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• W2063704060 abstract "Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that are mediated in part by its interactions with its cofactor protein S and the endothelial cell protein C receptor (EPCR). The protein C/APC Gla domain is implicated in both interactions. We sought to identify how the protein C Gla domain enables specific protein-protein interactions in addition to its conserved role in phospholipid binding. The human prothrombin Gla domain, which cannot bind EPCR or support protein S cofactor activity, has 22/45 residues that are not shared with the human protein C Gla domain. We hypothesized that the unique protein C/APC Gla domain residues were responsible for mediating the specific interactions. To assess this, we generated 13 recombinant protein C/APC variants incorporating the prothrombin residue substitutions. Despite anticoagulant activity similar to wild-type APC in the absence of protein S, APC variants APC(PT33-39) (N33S/V34S/D35T/D36A/L38D/A39V) and APC(PT36/38/39) (D36A/L38D/A39V) were not stimulated by protein S, whereas APC(PT35/36) (D35T/D36A) exhibited reduced protein S sensitivity. Moreover, PC(PT8/10) (L8V/H10K) displayed negligible EPCR affinity, despite normal binding to anionic phospholipid vesicles and factor Va proteolysis in the presence and absence of protein S. A single residue variant, PC(PT8), also failed to bind EPCR. Factor VIIa, which also possesses Leu-8, bound soluble EPCR with similar affinity to wild-type protein C, collectively confirming Leu-8 as the critical residue for EPCR recognition. These results reveal the specific Gla domain residues responsible for mediating protein C/APC molecular recognition with both its cofactor and receptor and further illustrate the multifunctional potential of Gla domains. Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that are mediated in part by its interactions with its cofactor protein S and the endothelial cell protein C receptor (EPCR). The protein C/APC Gla domain is implicated in both interactions. We sought to identify how the protein C Gla domain enables specific protein-protein interactions in addition to its conserved role in phospholipid binding. The human prothrombin Gla domain, which cannot bind EPCR or support protein S cofactor activity, has 22/45 residues that are not shared with the human protein C Gla domain. We hypothesized that the unique protein C/APC Gla domain residues were responsible for mediating the specific interactions. To assess this, we generated 13 recombinant protein C/APC variants incorporating the prothrombin residue substitutions. Despite anticoagulant activity similar to wild-type APC in the absence of protein S, APC variants APC(PT33-39) (N33S/V34S/D35T/D36A/L38D/A39V) and APC(PT36/38/39) (D36A/L38D/A39V) were not stimulated by protein S, whereas APC(PT35/36) (D35T/D36A) exhibited reduced protein S sensitivity. Moreover, PC(PT8/10) (L8V/H10K) displayed negligible EPCR affinity, despite normal binding to anionic phospholipid vesicles and factor Va proteolysis in the presence and absence of protein S. A single residue variant, PC(PT8), also failed to bind EPCR. Factor VIIa, which also possesses Leu-8, bound soluble EPCR with similar affinity to wild-type protein C, collectively confirming Leu-8 as the critical residue for EPCR recognition. These results reveal the specific Gla domain residues responsible for mediating protein C/APC molecular recognition with both its cofactor and receptor and further illustrate the multifunctional potential of Gla domains. Protein C is the key component of the anticoagulant protein C pathway (1Dahlback B. Villoutreix B.O. J. Thromb. Haemost. 