Mouse recombinant protein C variants with enhanced membrane affinity and hyper-anticoagulant activity in mouse plasma Michael J Krisinger1, Li Jun Guo1, Gian Luca Salvagno2, Gian Cesare Guidi2, Giuseppe Lippi2 and Bjorn Dahlback1 ă ă Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University, University Hospital, Malmo, Sweden ă Clinical Chemistry Section, Department of Morphological-Biomedical Sciences, University Hospital of Verona, Italy Keywords anticoagulation; Gla domain; mouse protein C; mouse plasma; protein–membrane interactions Correspondence B Dahlback, Department of Laboratory ¨ Medicine, Division of Clinical Chemistry, Wallenberg Laboratory, Entrance 46, Floor 6, Lund University, University Hospital, S-20502 Malmo, Sweden ă Fax: +46 40 337044 Tel: +46 40 331501 E-mail: bjorn.dahlback@med.lu.se (Received July 2009, revised September 2009, accepted September 2009) doi:10.1111/j.1742-4658.2009.07371.x Mouse anticoagulant protein C (461 residues) shares 69% sequence identity with its human ortholog Interspecies experiments suggest that there is an incompatibility between mouse and human protein C, such that human protein C does not function efficiently in mouse plasma, nor does mouse protein C function efficiently in human plasma Previously, we described a series of human activated protein C (APC) Gla domain mutants (e.g QGNSEDY-APC), with enhanced membrane affinity that also served as superior anticoagulants To characterize these Gla mutants further in mouse models of diseases, the analogous mutations were now made in mouse protein C In total, seven mutants (mutated at one or more of positions P10S12D23Q32N33) and wild-type protein C were expressed and purified to homogeneity In a surface plasmon resonance-based membranebinding assay, several high affinity protein C mutants were identified In Ca2+ titration experiments, the high affinity variants had a significantly reduced (four-fold) Ca2+ requirement for half-maximum binding In a tissue factor-initiated thrombin generation assay using mouse plasma, all mouse APC variants, including wild-type, could completely inhibit thrombin generation; however, one of the variants denoted mutant III (P10Q ⁄ S12N ⁄ D23S ⁄ Q32E ⁄ N33D) was found to be a 30- to 50-fold better anticoagulant compared to the wild-type protein This mouse APC variant will be attractive to use in mouse models aiming to elucidate the in vivo effects of APC variants with enhanced anticoagulant activity Introduction Protein C is a vitamin K-dependent c-carboxyglutamic acid-containing protein (Gla protein) found in human and mouse plasma at a concentration of approximately 70 nm [1] This zymogen is efficiently converted by the thrombin–thrombomodulin complex to the multifunctional serine protease activated protein C (APC) With its cofactor, protein S, APC degrades factors Va and VIIIa on anionic phospholipid membranes, thereby Abbreviations APC, activated protein C; Cmax, maximal concentration of thrombin; ETP, endogenous thrombin potential; Gla protein, c-carboxyglutamic acid-containing protein; DOPS, 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine]; FU, fluorescence units; PE, phosphatidylethanolamine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; PS, phosphatidylserine; Rmax, maximum surface coverage; RU, response units; SPR, surface plasmon resonance; Tmax, time required to reach maximum thrombin generation 6586 FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS M J Krisinger et al efficiently turning off the major driving force of coagulation Although historically known for its role in anticoagulation, APC was recently revealed to have cytoprotective, anti-inflammatory and anti-apoptotic functions These new functions of APC are related to the ability of APC to bind endothelial protein C receptor and activate protease activated receptor 1, triggering intracellular signaling [2–4] Moreover, recombinant human APC was recently shown to inhibit integrin-mediated neutrophil migration by a direct interaction with leukocyte b1 and b3 integrin receptors [5] APC appears to play a central role in the pathogenesis of sepsis and associated organ dysfunction In patients with sepsis, the APC system malfunctions at almost all levels First, plasma levels of the zymogen protein C are low or very low because of impaired synthesis, consumption and degradation by proteolytic enzymes such as neutrophil elastase [6] Furthermore, significant down-regulation of thrombomodulin caused by pro-inflammatory cytokines such as tumor necrosis factor-a and interleukin-1 has been demonstrated, resulting in diminished protein C activation [7] The protective effects of APC supplementation in patients with severe sepsis complicated with disseminated intravascular coagulation [8] remain to be fully elucidated and are likely the result of its ability to modulate multiple biochemical pathways [7] A prerequisite for Gla protein-membrane binding is the saturation of seven Ca2+ sites in the N-terminal Gla domain, which changes its tertiary structure from an unfolded and nonfunctional conformation to a tightly folded membrane-binding domain [9,10] This Ca2+ binding requires the presence of Gla residues The Gla domains within the protein C family comprise 44 amino acids and contain between nine and 11 Gla residues, which mediate the Ca2+ interaction In human protein C, a detailed analysis of the function of each of these Gla residues has been evaluated [11] Of the Gla residues, nine are strictly conserved throughout the Gla proteins From crystal structures of the Gla domain of prothrombin and factor VIIa, the placement of the seven Ca2+ in relation to their Gla ligands is almost identical in the two proteins [12] The conformational transition induced by the cooperative binding of Ca2+ turns the N-terminal part of the Gla domain inside out, exposing the hydrophobic x-loop to solvent and burying the majority of the Gla residues Of the seven Ca2+, the majority are buried and are integral to maintain the membrane-binding conformation [9,13] A few of the Gla-bound Ca2+ are accessible to solvent and may play a role in membrane binding A membrane-bound structure of a Gla protein does not exist, hampering our understanding of Anticoagulant mouse protein C variants how the Gla domain engages and reversibly binds to a membrane surface Thus, it is also unclear how the Gla domains of the previously engineered mutants (e.g human QGNSEDY-APC; see below) have been selectively altered to enhance membrane binding However, it does appear that electrostatic, hydrophobic and specific lipid headgroup interactions are all involved in mediating the interaction The nature of the phospholipid membrane also contributes to binding efficiency, with phosphatidylserine (PS) and, to a lesser extent, phosphatidylethanolamine (PE) being generally accepted as most important membrane phospholipids