced by APC (<1.3 fold with APC) |
| Cluster Incl AB028069:Homo sapiens mRNA for activator of S phase Kinase | 2.0 | 2.0 | 1.2 |
| Cluster Incl U85267:Homo sapiens down syndrome candidate region 1 (DSCR1) gene | 1.6 | 2.0 | 0.9 |
| Cluster Incl U78082:Human RNA polymerase transcriptional regulation mediator (h-MED6) mRNA | 1.9 | 1.3 | 1.1 |
| Cluster Incl D38537:Human mRNA for protoporphyrinogen oxidase | 1.3 | 1.6 | 1.0 |
| M83667 /FEATURE=mRNA /DEFINITION=HUMNFIL6BA Human NF-IL6-beta protein | 1.4 | 1.3 | 1.1 |
g/ml) and WEDE15 (used at 20 