2003; 1: 1525-1534Crossref PubMed Scopus (72) Google Scholar, 2Esmon C.T. Gu J.M. Xu J. Qu D. Stearns-Kurosawa D.J. Kurosawa S. Haematologica. 1999; 84: 363-368PubMed Google Scholar). Homozygous protein C deficiency is associated with severe neonatal thrombosis (3Seligsohn U. Berger A. Abend M. Rubin L. Attias D. Zivelin A. Rapaport S.I. N. Engl. J. Med. 1984; 310: 559-562Crossref PubMed Scopus (352) Google Scholar), whereas individuals with heterozygous deficiency are thrombophilic (4Reitsma P.H. Bernardi F. Doig R.G. Gandrille S. Greengard J.S. Ireland H. Krawczak M. Lind B. Long G.L. Poort S.R. et al.Thromb. Haemostasis. 1995; 73: 876-889Crossref PubMed Scopus (273) Google Scholar). Protein C is activated by the thrombin-thrombomodulin complex on endothelial cells (5Stearns-Kurosawa D.J. Kurosawa S. Mollica J.S. Ferrell G.L. Esmon C.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10212-10216Crossref PubMed Scopus (456) Google Scholar). Protein C activation is enhanced ∼5-fold by protein C binding to the endothelial cell protein C receptor (EPCR) 2The abbreviations used are: EPCR, endothelial cell protein C receptor; sEPCR, soluble EPCR; APC, activated protein C; RU, resonance unit; ETP, estimated thrombin potential; PE, phosphatidylethanolamine; FVIIa, factor VIIa; Gla, γ-carboxyglutamic acid. (5Stearns-Kurosawa D.J. Kurosawa S. Mollica J.S. Ferrell G.L. Esmon C.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10212-10216Crossref PubMed Scopus (456) Google Scholar). Activated protein C (APC) binds to anionic phospholipid surfaces, where it inactivates procoagulant cofactors factor Va (FVa) and factor VIIIa (FVIIIa), thereby attenuating thrombin generation (6Walker F.J. Sexton P.W. Esmon C.T. Biochim. Biophys. Acta. 1979; 571: 333-342Crossref PubMed Scopus (306) Google Scholar, 7Fay P.J. Smudzin T.M. Walker F.J. J. Biol. Chem. 1991; 266: 20139-20145Abstract Full Text PDF PubMed Google Scholar). Proteolysis of FVa and FVIIIa by APC is enhanced by the presence of the APC cofactor, protein S (8Walker F.J. Semin. Thromb. Hemostasis. 1984; 10: 131-138Crossref PubMed Scopus (118) Google Scholar). APC also has potent anti-inflammatory (9Bernard G.R. Vincent J.L. Laterre P.F. LaRosa S.P. Dhainaut J.F. Lopez-Rodriguez A. Steingrub J.S. Garber G.E. Helterbrand J.D. Ely E.W. Fisher Jr., C.J. N. Engl. J. Med. 2001; 344: 699-709Crossref PubMed Scopus (5068) Google Scholar, 10Joyce D.E. Gelbert L. Ciaccia A. DeHoff B. Grinnell B.W. J. Biol. Chem. 2001; 276: 11199-11203Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar, 11Taylor Jr., F.B. Chang A. Esmon C.T. D'Angelo A. Vigano-D'Angelo S. Blick K.E. J. Clin. Investig. 1987; 79: 918-925Crossref PubMed Scopus (770) Google Scholar) and anti-apoptotic properties (12Mosnier L.O. Griffin J.H. Biochem. J. 2003; 373: 65-70Crossref PubMed Scopus (204) Google Scholar, 13Cheng T. Liu D. Griffin J.H. Fernandez J.A. Castellino F. Rosen E.D. Fukudome K. Zlokovic B.V. Nat. Med. 2003; 9: 338-342Crossref PubMed Scopus (530) Google Scholar), which may be mediated in part by EPCR-bound APC activation of protease-activated receptor 1 (14Riewald M. Petrovan R.J. Donner A. Mueller B.M. Ruf W. Science. 2002; 296: 1880-1882Crossref PubMed Scopus (748) Google Scholar). Protein C is a vitamin K-dependent protein with a multidomain structure comprising a serine protease domain, two epidermal growth factor (EGF) domains, and a Gla domain (15Foster D. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4766-4770Crossref PubMed Scopus (132) Google Scholar). Situated at the protein C N terminus, the Gla domain consists of 45 amino acid residues. Of these, 9 glutamic acid (Glu) residues are post-translationally modified to γ-carboxyglutamic acid (Gla) residues. Gla residues facilitate Ca2+ ion coordination, which causes the domain to undergo a profound structural transition (16Christiansen W.T. Tulinsky A. Castellino F.J. Biochemistry. 