in promoting efficient binding, complex assembly and enzyme catalysis in vivo Membrane affinity of a Gla protein often correlates with its membrane localized activity Strategies used to increase the affinity of the Gla protein–membrane interaction involve Gla-domain mutation (for human protein C) [14–16], Gla domain substitution [17] and covalent dimerization of the Gla protein [18] We have previously created several Gla-domain mutated human protein C variants with enhanced anticoagulant activity One of these variants with several Gla domain mutations, QGNSEDY-human APC (H10Q ⁄ S11G ⁄ S12N ⁄ D23S ⁄ Q32E ⁄ N33D ⁄ H44Y), bound phospholipid membranes with increased (approximately seven-fold) affinity compared to wild-type [16] QGNSEDYhuman APC was shown to be potent in both a human plasma-based clotting assay (20-fold better) [16] and a FVa-degradation assay, cleaving R306 (18-fold) and R506 (four-fold) more efficiently [19] However, the variant had no antithrombotic effect when used in a rat model of arterial thrombosis [20,21] The lack of effect was possibly a result of species–species differences between human protein C and the rat hemostatic system The reason for the poor anticoagulant effect of human APC in rat plasma remains unknown but may be a result of rat FVa ⁄ FVIIIa being poor substrates for human APC [21] APC variants with enhanced anticoagulant activity resulting from improved membrane-binding ability may prove more efficient than wild-type APC in the treatment of different diseases (e.g thromboembolism and sepsis) [13] The low-affinity binding of APC to negatively-charged phospholipid membranes may be adequate under normal healthy conditions because protein S serves as a specific cofactor to increase the membrane binding of APC at certain locations The situation may be different under pathologic conditions such as sepsis, where a higher membrane-binding ability of APC could potentially be beneficial, in particular because protein S and FV may be consumed under these conditions High affinity mouse APC variants FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS 6587 Anticoagulant mouse protein C variants M J Krisinger et al will allow the in vivo elucidation of the biologic consequences of the enhanced membrane-binding ability of protein C and may open a path for the development of APC variants with improved therapeutic potential in sepsis, as well as other thromboembolic disorders Although human protein C variants with enhanced affinity and function can be created, interspecies incompatibility in functional assays using human protein C in animal model systems, prompted us to characterize several mouse protein C variants in the present study Individual amino acids residues within the Gla domain contribute to the membrane affinity differences reported for the Gla proteins In the 44-residue Gla domain of human and mouse protein C, there are eight amino acid differences Thus, wild-type mouse protein C as well as seven variants mutated at three regions (positions 10, 12, 23, 32 and 33) of the Gla domain were purified and characterized The results obtained indicate that the functional improvements were closely related to enhanced membrane affinity The mutant with highest function, mutant III (P10Q ⁄ S12N ⁄ D23S ⁄ Q32E ⁄ N33D), showed reduced Ca2+ dependence for membrane binding and a 30-50fold inhibition improvement over wild-type in tissuefactor-dependent thrombin generation in mouse plasma Overall, the proteins described in the present study provide insight into the Gla protein–membrane interaction and identify new reagents with varying degrees of anticoagulant potency that may be of use for testing in murine models of sepsis and thromboembolic disorders Fig SDS-PAGE analysis of recombinant mouse protein C and APC Purified proteins (8 lg) were incubated with human thrombinthrombomodulin for h (odd lanes) or 24 h (even lanes) Thrombin catalysis was stopped with excess hirudin and subjected to 12% SDS-PAGE under reducing conditions Approximately 0.1 lg of protein C (odd numbered lanes) or APC (even numbered lanes) was applied to each lane and visualized by silver staining Protein C variants and molecular weight markers (MWM) ran in each lane are indicated The location of heavy chain (HC), light chain (LC) and thrombin (IIa) is also indicated Results Expression and characterization of mouse protein C variants To determine whether the mutations previously made in human protein C result in a similar enhancement of both membrane affinity and anticoagulant activity in a mouse system, the analogous mutations were made in mouse protein C Wild-type and seven variants of mouse protein C (Fig 1) were expressed and purified SDS-PAGE analysis of the purified proteins (Fig 2) demonstrated slightly different mobilities of the light chains, an effect caused by the mutations, whereas the Fig Gla domain sequence alignment from different species and mouse protein C variants used in the present study N-terminal Gla sequence (1–44) is shown and defined between the propeptidase and chymotrypsin cleavage sites Positions in the sequence at which c-carboxylation of glutamic acid residues is either known to occur or may occur are indicated by X The numbering at the top refers to the mouse protein C sequence Highlighted residues are different with respect to wild-type mouse protein C Sequences used for comparison were obtained from NCBI with accession numbers: protein C for mouse (NP_032960.2), human (NP_000303.1), rat (NP_036935.1), bovine (XP_585990.3) and human prothrombin (NP_000497.1) 6588 FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS M J Krisinger et al heavy chains migrated to similar positions All proteins were fully activated by the human thrombin–TM complex, as demonstrated by the shift of the heavy chains to slightly lower molecular weight positions The amidolytic activities of activated protein C mutants were comparable with that of wild-type protein C (data not shown) The proteins bound Ca2+ similar to their human counterparts, as judged by the shift in mobilities in native agarose gel electrophoresis in the presence of Ca2+ compared to EDTA (data not shown) The proteins were found to be c-carboxylated, as judged by western blotting using a Gla-specific antibody (Fig S1) Membrane binding ability of wild-type and variants of mouse protein C To determine the functional significance of the substituted Gla domain residues, we measured membrane binding properties by surface plasmon resonance (SPR) Chips were coated with 0-20-80, 0-10-90 and 20-10-70 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine ⁄ 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] ⁄ 1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine (POPE-DOPS-POPC) liposomes, whereas a control surface was either left blank or coated with 100% POPC We first measured the binding of each protein