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Blood, Vol. 113, Issue 23, 5970-5978, June 4, 2009
Hyperantithrombotic, noncytoprotective Glu149Ala-activated protein C mutant
Blood Mosnier et al.
113: 5970
Supplemental materials for: Mosnier et al
Purification of human and mouse protein C – For the collection of conditioned media, stable transfected human kidney 293 (HEK-293) cells expressing the various protein C’s were seeded in a cell factory (6320 cm2; Nunc) in Dulbeco’s modified Eagle media (DMEM): Ham’s F-12 (Invitrogen) supplemented with penicillin-streptomycin-glutamine (PSG) (Gibco), 10% fetal bovine serum (FBS) (Omega), 0.6 mg/ml G418 and 10 µg/ml vitamin K1 in a humidified atmosphere at 37°C and 5% CO2. Confluent cells were washed with PBS and switched to serum free expression media DMEM:F-12 without phenol red (CellGro) supplemented with PSG and 10 µg/ml vitamin K1. Conditioned media was harvested every 2 days, supplemented with 10 mM benzamidine, filtered, and stored at −80°C until purification. Typical protein C expression levels of 2–5 mg/L were obtained for both human and mouse protein C mutants. Protein C was purified by two passes over fast flow Q-Sepharose (Pharmacia) using first CaCl2 for elution and subsequent a NaCl gradient for elution (0–600 mM NaCl in 20 mM Tris pH 7.4) as described.2–4 Protein C concentrations were estimated by absorbance using an extinction coefficient of 14.5 (280 nm, 1%, cm−1). Activation of protein C – Purified protein C was activated by thrombin (1/50 w/w) in the presence of 2 mM EDTA to maximal activity (2.5–3 h at 37°C (human) or 1 h at room temperature (mouse)), followed by the addition of hirudin (Sigma) to inactivate the thrombin and then the use of fast flow Q-Sepharose chromatography to remove thrombin.3 APC concentrations were determined by active-site titration according to Chase and Shaw with minor modifications.5,6 Catalytic activity against small substrates – Amidolytic assays (S-2366, Chromogenix; Spectrozyme aPC, American Diagnostica and Pefachrome PCa, Pentapharm) were performed as described.6,7 Coagulation assays – A-modified APTT clotting assay was used to measure the anticoagulant activity of mAPC. Briefly, 5 µl of pooled normal mouse plasma was incubated with 25 µl human fibrinogen (2 mg/ml), 25 µl of the APTT reagent Platelin LS (Biomérieux) and 25 µl mAPC in 50 mM Tris pH 7.4, 100 mM NaCl and 0.5% BSA for 3 min at 37°C after which clotting was initiated by the addition of 25 µl of 30 mM CaCl2 in TBS. Clotting times were determined using a KC4a micro (Amelung). Cytoprotective activity assays – APC anti-inflammatory activity was determined as the inhibition of cytokine release (TNFα; mouse TNFα ELISA from Invitrogen) by APC of LPS-stimulated (10 ng/ml LPS E. coli 055:B5 for 4 h) mouse Raw264.7 macrophage-like cells, as previously described.8 Constructs – EPCR was cloned from EA.hy926 endothelial cells following RNA isolation (RNAqueous-4PCR, Ambion), RT-PCR with oligo dT primers (Retroscript, Ambion) and PCR with EPCR specific primes (forward: 5′-TAGACTGCAGCTAGCGGAG-3′ and reverse: 5′-TGCCAGCCTCCATCAATCCAG-3′) using SuperTaq (Ambion). The obtained PCR product for EPCR was introduced into pCR 2.1-TOPO (Invitrogen) and transferred to pcDNA 3.1 (+) zeo (Invitrogen) using the HindIII and XhoI restriction sites. Since the EA.hy926 endothelial cells were found to encode for the A3 EPCR haplotype, residue Gly219 was mutated to Ser found in the more common human A1 haplotype using Quickchange mutagenesis. The sequence of the resulting wt-EPCR was verified and transfected into HEK-K293 cells. Soluble EPCR (sEPCR) was generated by introducing an AgeI restriction site in place of Leu211, the start of the transmembrane domain, and replacing the downstream EPCR sequence with synthetic double stranded oligomer encoding a short flexible spacer, 6xHis tag, stop codon and the appropriate AgeI and NotI (3′ MCS) overhang (forward: 5′-CCGGGCCCGCGGTTCGAAGGTCATCATCATCATCATCATTGA-3′ and reverse: 5′-GGCCTCAATGATGATGATGATGATGACCTTCGAACCGCGGGC-3′). The sequence of the resulting sEPCR was verified and transfected into HEK-293 cells after which sEPCR was purified from conditioned media using NTA-Sepharose (Pharmacia) followed by extensive dialysis against HBS buffer (20 mM Hepes, pH 7.4, 147 mM NaCl and 3 mM KCl) with 5 mM CaCl2. The SEAP coding sequence was obtained from pSEAP2-basic (Clonetech) and transferred to pcDNA3.1(+)neo using the restriction sites XbaI and HindIII. The reading frame downstream of SEAP was adjusted by ligation of a synthetic spacer (5′-CTAGAGGGCC-3′) between