1994; 33: 14993-15000Crossref PubMed Scopus (30) Google Scholar). In this conformation, the protein C Ca2+-bound Gla domain binds to anionic phospholipids exposed on the surface of activated endothelial cells and platelets, albeit with lower affinity than other vitamin K-dependent proteins (17McDonald J.F. Shah A.M. Schwalbe R.A. Kisiel W. Dahlback B. Nelsestuen G.L. Biochemistry. 1997; 36: 5120-5127Crossref PubMed Scopus (105) Google Scholar, 18Smirnov M.D. Safa O. Regan L. Mather T. Stearns-Kurosawa D.J. Kurosawa S. Rezaie A.R. Esmon N.L. Esmon C.T. J. Biol. Chem. 1998; 273: 9031-9040Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 19Sun Y.H. Shen L. Dahlback B. Blood. 2003; 101: 2277-2284Crossref PubMed Scopus (26) Google Scholar). The mechanism by which protein S exerts its cofactor activity on APC is not fully understood. Protein S enhances APC affinity for phospholipids and is required for optimal alignment of the APC active site for substrate cleavage (8Walker F.J. Semin. Thromb. Hemostasis. 1984; 10: 131-138Crossref PubMed Scopus (118) Google Scholar, 20Yegneswaran S. Wood G.M. Esmon C.T. Johnson A.E. J. Biol. Chem. 1997; 272: 25013-25021Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Protein S has also been reported to remove factor Xa (FXa) protection of FVa in the prothrombinase complex (21Hackeng T.M. Yegneswaran S. Johnson A.E. Griffin J.H. Biochem. J. 2000; 349 (Pt. 3): 757-764Crossref PubMed Scopus (18) Google Scholar). Several protein S domains have been shown to contribute to APC cofactor activity, including the thrombin-sensitive region, EGF1, EGF2, and the Gla domain (21Hackeng T.M. Yegneswaran S. Johnson A.E. Griffin J.H. Biochem. J. 2000; 349 (Pt. 3): 757-764Crossref PubMed Scopus (18) Google Scholar, 22Dahlback B. Hildebrand B. Malm J. J. Biol. Chem. 1990; 265: 8127-8135Abstract Full Text PDF PubMed Google Scholar, 23Giri T.K. de Frutos P.G. Yamazaki T. Villoutreix B.O. Dahlback B. Thromb. Haemostasis. 1999; 82: 1627-1633Crossref PubMed Google Scholar, 24Giri T.K. Villoutreix B.O. Wallqvist A. Dahlback B. de Frutos P.G. Thromb. Haemostasis. 1998; 80: 798-804Crossref PubMed Scopus (21) Google Scholar, 25He X. Rezaie A.R. J. Biol. Chem. 1999; 274: 4970-4976Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 26He X. Shen L. Villoutreix B.O. Dahlback B. J. Biol. Chem. 1998; 273: 27449-27458Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 27Stenberg Y. Drakenberg T. Dahlback B. Stenflo J. Eur. J. Biochem. 1998; 251: 558-564Crossref PubMed Scopus (27) Google Scholar, 28Saller F. Villoutreix B.O. Amelot A. Kaabache T. Le Bonniec B.F. Aiach M. Gandrille S. Borgel D. Blood. 2005; 105: 122-130Crossref PubMed Scopus (31) Google Scholar). A direct role for the APC Gla domain in mediating protein S cofactor activity was suggested by a study in which a recombinant protein C chimera was generated and the protein C Gla domain (residues 1-45) was replaced by that of prothrombin (18Smirnov M.D. Safa O. Regan L. Mather T. Stearns-Kurosawa D.J. Kurosawa S. Rezaie A.R. Esmon N.L. Esmon C.T. J. Biol. Chem. 1998; 273: 9031-9040Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). This chimera was resistant to protein S cofactor stimulation. Furthermore, the anticoagulant activity of an APC chimera in which the APC Gla domain residues 1-22 were substituted with the corresponding prothrombin residues was enhanced by protein S, suggesting a role for APC residues 22-45. However, the precise residues responsible, present in the protein C/APC Gla domain but not in prothrombin, were not determined. The protein C Gla domain is also required for EPCR binding (29Regan L.M. Mollica