at equi-molar concentration (100 nm) to estimate their relative membrane binding abilities (Fig 3A–C) Noticeably, mutants II and III stand out from the other proteins analyzed, obtaining the highest responses for all membrane types Mutants V and VII also show a significant binding-response enhancement, whereas mutations introduced into mutants IV and VI had little effect relative to the wild-type protein (Fig 3A–C, insets) Figure 3D–F shows the equilibrium binding analysis of the protein– membrane interactions, and the KD values determined from the curve fitting are summarized in Table The affinities of wild-type mouse protein C for 0-20-80 and 20-10-70 liposomes are comparable (KD  lm), with a value lower than that of the human ortholog (KD = 2.1 lm) [15] and comparable with the bovine ortholog (KD = 9.2 lm) [14], as assessed previously under similar experimental conditions Using 0-20-80 or 20-10-70 membranes, mouse protein C variants that show a considerable improvement in membrane affinity over wild-type are mutant II (12-fold KD decrease), mutant III (six-fold KD decrease) and mutants V and VII (three- to four-fold KD decrease) Equilibrium binding dissociation constants, using 0-10-90 membranes, could only be determined for the high affinity proteins A further improvement in membrane binding of the variants is shown in terms of membrane bind- Anticoagulant mouse protein C variants ing occupancy at the saturating protein concentration, a parameter experimentally determined as Rmax For 0-20-80 membranes, the respective binding Rmax determined for wild-type [722 response units (RU)], and mutants II (3569 RU), III (4380 RU) and VII (2060 RU), is clearly different, as is also evident from an inspection of Fig 3D (or the other membranes in Fig 3E,F) All variants were tested using the same immobilized membrane preparation Thus, different variants are able to utilize a different number of binding sites on the membrane surface For example, mutant II can utilize approximately five times as many binding sites on a 0-20-80 membrane as wild-type protein C Importance of the liposome phospholipid composition on membrane binding Simple model membranes composed of one, two or three synthetically-derived phospholipids were used to assess membrane binding Membranes composed entirely of POPC were inert to binding, whereas DOPS or DOPS with POPE-containing liposomes were necessary to obtain a binding response By varying the DOPS composition, we were able to show binding specificity in terms of DOPS content Doubling the DOPS content from 10 to 20 mol % resulted in increased binding sites with enhanced affinity (Fig 3D,E and Table 1) For example, mutant II binds to 0-10-90 with KD = 2.45 lm ⁄ Rmax = 3005 RU, whereas binding to 0-20-80 is improved with KD = 0.66 lm ⁄ Rmax = 3569 RU PE has been shown to enhance the assembly and function of several clotting factor complexes [22,23] We also show that POPE influences the binding of mouse protein C Comparing the binding data of 20-10-70 and 0-10-90 membranes (Fig 3E,F and Table 1), we observe the effect that POPE has on membrane binding when holding DOPS at a fixed concentration Substantial improvements in both the number of binding sites and average affinity are observed with the POPE containing membrane Importance of Ca2+ on membrane binding Because the Gla protein–membrane interaction is highly dependent on Ca2+, we also investigated how the introduced mutations affect membrane binding as a function of Ca2+ concentration Figure 4A presents a representative sensorgram showing the effect of Ca2+ on the interaction of mutant II, at fixed concentration, with 0-10-90 membranes Wild-type protein C at 20 mm Ca2+ is included as a standard for comparison As expected, the Gla protein–membrane interaction FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS 6589 Anticoagulant mouse protein C variants M J Krisinger et al Fig Protein–membrane interaction of wild-type and variant mouse protein C to liposomes of varying phospholipid composition Protein C variants (wild-type, I–VII at 0.1 lM) or running buffer was injected for (association), over either the (A) 0-20-80 or (B) 0-10-90 or (C) 20-10-70 POPE-DOPS-POPC membrane bilayer surface, to determine their relative binding efficiencies Dissociation under running buffer conditions was followed for an additional Ca2+ concentrations used throughout were mM The SPR response curves are shown after background correction using a blank control flow cell Binding to the control surface was not apparent and no evidence of nonspecific binding was evident from an injection of Gla-less, prethrombin-1 (10 lM, not shown) Similar amounts of 0-20-80 (5539 RU), 0-10-90 (5738 RU) and 20-10-70 (5997 RU) liposomes were immobilized allowing comparisons Protein labeling is shown Insets show the same data on a smaller scale highlighting the low affinity binders Note y-axis scale differences in (B) Steady-state binding of mouse protein C wild-type ( ), mutant II ( ), III (.) and VII ( ), over either the (D) 0-20-80 or (E) 0-10-90 or (F) 20-10-70 POPE-DOPS-POPC membrane bilayer surface, was measured using the indicated protein concentrations Responses obtained at equilibrium were used to generate a binding isotherm fitted to a one-site binding hyperbola using nonlinear least squares analysis Binding isotherms were used to determine KD reported in Table and Rmax Additional details are provide in the Experimental procedures is highly dependent on Ca2+, with maximum binding occurring at approximately 10 mm Ca2+ Figure 4B– D shows the equilibrium binding analysis of the protein–membrane interactions at different Ca2+ concentrations, and [Ca2+]1 ⁄ max determined from the curve fitting are summarized in Table Employing a 6590 20-10-70 membrane, half-maximum Ca2+ concentrations required for mutant II (3.7 mm) and III (3.7 mm) are much improved compared to wild-type ( 11 mm) and approach that of plasma-derived human prothrombin (1.8 mm), comprising an efficient membrane-binding Gla protein FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS M J Krisinger et al Anticoagulant mouse protein C variants Table Effect of Gla-domain mutations on mouse protein C ⁄ APC Membrane dissociation constants (KD) at various membrane compositions were determined by SPR for mouse protein C Cmax and ETP generated in mouse plasma were determined using mouse APC Further details on methodology and experimental conditions are provided in Fig (membrane binding) and Fig (thrombin generation) Protein C Membrane affinity (POPE-DOPS-POPC) Activated protein C Thrombin generation in mouse plasma KD (lM)a Cmax (Fmin)1)e ETP (FU)e,f nMg 0-20-80b Buffer control Wild-type Mutant I Mutant II Mutant III Mutant IV Mutant V Mutant VI Mutant VII 0-10-90 20-10-70 nM g – 7.26 5.55 0.66 1.32 7.10 2.05 7.47 2.55 – NAd NA 2.45 8.70 NA 8.40 NA 8.77 – 8.77 4.96 0.67 1.44 6.79 