the XbaI and ApaI restriction sites. PAR-1 was cloned from liver cDNA (Invitrogen) using initial primers (forward: 5′-AGAAGTCAGGAGAGAGGGTGAAG-3′ and reverse: 5′-TAGAATCCTCAGGTTATTC-3′) and nested primers (forward: 5′-AGCCCGAGGCTAGCCAGCCT-3′ and reverse: 5′-GAATCCTCGAGTTATTCAC-3′). The obtained PCR fragment was introduced into pCR 2.1-TOPO and transferred to pcDNA 3.1 (+) neo using the KpnI and XbaI restriction sites. Subsequently, the PAR-1 coding region of PAR-1 derived from wt-PAR-1-pcDNA3.1 was fused to SEAP using the ApaI restriction site. The sequence of the resulting full length SEAP-PAR1 fusion, encoding for the pSEAP2-basic signal peptide, SEAP protein (residues 1–489), a short Tyr-Ser linker followed by Arg1 (residue 25 of the propeptide-PAR-1 sequence) of the mature PAR-1 protein (residues 1–401), was verified and stably transfected into HEK-293 cells. In addition, wt-EPCR was transfected into SEAP-PAR1 HEK-293 cells to obtain stable cells expressing wt-EPCR/SEAP-PAR1. REFERENCES
(1) Mosnier LO, Yang XV, Griffin JH. Activated protein C mutant with minimal anticoagulant activity, normal cytoprotective activity, and preservation of thrombin activable fibrinolysis inhibitor-dependent cytoprotective functions. J Biol Chem. 2007;282:33022–33033.
(2) Zhang L, Castellino FJ. A gamma-carboxyglutamic acid (gamma) variant (gamma 6D, gamma 7D) of human activated protein C displays greatly reduced activity as an anticoagulant. Biochemistry. 1990;29:10828–10834.
(3) Yan SC, Razzano P, Chao YB, Walls JD, Berg DT, McClure DB, Grinnell BW. Characterization and novel purification of recombinant human protein C from three mammalian cell lines. Biotechnology. 1990;8:655–661.
(4) Fernández JA, Xu X, Liu D, Zlokovic BV, Griffin JH. Recombinant murine-activated protein C is neuroprotective in a murine ischemic stroke model. Blood Cells Mol Dis. 2003;30:271–276.
(5) Chase T, Shaw E. p-Nitrophenyl-p'-guanidinobenzoate HCl: A new active site titrant for trypsin. Biochem Biophys Res Commun. 1967;29:508–514.
(6) Gale AJ, Heeb MJ, Griffin JH. The autolysis loop of activated protein C interacts with factor Va and differentiates between the Arg506 and Arg306 cleavage sites. Blood. 2000;96:585–593.
(7) Yang L, Bae JS, Manithody C, Rezaie AR. Identification of a specific exosite on activated protein C for interaction with protease activated receptor 1. J Biol Chem. 2007;282:25493–25500.
(8) Kerschen EJ, Fernandez JA, Cooley BC, Yang XV, Sood R, Mosnier LO, Castellino FJ, Mackman N, Griffin JH, Weiler H. Endotoxemia and sepsis mortality reduction by non-anticoagulant activated protein C. J Exp Med. 2007;204:2439–2448.
Files in this Data Supplement:
- Table S1. Kinetic constants for human and mouse wt-APC and E149A-APC amidolytic activity vs. chromogenic peptide substrates (PDF, 83.9 KB)
- Figure S1. Characterization of E149A-mAPC (JPG, 75 KB)
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(A) SDS-PAGE analysis without reducing agent of purified human and mouse APC visualized by silver staining, indicating the proteins are purified to homogeneity. The arrow pointing to the band at ~120kDa observed in mAPC corresponds to a presumed dimer of mAPC based on its molecular weight, disappearance under reducing conditions and its immunoreactivity with anti-mouse protein C antibodies on Western blot. (B) SDS-PAGE analysis with reducing agent of purified human (h) and mouse (m) protein C and APC visualized by silver staining. The migration of single chain protein C (sc), the protein C heavy chain (hc) and light chain (lc) are indicated on the right. The slightly faster migration of the APC heavy chain band vs. the protein C heavy chain band is consistent with the removal of the 12 amino acid activation peptide from protein C. The band at ~120 kDa attributed to a dimer of mouse APC under non-reducing conditions disappears following reduction without the generation of bands other than the expected single chain, heavy chain and light chain protein C or APC. (C) Amidolytic activity of wt-mAPC (□) and E149A-mAPC (◇) for the small chromogenic substrate (S-2366). (D) APC anticoagulant activity of E149A-mAPC (◇) and wt-mAPC (□) in an APTT clotting assays based on mouse plasma, showing that E149A-mAPC has increased anticoagulant activity compared to wt-mAPC (2.7-fold). (E) APC anti-inflammatory activity determined as inhibition of TNFα secretion of LPS-challenged mouse Raw264.7 macrophage-like cells by wt-mAPC (□) and E149A-mAPC (◇). Similar to E149A-hAPC, E149A-mAPC is devoid of anti-inflammatory activity.
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