J.S. Rezaie A.R. Esmon C.T. J. Biol. Chem. 1997; 272: 26279-26284Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). A crystal structure of part of the protein C Gla domain (residues 1-33) bound to soluble EPCR (sEPCR) indicated that hydrophobic residues Phe-4 and Leu-8 contained within the ω-loop of the protein C Gla domain and Gla residues 7, 25 and 29, bind EPCR (30Oganesyan V. Oganesyan N. Terzyan S. Qu D. Dauter Z. Esmon N.L. Esmon C.T. J. Biol. Chem. 2002; 277: 24851-24854Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Despite the significant amino acid sequence conservation between the EPCR binding residues of the protein C/APC Gla domain and that of other vitamin K-dependent coagulation proteins, surprisingly only protein C/APC has been reported to bind EPCR. Binding competition experiments using FX, FXa, and protein S failed to prevent high affinity protein C-EPCR binding (31Fukudome K. Esmon C.T. J. Biol. Chem. 1994; 269: 26486-26491Abstract Full Text PDF PubMed Google Scholar). Consequently, the residues that enable the EPCR to recognize and bind specifically to the protein C/APC Gla domain are currently unknown. In this study, we evaluated 13 protein C/APC recombinant variants where protein C residues were substituted for those of prothrombin at non-identical positions (22/45 Gla domain residues). We assessed the binding of each variant for both phospholipids and sEPCR and evaluated the APC variants in anticoagulant activity assays in the presence and absence of protein S in both purified protein and plasma systems. We describe the critical residues in the APC Gla domain that mediate protein S cofactor activity. Moreover, we have identified the protein C Gla domain residue responsible for the specific “recognition” of EPCR and how its presence in factor VII (FVII) also enables it to bind EPCR. Generation of Protein C Variants—The pRc/CMV vector (Invitrogen) containing full-length protein C cDNA was used to generate recombinant wild-type protein C and as a template for PCR site-directed mutagenesis (QuikChange mutagenesis kit; Stratagene) using mutagenic oligonucleotide primers (available on request) as before (32Preston R.J. Villegas-Mendez A. Sun Y.H. Hermida J. Simioni P. Philippou H. Dahlback B. Lane D.A. FEBS J. 2005; 272: 97-108Crossref PubMed Scopus (37) Google Scholar). Expression vectors for each protein C variant were used to transfect human embryonic kidney 293 cells (European Collection of Cell Cultures, Wiltshire, UK), and G418-selected colonies expressing protein C at high levels were picked for further expansion. Serum-free conditioned medium containing each recombinant protein C was buffer exchanged against 20 mm Tris-HCl, pH 7.4, 150 mm NaCl. Protein C was purified by ion exchange chromatography using a Q-Sepharose Fast Flow column (Amersham Biosciences) with a gradient elution using 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 30 mm CaCl2 as previously described (19Sun Y.H. Shen L. Dahlback B. Blood. 2003; 101: 2277-2284Crossref PubMed Scopus (26) Google Scholar, 33Esmon C.T. Esmon N.L. Le Bonniec B.F. Johnson A.E. Methods Enzymol. 1993; 222: 359-385Crossref PubMed Scopus (57) Google Scholar, 34Castellino F.J. Geng J.P. Methods Enzymol. 