2.28 5.99 2.67 276 241 105 165 15 247 24 ± ± ± ± ± ± ± ± 0.50c 0.26 0.04 0.16 0.71 0.20 0.46 0.25 ± 0.13 ± 0.98 ± 1.15 ± 0.75 ± ± ± ± ± ± ± ± 0.75 0.16 0.05 0.18 0.45 0.17 0.45 0.23 ± ± ± ± ± ± ± ± ± 32 20 28 25 18 18 26 5677 5380 2813 737 658 4094 927 5467 1175 ± ± ± ± ± ± ± ± ± 171 50 682 118 88 674 401 67 531 a KD is a representative determination from three experiments b Membranes used were 100 nm extruded liposomes with synthetic phospholipids: POPE-DOPS-POPC (mol %) c SE from one-site binding hyperbola fitting d NA, not available Concentrations tested did not allow determination of KD e SD from three independent experiments f ETP determined after 60 g Concentration of APC added to assay Wild-type protein C and mutant II have different maximum surface coverage (Rmax) and therefore any attempt to draw conclusions based on absolute response values is erroneous Furthermore, because human prothrombin (72 kDa) has a different molecular mass than that of mouse protein C (56 kDa), absolute response values cannot be directly compared However, calculating the fraction of binding relative to Rmax (equilibrium response at indicated Ca2+ divided by Rmax at saturating Ca2+, i.e  20 mm Ca2+), as shown in Fig 5, reveals the Ca2+-dependent membrane binding differences amongst the proteins Mutant II and III display a much improved fractional membrane occupancy at physiologically relevant Ca2+ concentrations (1–5 mm) [24,25] compared to wildtype For example, at mm Ca2+, prothrombin already obtains over 70% of its potential binding and protein C mutants II and III each have approximately 50%, whereas wild-type protein C has obtained a mere 19% of its potential binding This indicates that the mutations introduced in mutants II and III lower the Ca2+ concentration requirement for effective binding, thereby improving membrane affinity A similar trend in fractional binding site occupancy is observed for the 0-20-80 and 0-10-90 membranes (data not shown) Mouse APC variants with hyper-anticoagulant activity in mouse plasma The generation of thrombin is severely diminished in mouse plasma when an APC variant with high affinity for membranes is included in the reaction (Fig 6A and Table 1) Compared at equivalent concentrations (0.5 nm), mutant III completely abolished thrombin generation, whereas mutants I, II, V and VII and, to a lesser extent, mutant IV caused a down-regulation of thrombin generation compared to wild-type APC, which did not have an anticoagulant effect at this concentration Although wild-type recombinant APC can function as an effective anticoagulant (Fig 6B), as also shown by Tchaikovski et al [26], strikingly lower concentrations of APC mutants II and III (Fig 6C,D) were required to achieve an identical anticoagulant result For example, to observe a similar measurable down-regulation difference of thrombin generation relative to thrombin generation in the absence of added APC [as assessed by either maximal concentration of thrombin (Cmax) or endogenous thrombin potential (ETP)], the concentrations required for wild-type, mutant II and mutant III were 1, 0.08 and 0.02 nm, respectively Similarly, thrombin generation was completely inhibited at the concentrations tested for wildtype (16 nm), mutant II (1.28 nm) and mutant III (0.5 nm) Figure reflects these findings and summarizes how each of the recombinant APC variants at several concentrations influences the generation of thrombin in mouse plasma Interestingly, thrombin generation parameters of lag-phase and time required to reach maximum thrombin generation (Tmax) are not significantly altered by the addition of the tested APC molecules Furthermore, a three-fold higher concentration of mutant III was required to obtain a similar FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS 6591 Anticoagulant mouse protein C variants A B C M J Krisinger et al Fig Effect of Ca2+ on the mouse protein C–membrane interaction (A) Protein C mutant II (1 lM) was injected for (association) over a 0-10-90 POPE-DOPS-POPC membrane bilayer surface at variable Ca2+ concentration to determine binding efficiency as a function of Ca2+ concentration for this high affinity binder Dissociation under running buffer conditions was followed for an additional Ca2+ concentration (mM) is indicated near the appropriate curve The SPR response curves are shown after background correction using a 100% POPC control flow cell Binding to the control surface was not apparent and no evidence of nonspecific binding was evident from an injection of Gla-less, prethrombin-1 (10 lM, not shown) Wild-type protein C (1 lM) at 20 mM Ca2+ was also included for comparison (indicated with an arrow: 20 wild-type) (B–D) Steady-state binding of mouse protein C wild-type ( ), mutant II ( ), III (.), V (+) and human prothrombin (h), each at lM, over either the (B) 0-20-80 or (C) 0-10-90 or (D) 20-10-70 POPE-DOPS-POPC membrane bilayer surface was measured using the indicated Ca2+ concentrations Responses obtained at equilibrium were used to generate a binding isotherm fitted to a one-site binding hyperbola using nonlinear least squares analysis Note that the 50 mM Ca2+ data points were excluded from fitting because high Ca2+ concentrations appear to inhibit the membrane interaction Binding isotherms were used to determine half-maximal Ca2+ concentration or [Ca2+]½ max reported in Table Data are representative of three experiments Additional details are provide in the Experimental procedures anticoagulant activity (assessed by either Cmax or ETP) in human plasma compared to mouse plasma (data not shown), further highlighting the importance of using proteins from the same species Discussion D 6592 Recombinant APC has been used to treat patients with reduced protein C levels suffering from severe sepsis (PROWNESS study) [8] The results of this and several other proceeding clinical trials [27] lead us to develop an APC molecule with enhanced anticoagulant activity with the purpose of investigating the effect of APCs with increased anticoagulant activity in vivo on different thromboembolic diseases APC has species specificity in its anticoagulant function that may, to a certain extent, be a result of its interaction with protein S [21,28,29] Therefore, we were unable to continue work in mouse models of sepsis or disseminated intravascular coagulation with our previously developed human APC variants (e.g QGNSEDY-APC) In the present study, we created several mouse APC variants with improved membrane binding characteristics and hyperanticoagulant activity Binding ability was established using a SPR membrane binding assay and anticoagulant activity was assessed by a thrombin generation assay The most active variant mutant III (P10Q ⁄ S12N ⁄ D23S ⁄ Q32E ⁄ N33D or QNSED), which is the FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS M J Krisinger et al Anticoagulant mouse protein