1997; 282: 369-384Crossref PubMed Scopus (1) Google Scholar). Protein C concentrations were determined either by absorbance at 280 nm or by enzyme-linked immunosorbent assay (32Preston R.J. Villegas-Mendez A. Sun Y.H. Hermida J. Simioni P. Philippou H. Dahlback B. Lane D.A. FEBS J. 2005; 272: 97-108Crossref PubMed Scopus (37) Google Scholar). The purity of all recombinant proteins was confirmed by SDS-PAGE and Coomassie staining, and Western blotting was performed using a horse-radish peroxidase-conjugated polyclonal anti-protein C antibody (Dako, Ely, UK). To generate APC, wild-type protein C and protein C variants were activated with Protac as previously described (32Preston R.J. Villegas-Mendez A. Sun Y.H. Hermida J. Simioni P. Philippou H. Dahlback B. Lane D.A. FEBS J. 2005; 272: 97-108Crossref PubMed Scopus (37) Google Scholar). Kinetic values were used to active site titrate each recombinant APC variant against wild-type APC. Phospholipid Binding of Protein C Variants—A BIAcore X system (BIAcore, Uppsala, Sweden) was used to evaluate protein C binding to anionic phospholipids. 10 μg/ml of a Ca2+-dependent anti-protein C monoclonal antibody (Hematologic Technologies Inc.) was immobilized onto both flow cells of a CM5 sensor chip (BIAcore), corresponding to ∼12,000 RU on each flow cell. 50 μl of wild-type or variant protein C was then passed over the surface of one flow cell at a flow rate of 30 μl/min until a response of ∼2,000 RU was achieved for each protein C species tested. The flow cell with no protein C bound was used to detect nonspecific binding. Phospholipids phosphatidylcholine/phosphatidylserine (PC/PS, 80%:20%) were prepared as described previously (32Preston R.J. Villegas-Mendez A. Sun Y.H. Hermida J. Simioni P. Philippou H. Dahlback B. Lane D.A. FEBS J. 2005; 272: 97-108Crossref PubMed Scopus (37) Google Scholar) in HBS-N buffer (100 mm HEPES, pH 7.4, 150 mm NaCl) (BIAcore) containing 3 mm CaCl2 and 0.6 mm MgCl2 and then passed over the surface of the sensor chip at 30 μl/min for 1 min. Phospholipids were dissociated from the surface using 5 μl of 40 mm octyl glucoside (Sigma) at the same flow rate. The maximum binding response at each phospholipid concentration was assessed using BIA-evaluation software (3.0) (BIAcore). The sensor chip surface was regenerated using HBS-EP buffer (100 mm HEPES, pH 7.4, 150 mm NaCl, 3 mm EDTA, 0.005% v/v surfactant P20) (BIA-core), for 5 min at 30 μl/min. EPCR Binding Affinity of Protein C Variants—Protein C binding to sEPCR was quantitatively assessed as previously described (32Preston R.J. Villegas-Mendez A. Sun Y.H. Hermida J. Simioni P. Philippou H. Dahlback B. Lane D.A. FEBS J. 2005; 272: 97-108Crossref PubMed Scopus (37) Google Scholar). Briefly, 300 ng of RCR-2 (a kind gift from K. Fukudome, Saga Medical School, Japan; characterized in Ref. 32Preston R.J. Villegas-Mendez A. Sun Y.H. Hermida J. Simioni P. Philippou H. Dahlback B. Lane D.A. FEBS J. 2005; 272: 97-108Crossref PubMed Scopus (37) Google Scholar) was injected for 6 min across both flow cells of a CM5 sensor chip, generating a response of 8,000-9,000 RU. sEPCR was expressed as described previously (32Preston R.J. Villegas-Mendez A. Sun Y.H. Hermida J. Simioni P. Philippou H. Dahlback B. Lane D.A. FEBS J. 2005; 272: 97-108Crossref PubMed Scopus (37) Google Scholar). sEPCR in HBS-P buffer (100 mm HEPES, pH 7.4, 150 mm NaCl, 0.005% v/v surfactant p20) containing 3 mm CaCl2 and 0.6 mm MgCl2 was injected and equilibrated. Protein C concentrations (12.5-200 nm) were sequentially injected over both flow cells at a flow rate of 30 μl/min for 80 s. The flow cell immobilized with RCR-2 without sEPCR bound was used to detect nonspecific binding. Any influence of mass transport effects was discounted from results of binding and dissociation at different flow rates. HBS-EP buffer (BIAcore) was used to dissociate the protein C-sEPCR complex. The RCR-2 surface was regenerated with 10 mm glycine-HCl, pH 2.5, after each set of experiments. Determination of APC-mediated Factor Va Proteolysis—To determine FVa degradation by APC, 0.32 nm APC was incubated at 37 °C with phospholipid vesicles (PC/PS, 80%:20%) and 4 nm FVa (Hematologic Technologies Inc.) in 40 mm Tris-HCl, 140 mm NaCl, 3 mm CaCl2, and 0.3% w/v bovine serum albumin (0.08 nm APC, 19 μm phospholipids, and 1 nm FVa, final concentration). At specified time points over 20 min, 2-μl aliquots were removed and added to a prothrombinase mixture consisting of 75 μm phospholipids, 3 nm factor Xa, and 1.5 μm prothrombin (25 μm phospholipids (PC/PS, 80%:20%), 1 nm factor Xa, and 0.5 μm prothrombin, final concentrations) (Hematologic Technologies Inc.) for 3 min. Each reaction was then stopped using 5 μl of ice-cold 0.5 m EDTA. 100 μl of the reaction mixture was removed and incubated with 50 μl of chromogenic substrate S-2238 (Chromogenix, Milan, Italy) to assess thrombin generation. The rate of S-2238 cleavage was measured at 405 nm using an iEMS plate reader MF (Labsystem, Basingstoke, UK). Protein S-enhanced Proteolysis of FVa by APC—Human protein S (2.5-25 nm) was incubated with 0.8 nm APC, 8 nm FVa, and 75 μm phospholipid vesicles (PC/PS/phosphatidylethanolamine (PE), 60%:20%:20%) in 40 mm Tris-HCl, pH 7.4, 140 mm NaCl, 3 mm CaCl2, 0.3% (w/v) bovine serum albumin (0.2 nm APC, 2 nm FVa, and 19 μm phospholipid vesicles, final concentrations) for 2 min at 37 °C. A 2-μl aliquot was added to 0.3 nm FXa, 1.5 μm prothrombin, and 75 μm phospholipid vesicles (0.1 nm FXa, 0.5 μm prothrombin, and 25 μm phospholipid vesicles, final concentrations) at 37 °C for 3 min. 5 μl of ice-cold 250 mm EDTA stopped the reaction. 50 μl of S-2238 was diluted 1:2 with 50 mm Tris-HCl, 150 mm NaCl, pH 8.3, and added to 50 μl of the reaction mixture. The rate of S-2238 cleavage was then measured as before. Protein S-dependent APC Anticoagulant Activity in Plasma— Thrombin generation was assessed using a Fluoroskan Ascent Plate Reader (Thermo Lab System, Helsinki, Finland) in combination with Thrombinoscope software (SYNAPSE BV) as previously described (35Hemker H.C. Beguin S. Thromb. Haemostasis. 1995; 74: 134-138Crossref PubMed Scopus (264) Google Scholar, 36Sere K.M. Rosing J. Hackeng T.M. Blood. 2004; 104: 3624-3630Crossref PubMed Scopus (59) Google Scholar). 80 μl/well of protein C-deficient plasma (Affinity Biologicals Inc., Ontario, Canada) was incubated with 240 μg/ml of anti-protein S polyclonal antibody or the equivalent volume of water for 15 min at room temperature. Each plasma pool was then incubated with 16 pm recombinant tissue factor (Dade Innovin, Dade Behring, Marburg, Germany) and 17.5 μm phospholipid vesicles (PC/PS/PE, 60%:20%:20%). 42 μg/ml corn trypsin inhibitor (Hematologic Technologies Inc.) was added to prevent contact activation. APC (3.5-7 nm) was added to microtiter plate wells and the plasma mix with or without anti-protein S polyclonal antibody subsequently added, making a final volume of 100 μl/well. Thrombin generation was initiated by automatic dispensation of 2.5 mm Z-Gly-Gly-Arg-AMC·HCl, 2.5% Me2SO, 18 mm HEPES, pH 7.4, 54 mg/ml bovine serum albumin, and 100 mm CaCl2 into each well. Thrombin generation was quantitated using a thrombin calibration standard (Synapse, Maastricht, Netherlands). Measurements were taken at 20-s intervals for 40 min at wavelengths 390 nm (excitation) and 460 nm (emission). Generation and Characterization of Protein C Variants—To identify protein C Gla domain residues that enable protein S cofactor activity and EPCR recognition, a series of protein C variants with prothrombin residue substitutions at non-identical positions were generated (Fig. 1). These variants were divided into two groups, those situated at the N-terminal end of the protein C Gla domain (residues 1-25) and those located at the C-terminal end incorporating the hydrophobic stack (residues 26-45). The purity of each recombinant protein C variant was confirmed by Coomassie staining and exhibited identical characteristic double bands under non-reducing conditions