C variants Table Half-maximal binding [Ca2+] as functions of various membrane compositions were determined by SPR for mouse protein C Ca2+ titration determined at fixed (1 lM) protein concentration Membrane (POPE-DOPS-POPC)a [Ca2+]1 ⁄ max 0-20-80 mPC wild type mPC mutant II mPC mutant III mPC mutant V Human prothrombin 15.9 3.7 3.6 9.0 1.4 ± ± ± ± ± 7.0 1.6 1.6 5.0 0.6 (mM)b 0-10-90 c 20-10-70 15.2 7.2 6.7 16.7 2.9 10.8 3.7 3.7 8.0 1.8 ± ± ± ± ± 11.1 3.4 3.5 12.0 0.9 ± ± ± ± ± 3.43 1.5 1.6 3.7 0.6 a Membranes used were 100 nm extruded liposomes with synthetic phospholipids: POPE-DOPS-POPC (mol % indicated) b Half-maximum binding [Ca2+] determined from one-site binding hyperbola fitting Representative determination from three experiments c SE determined from one-site binding hyperbola fitting Fig Fractional membrane binding site occupancy by the various protein C variants at different Ca2+ concentrations Equilibrium binding responses for mouse protein C wild-type ( ), mutant II ( ), III (.), V (+) and human prothrombin (h), using a 20-10-70 POPEDOPS-POPC membrane, were obtained at the indicated Ca2+ concentration as described in Fig 4D Equilibrium binding responses were normalized for fractional occupancy for each individual protein % Rmax is expressed as the equilibrium response at the indicated Ca2+ concentration divided by Rmax at saturating Ca2+ concentration (20 mM Ca2+) mouse equivalent of the human QGNSEDY-APC variant, are discussed in more detail below In the ETP assay using mouse plasma, mutant III served as a far superior anticoagulant compared to wild-type APC Mutant III had a 50-fold higher activity, thereby efficiently down-regulating thrombin generation, such that a mere 0.02 nm concentration was required to reduce either Cmax or ETP Complete inhibition of thrombin generation was obtained at a mutant III concentration of 0.5 nm, which is 30-fold lower than the concentration of wild-type APC required to give similar inhibition Previous work based on a tissue factor-dependent clot-based assay in normal human plasma showed that human APC variant QGNSEDY had a 20-fold higher anticoagulant potential than human wild-type APC [16] Although this was a simple end point assay, the anticoagulant potency of human APC variant QGNSEDY in human plasma parallels that of mouse APC mutant III in mouse plasma Mutant III, although only containing five mutations, is equivalent to the human QGNSEDY variant because the wild-type mouse Gla domain already has G at position 11 and Y at position 44 Thus, the seven Gla domain residues introduced in human APC (Q10, G11, N12, S23, E32, D33 and Y44) are all present in mouse APC mutant III The high sensitivity of SPR detection allowed us to accurately analyze even low affinity proteins, such as wild-type mouse protein C, to membrane binding site saturation The results obtained are thus based on the combined analysis of binding affinity, Rmax and qualitative kinetics Efficient binding of protein C, as well as other Gla proteins, to membrane is dependent on three complimenting factors: an optimal Ca2+ concentration, an optimal phospholipid composition and, lastly, structures intrinsic to the protein (e.g optimal arrangement of residues in the Gla domain) For any given Gla protein, a good membrane can compensate for binding at sub-optimal Ca2+ concentrations Conversely, an optimal Ca2+ concentration can compensate for binding at sub-optimal membrane compositions Thus, the results obtained in the present study indicate that any one of these three factors can compensate for two of the others if they are presented in a sub-optimal manner The protein C mutations act as excellent reporters of how these residues influence the membrane interaction Single and double amino acid substitutions at three separate regions of the protein C Gla domain were introduced at positions 10 ⁄ 12, 23 and 32 ⁄ 33 Mutagenesis at position 10 ⁄ 12, as in mutant I (QN), caused a small (1.5-fold) gain in affinity compared to wild-type The major affinity improvement came from combined mutagenesis at position 32 ⁄ 33 with mutant V (ED) having a 3.5-fold higher affinity than wildtype Similarly, this double mutagenesis caused an eight-fold higher affinity in mutant II (QNED) relative to mutant I (QN) A Glu residue (converted to Gla residue upon post-translational modification) introduced at position 32 in factor VII has been suggested to bind an additional Ca2+ during membrane binding [30], although it does not appear to serve the same role FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS 6593 Anticoagulant mouse protein C variants A B C D 6594 M J Krisinger et al Fig Inhibition of thrombin generation in mouse plasma by mouse APC and variants (A) Mouse plasma was incubated with either 0.5 nM extrinsically added wild-type mouse APC, or variant (I–VII), or in the absence of added protein (as indicated on curves) Thrombin generation was initiated with 0.25 pM tissue factor, 10 lM phospholipid liposomes and 16.7 mM CaCl2 and followed continuously with the fluorogenic substrate I-1140 (Z-Gly-Gly-Arg-7amino-4-methylcoumarinỈHCl, 300 lM) in 25 mM Hepes, 175 mM NaCl (pH 7.4) containing 0.5% BSA at 37 °C All indicated concentrations are final concentrations Mouse plasma (10 lL) was used in a final reaction volume of 120 lL The first derivative of a typical experiment (n = 3) is shown (B–D) Concentration-dependent inhibition of thrombin generation in mouse plasma by mouse APC is shown Mouse plasma was incubated with the indicated concentration (nM) of extrinsically added mouse APC (B) wild-type, (C) mutant II or (D) mutant III using the conditions described above in mouse protein C (see discussion on Ca2+ below) An acidic residue at position 23, such as Asp in protein C, has been speculated to instill low affinity to Gla proteins However, the D23S mutation had little effect on binding affinity; for example, when comparing mutant VI (S) and wild-type The D23S mutation appears even inhibitory if mutant II (QNED) and III (QNSED) are compared Similarly, the gain of function observed with mutant I (QN) was reversed by having the D23S mutation present, as in mutant IV (QNS) It is noteworthy that the multi-site mutations introduced often resulted in synergistic affinity effects and were not simply the additive sum of individual mutations As such, there appears to be an intramolecular synergism between the 10 ⁄ 12 (QN) and 32 ⁄ 33 (ED) sites in mouse protein C Our previous work with human protein C showed that variant QGNSEDY had increased membrane affinity (3.5- to seven-fold) and was more potent as an anticoagulant in a TF-dependent clotting (PT) assay (approximately five-fold longer prolonged clot time) than wild-type [16] Mutagenesis at less sites in human protein C, as in variants GNED (approximately one-fold ⁄ 1.5-fold), QGN (1.1-fold ⁄ one-fold) and SEDY (1.6-fold ⁄ onefold), had negligible or minor improvements in affinity and anticoagulant activity, respectively [16] Although a single amino acid in some Gla proteins can significantly influence affinity upon mutagenesis, there appears to be other additional mechanisms that can control the affinity of Gla proteins, as illustrated with Pro10 of the human, bovine and mouse protein C orthologs A Pro at position 10, occurring naturally or introduced by site-directed mutagenesis was shown to significantly lower the membrane affinity of Gla proteins In bovine protein C, P10H mutagenesis results in a ten-fold affinity increase, whereas, with human protein C, H10P mutagenesis results in a five-fold affinity FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS M J Krisinger et al Fig Concentration-dependent inhibition of thrombin generation in mouse plasma by mouse APC Mouse plasma was incubated in the absence of protein or with the indicated concentration of extrinsically added wild-type mouse APC or variant (I–VII) Thrombin generation was initiated as described in Fig % Cmax is expressed as the maximum concentration of thrombin generated in the presence of APC divided by the maximum concentration of thrombin generated in the absence of APC Mean values from duplicate determinations from a single experiment (data for 0.1, and 10 nM) and the mean ± SD from three independent experiments each with duplicate determinations (data for 0.5 and nM) are shown (log2 scale) decrease [14] Mouse protein C contains the ‘low affinity’ Pro10, but did not gain a significant increase in affinity by mutagenesis to P10Q because mutant I (QN) had only a modest 1.5-fold affinity increase compared to wild-type protein Thus, work on human, bovine and mouse protein C mutagenesis illustrates the intricacy of improving the Gla protein–membrane interaction Mutant II has a modest (two-fold), but significant, affinity enhancement compared to mutant III, although, unexpectedly, mutant III has better anticoagulant activity The only differences between mutant II and mutant III is the amino acid at position 23 (D in mutant II and S in mutant III) It is conceivable that the amino acid at position 23 may affect anticoagulant activity that is not dependent on membrane binding In human proteins, protein S binds to the protein C Gla domain Although this interaction has been mapped to Gla regions C-terminal to the 23 site [31,32], it may still influence the interaction to the cofactor and thus influence the anticoagulant activity of APC An influence of position 23 on the interaction to APC substrates FVa and FVIIIa also cannot be ruled out The same reasoning can be applied when comparing membrane binding and anticoagulant activity data of mutant V and mutant VII, which also only vary at position 23 Thus, 23S appears to be better for membrane binding, but 23D appears to instill a better anticoagulant property in mouse APC Anticoagulant mouse protein C variants Membrane binding capacity is a measure of the Gla protein packing density to sites provided by the membrane at saturating protein concentrations Of relevance for this discussion is the variation of membrane binding site occupancy amongst the different mouse protein C variants (Fig 3D–F) For example, steadystate Rmax for wild-type and mutant II differ by approximately five-fold to a 0-20-80 membrane surface In the classical view of a simple bimolecular (1 : 1) interaction, a receptor site can be saturated with different affinity analytes (of equal molecular weight), which, by definition, will all have an equivalent saturation response (i.e occurring when all receptor sites are occupied) that will be approached by a specific analyte concentration specified by the KD of the receptor–analyte interaction This was clearly not observed when the saturation binding levels of the protein C variants were compared, for any of the phospholipid membranes tested, implying a different mode of binding between the proteins This indicates that the membrane, even in simple model membranes, provides a number of binding sites that are not isolated and homogenous in nature, but rather heterogeneous For example, it can be envisioned that a Gla protein can engage with a membrane site containing a variable number of PS molecules each displaying a different affinity The results obtained in the present study indicate that the high affinity mutants (e.g II and III) can utilize ‘poor’ binding sites that low affinity mutants (e.g wild-type) cannot engage with, and, thus, the high affinity mutants have access to more total binding sites resulting in a higher Rmax The membrane binding capacity difference observed amongst the variants argues for the existence of several binding sites composed of a variable number of PS molecules Thus, we conclude that two processes make the binding of mouse protein C mutants more efficient to membrane than wild-type First, variants such as mutants II and III are able to utilize a higher number of membrane binding sites Second, these variants are also able to engage with these membrane binding sites with an overall higher averaged affinity then wild-type mouse protein C These two processes allow substantially more protein to bind membrane when eqimolar concentrations of these proteins are compared Membrane binding on- and off-rates are probably important specifications for the functions of protein C ⁄ APC as well as the other Gla proteins Association with the membrane is likely a dynamic process involving several bound intermediates An initial membrane engagement step (termed electrostatic docking) [33], followed by the association of an unknown number of PS headgroups, hydrophobic x-loop insertion into the FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS 6595 Anticoagulant mouse protein C variants M J Krisinger et al membrane core and, finally, the chelation of additional calcium ions between membrane and protein, all provide independent free-energy contributions to bound intermediates It is not surprising that the kinetic binding data describing the sum of these kinetic events could not be adequately simulated by simple binding models commonly used in the kinetic fitting of protein–protein interaction data On a qualitative basis, the kinetic shapes of the interaction profiles (Fig 3A– C) were not drastically different amongst the protein C variants analyzed Thus, from the data obtained in the present study, we conclude that, in addition to utilizing a higher number of binding sites, mutants II and III gained their modest (approximately ten-fold) membrane binding affinity enhancement by minor increases in the overall on-rate and ⁄ or minor decreases in the overall off-rate The results obtained in the present study support that the binding of a Gla protein is to a few PS molecules rather than a PS microdomain (a membrane PSrich regions containing over 20 grouped PS