upon Western blotting with an anti-protein C polyclonal antibody (data not shown). Phospholipid Binding of Protein C Variants—To characterize the Gla domain integrity of the recombinant protein C variants, the ability of each variant to bind to phospholipids was assessed. Protein C was bound to the surface of a CM5 sensor chip by a Ca2+-dependent anti-protein C monoclonal antibody directed against the heavy chain, and not the Gla domain-containing light chain, of protein C. A protein C variant (E16D), possessing a residue substitution that causes aberrant Ca2+ binding and Gla domain misfolding (32Preston R.J. Villegas-Mendez A. Sun Y.H. Hermida J. Simioni P. Philippou H. Dahlback B. Lane D.A. FEBS J. 2005; 272: 97-108Crossref PubMed Scopus (37) Google Scholar), was found to bind to the immobilized antibody with equal affinity to human protein C, indicating that the antibody epitope was not on the Gla domain (data not shown). Increasing concentrations of anionic phospholipid vesicles were passed over the protein C surface and specific binding was detected (Fig. 2A). Due to the slow dissociation rate of the phospholipid-protein C interaction, thorough analysis of the binding kinetics was not possible. The maximum binding response at each phospholipid concentration was therefore measured and used to qualitatively assess phospholipid binding. The binding response of both human protein C and recombinant wild-type protein C at each phospholipid concentration tested was essentially identical (Fig. 2B). When the E16D protein C variant (which does not bind phospholipids) (32Preston R.J. Villegas-Mendez A. Sun Y.H. Hermida J. Simioni P. Philippou H. Dahlback B. Lane D.A. FEBS J. 2005; 272: 97-108Crossref PubMed Scopus (37) Google Scholar) was immobilized, a minimal response to phospholipids was detected (Fig. 2B, top panel), demonstrating the specificity of this approach. The phospholipid binding of the remaining variants was found to be very similar to that of wild-type protein C (Fig. 2B, middle and lower panels), with the exception of PC(PT21/23/24) and PC(PT31-35). These variants exhibited markedly reduced binding to phospholipids (Fig. 2B, middle and lower panel, respectively), suggesting that these variants most likely possess a misfolded Gla domain due to suboptimal γ-carboxylation. The defective phospholipid binding of both variants also resulted in severely impaired anticoagulant activity in protein C-deficient plasma (data not shown). Consequently, these variants were not investigated further. Protein S-independent FVa Proteolysis by APC Variants— Each protein C variant was activated by Protac, and the amidolytic activity of each APC variant against a short chromogenic substrate (S-2366) was tested and found to be identical to that of wild-type APC (data not shown). To determine whether the introduced prothrombin residue substitutions influenced FVa proteolysis in the absence of protein S, each APC variant was investigated for its ability to proteolyze FVa in a phospholipid-dependent time course reaction (Fig. 3). Under these conditions, wild-type APC reduced FVa cofactor activity to 21 ± 5% after 20 min. APC variants were found to inactivate FVa at effectively the same rate as wildtype APC. Some variants exhibited mildly increased FVa proteolysis, including APC(PT11/12) (S11G/S12N), APC(PT33-39) (N33S/V34S/D35T/D36A/L38D/A39V), and APC(PT35/36) (D35T/D36A) (Fig. 3, A and B), possibly as a result of the slightly increased affinity for phospholipids of these variants (Fig. 2). Protein S Does Not Enhance FVa Proteolysis by APC Variants APC(PT33-39), APC(PT35/36), and APC(PT36/38/39)—To examine the influence of substituted protein C Gla domain residues in mediating protein S cofactor activity, the ability of each APC variant to proteolyze FVa in the presence of varying protein S concentrations was determined. Wild-type APC reduced FVa cofactor activity to 27 ± 8% of its original activity in the presence of 25 nm protein S (Fig. 4). All N-terminal Gla domain APC variants tested were stimulated by protein S normally (Fig. 4A), as were half of the C-terminal variants (Fig. 4B). However, FVa proteolysis by APC variants APC(PT33-39) and APC(PT36/38/39) (D36A/L38D/A39V) was not enhanced by protein S. The remaining FVa activity in the presence of 25 nm protein S with variants APC(PT33-39) and APC(PT36/38/39) was 92 ± 7 and 91 ± 2%, respectively (Fig. 4B). The APC(PT35/36) variant also exhibited a significantly impaired response to protein S compared with wild-type APC, only reducing FVa cofactor activity to 73 ± 9% of its original value in the presence of 25 nm protein S (Fig. 4B). APC Variants Containing Residue Substitutions between Positions 33-39 Are Not Enhanced by Protein S in Plasma—To assess whether the observed reduced sensitivity to protein S exhibited by APC variants APC(PT33-39), APC(PT36/38/39), and APC(PT35/36) using purified plasma proteins was replicated in the plasma milieu, an assay measuring tissue factor-induced thrombin generation in protein C-deficient plasma was used. Thrombin generation in protein C-deficient plasma was initiated by the presence of tissue factor and anionic phospholipid vesicles, generating an estimated thrombin potential (ETP) of 1720 ± 82 nm. When wild-type APC was incubated in the protein C-deficient plasma, the ETP diminished in a concentration-dependent manner (Fig. 5A, top panel). At the highest APC concentration tested (7 nm), the ETP was reduced to 15 ± 1% of the ETP determined with no APC present (Fig. 5, A and C). However, when a polyclonal anti-protein S antibody known to inhibit APC-dependent protein S cofactor function (36Sere K.M. Rosing J. Hackeng T.M. Blood. 2004; 104: 3624-3630Crossref PubMed Scopus (59) Google Scholar) was incubated with 7 nm wild-type APC, the ETP was not diminished and the anticoagulant effect of wild-type APC was effectively lost (Fig. 5B). In this way, the assay was shown to be completely dependent upon protein S to facilitate APC down-regulation of thrombin generation. Variants APC(PT33-39), APC(PT35/36), and APC(PT36/38/39) were tested in both the presence and absence of the inhibitory anti-protein S antibody. In the presence of the anti-protein S antibody, minimal ETP reduction (<10%) was observed for each of the APC variants tested (data not shown). In the absence of the anti-protein S antibody, APC(PT35/36) reduced thrombin generation ∼2-fold less effectively than wild-type APC (Fig. 5, A and C). Variants APC(PT33-39) and APC(PT36/38/39) failed to inhibit thrombin generation at any of the APC concentrations tested (Fig. 5, A and C), highlighting their inability to interact with protein S to reduce thrombin generation. The phospholipid PE has been previously shown to significantly enhance APC anticoagulant activity (18Smirnov M.D. Safa O. Regan L. Mather T. Stearns-Kurosawa D.J. Kurosawa S. Rezaie A.R. Esmon N.L. Esmon C.T. J. Biol. Chem. 1998; 273: 9031-9040Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). To confirm that the results observed for variants APC(PT33-39), APC(PT35/36), and APC(PT36/38/39) were not a consequence of defective interaction with PE, the same experiments were performed in the presence of phospholipid vesicles composed of PC/PS only. The results of these experiments we" @default.
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• W2063704060 title "Multifunctional Specificity of the Protein C/Activated Protein C Gla Domain" @default.
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