molecules) We have shown that doubling the PS concentration in the membrane results in an increase in the number of binding sites (compare Fig 3D and 3E) and also in an average site with increased affinity If Gla proteins were to bind exclusively to a PS microdomain, then the affinity is expected to remain conserved for liposomes with varying PS concentration That is, the identical patch would exist on a 10% and 20% PS-containing membrane, with the only difference being that the surface area of the PS patch would be two-fold greater in the 20% PS liposomes Consistent with the binding to a few PS molecules, it has been estimated previously, in experiments using phospholipid containing nanodiscs [34] or liposomes [33,35], that approximately up to five PS molecules can be utilized to bind a Gla domain to a membrane surface These derived values are in line with the physical dimensions of a Gla domain having a membrane footprint that occupies an area of approximately 3.9 nm2 [36] or the equivalent of six phospholipid headgroups (one phospholipid headgroup has a surface area of  0.7 nm2) [37] In addition, bovine Gla proteins, protein Z and factor X, were shown to cluster only a small number (approximately four) of fluorescentlabeled anionic phospholipids [38] Thus, the simple model membranes used in the present study, composed of even two types of synthetic phospholipids (DOPS and POPC), likely provide several compositionally different binding sites that Gla proteins can engage with Because binding to 100% POPC liposomes is insignificant, the different binding sites utilized by the different variants must be a function of DOPS content Thus, 6596 an enticing hypothesis is that the high affinity variants can utilize membranes sites with lower PS content that low affinity variants cannot engage with PE improved the binding of the mouse protein C variants when the DOPS concentration was held constant (Fig 3E,F) We not know at present whether the affinity and binding site enhancement is mediated directly by POPE (e.g POPE binding sites) or whether POPE causes indirect effects on DOPS (e.g membrane rearrangement) The free Ca2+ concentration in plasma is 1–1.5 mm [24,25] Protein C ⁄ APC binds to PS in membranes released by activated platelets in the platelet plug, within which the Ca2+ concentration increases to 3–5 mm [24] These variations in Ca2+ concentration, in vivo, may serve a regulatory function in hemostasis [12] The Gla domain structure is heavily dependent on seven Ca2+ coordinated by several Gla residues at the core of the domain The Ca2+ concentration required to cause 50% transition of protein C molecules is approximately 0.5 mm [39] and, thus, under in vivo Ca2+ concentrations, approximately 65–90% of protein C is expected to be in a membrane binding conformation An ordered Gla domain is a prerequisite enabling a subsequent membrane interaction Gla domain destabilizing mutations thus also reduce membrane affinity Furthermore, externally bound calcium ions are considered to form a direct link between protein and membrane, thereby providing an additional mode of interaction [40] Some putative conclusions can be made on the mutations made in the present study and their involvement in Ca2+-dependent membrane binding Because wild-type mouse protein C and mutant V (ED) have very similar Ca2+-dependent membrane binding properties, the QN to ED mutations at the 32 ⁄ 33 site are not suspected to significantly stabilize the Gla domain in a Ca2+ limiting environment or help in providing additional Ca2+ bridging anchoring points to the membrane However, further mutagenesis at the 10 ⁄ 12 site (PS to QN; e.g as in mutants II and III) improved the Ca2+-dependent membrane binding by up to four-fold According to the X-ray structure of bovine prothrombin fragment in complex with Ca2+ [41], the 10 ⁄ 12 site resides near the x-loop and the linear array of bound calcium ions of the Gla domain Indeed, the importance of the 10 ⁄ 12 site is also apparent when comparing the Ca2+dependent membrane binding data of mutants V and II (Fig 5) Differing only at the 10 ⁄ 12 site, mutant II (Q10 and N12) has a [Ca2+]1 ⁄ max that is two-fold lower than mutant V (P10 and S12) Similar findings for the 10 ⁄ 12 site were observed when comparing wildtype and mutant I (data not shown) A recent model FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS M J Krisinger et al of the membrane bound Gla domain of FVIIa showed that the inner calcium ions are required for optimal folding, allowing membrane insertion, and the outer calcium ions provide direct anchoring points to the membrane [40] The residues at positions 10 and 12 are expected to either influence the stability of the Gla domain at Ca2+ limiting concentrations and ⁄ or provide additional direct membrane anchoring points via Ca2+ Interestingly, human prothrombin, which also lacks a Pro at position 10 and has an Asn at position 12, also has excellent membrane binding properties at low Ca2+ concentrations In conclusion, several high affinity, high binding capacity, Gla domain mutated mouse protein C variants were established, which, upon activation, served as superior anticoagulants during tissue factor-dependent coagulation These activated protein C variants may therefore be beneficial for the treatment of severe sepsis and should be studied further in appropriate mouse models Experimental procedures Reagents A full-length mouse protein C cDNA clone was the generous gifts from E M Conway (University of Leuven, Leuven, Belgium) The TM cDNA clone was purchased from ATCC (Rockville, MD, USA) and soluble TM was produced as described previously [42] Human prothrombin was purchased from Enzyme Research Laboratories, Kordia (Leiden, The Netherlands) Human prethrombin-1 was prepared as described previously [43] Human thrombin was purchased from Haematological Technologies (Essex Junction, VT, USA) All proteins were judged to be greater than 98% pure from an overloaded Coomassie blue stained SDS-PAGE gel Protein concentrations were determined by absorbance using the extinction coefficients (E280 1%, cm) and molecular weights given by the supplier: human prothrombin: 13.8, 72 000; human prethrombin-1: 17.8, 49 900; mouse protein C: 14.5 (inferred from human protein C), 56 000 (estimate) The Gla-specific monoclonal antibody M3B was a kind gift from J Stenflo (Lund University, Malmo, Sweden) and was described previously ă [44] Hirudin was purchased from Pentapharm (Basel, Switzerland) Chromogenic APC substrate (S-2366) was purchased from DiaPharma (Molndal, Sweden) Synthetic ă phospholipids; POPE, DOPS and POPC were purchased from Avanti Polar Lipids, Inc (Alabaster, AL, USA) Recombinant tissue factor (thromboplastin) was from Dade InnovinÒ (Marburg, Germany) and dissolved according to the manufacturer’s instructions and stored at °C The tissue factor concentration in the thromboplastin stock is 400 ngỈmL)1 (8.5 nm) according to T M Hackeng (Cardio- Anticoagulant mouse protein C variants vascular Research Institute, Maastricht, The Netherlands, personal communication) The thrombin fluorogenic substrate I-1140 (Z-Gly-Gly-Arg-7-amino-4-methylcoumarinỈHCl, 300 lm) was obtained from Bachem (Bubendorf, Switzerland) and stored as 2.5 mm stock in 2.5% dimethylsulfoxide at )80 °C Citrated platelet-free plasma pooled from 48 CD-1 mice was from Sera Laboratories International Ltd (Haywards Heath, UK) Mouse plasma was centrifuged for at 17 000 g to remove any precipitate, stored in aliquots at )70 °C and thawed once at 37 °C just prior to use One out of every two samples from the supplier was discarded because it did not contain any clotting activity Inter-lot variability of functional mouse plasma was less than 5% by peak height and almost indistinguishable by lag-phase and time to peak parameters (results not shown) Hepes buffer and BSA were obtained from Sigma (St Louis, MO, USA) All SPR reagents including L1 sensor chips were purchased from GE Healthcare (Uppsala, Sweden) All other reagents and buffer components were commercially available and of highest the purity Mutagenesis, construction and stable expression of recombinant mouse protein C and variants Mutagenesis was performed using QuikChange (Stratagene, La Jolla, CA, USA) in accordance with the manufacturer’s instructions (primer sequences and mutagenesis strategy are described in the Supporting information) [14] In total, the following seven mutants were created: mutant I, P10Q ⁄ S12N (QN); mutant II P10Q ⁄ S12N ⁄ Q32E ⁄ N33D (QNED); mutant III, P10Q ⁄ S12N ⁄ D23S ⁄ Q32E ⁄ N33D (QNSED); mutant IV, P10Q ⁄ S12N ⁄ D23S (QNS); mutant V, Q32E ⁄ N33D (ED); mutant VI, D23S (S); and mutant VII, D23S ⁄ Q32E ⁄ N33D (SED) All mutations were confirmed by DNA sequencing before transfection The cDNAs corresponding to wild-type protein C and protein C variants (QN, QNED, QNSED, QNS, ED, S and SED) were all inserted into the eukaryotic expression vector the pcDNA3, transfected into HEK 293 cells (CRL-1573 ATCC), high-expressing colonies selected, and recombinant protein C purified, as previously described [45] Briefly, the transfected cells were selected in DMEM ⁄ F-12 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum containing 0.25 mgỈmL)1 Geneticin G418 (Invitrogen) for 3–5 weeks G418 resistant colonies were picked and grown in serum free medium containing 10 lgỈmL)1 vitamin K1 (Sigma) Colonies expressing protein C at high levels, assessed by dot blot assay using polyclonal sheep anti-(mouse protein C) serum (Haematological Technologies), were picked for further expression, as described previously [14,45] High-producing clones were isolated and grown until confluence in the presence of 10 lgỈmL)1 vitamin K1 HEK 293 cells expressing human protein C were previously shown to faithfully perform c-carboxylation on all glutamic acid residues within the Gla domain [15] FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS 6597 Anticoagulant mouse protein C variants M J Krisinger et al Mouse protein C purification Phospholipid liposome preparation The purification of mouse recombinant protein C and variants was based on the method described previously for human and bovine recombinant protein C [14] with some modifications Serum-free conditioned medium (5 L) from stably transfected cells containing each recombinant mouse protein C was supplemented with mm benzamidine and mm EDTA, centrifuged at 6000 g at °C for 10 and batch absorbed with 40 mL of Q Sepharose Fast Flow matrix (Amersham Biosciences, Uppsala, Sweden) by overnight incubation at °C The gel slurry was packed in a column, extensively washed with 20 mm Tris-HCl, 150 mm NaCl (pH 7.4) and eluted with buffer plus 10 mm CaCl2 Protein C containing fractions were collected and CaCl2 was removed by overnight dialysis against 20 mm Tris, 50 mm NaCl (pH 7.4) using Dialys Spectra ⁄ Por dialysis membranes with MW cut-off: 12–14 000 (Spectrum Laboratories Inc., Rancho Dominguez, CA, USA) and the proteins reabsorbed to a second HiprepÔ 16 ⁄ 10 Q FF column (Amersham Biosciences) after which it was eluted with a NaCl gradient (starting solution: 20 mm Tris, 50 mm NaCl, pH 7.4, limiting solution: 20 mm Tris, m NaCl, pH 7.4) Proteins were concentrated using Amicon Ultra centrifugal filter devices with a 10 000 MW cut-off (Bedford, MA, USA) and then stored at )80 °C The concentrations of proteins were established by measuring A280 After purification, the purities of all mouse recombinant protein C preparations were determined by SDS-PAGE followed by silver staining Liposomes were prepared as described previously [46] with minor revisions Briefly, lyophilized lipids were suspended in chloroform to approximately 30–100 mm and concentrations were established by inorganic phosphate determination, as described previously [47] The appropriate molar ratios of phospholipid in chloroform were dried, first under a stream of nitrogen and then under vacuum, for at least h The resultant lipid residue was resolubilized in HBS (10 mm Hepes, 150 mm NaCl, pH 7.4) to a final concentration of approximately 10 mm The resultant multilamellar vesicle suspension was subjected to a rapid freeze-thaw technique five times by cycling in liquid nitrogen and warm water Large unilamellar vesicles were generated by extrusion of multilamellar vesicles under pressure through two stacked Nucleopore polycarbonate filters (Avestin Inc., Ottawa, Canada) with a 100 nm pore size (19 passes) using a LiposoFast microextruder (Avestin Inc.) Membrane composition is stated as mol % POPE-DOPS-POPC The mean diameter of large unilamellar vesicles by this method is approximately 110 ± 25 nm Liposomes were stored at °C and were used within week of production Preparation of mouse activated protein C Incubation of mouse protein C with human or bovine thrombin using conditions known to work well for human protein C [14] were found to result in extra inappropriate cleavages in the light chain Many conditions were tried and finally we determined that the thrombin–TM complex was required to obtain reliable activation of mouse protein C Thus, to generate mouse APC with appropriate cleavage pattern and full activation, recombinant mouse protein C mutants (100 lgỈmL)1) were incubated with human thrombin (2 lgỈmL)1) in the presence of human soluble thrombomodulin (1 lgỈmL)1) and incubated for 24 h in 50 mm Tris-HCl, 150 mm NaCl, mm CaCl2 (pH 7.4) at 37 °C After incubation, 100 mL)1 hirudin was added and the APC reaction products were assessed by SDS-PAGE followed by silver staining Either the preparation was used as is, or the thrombin-hirudin-TM was removed by chromatography on a HiprepÔ 16 ⁄ 10 Q FF column as described for protein C purification Enzymatically active APC concentrations were determined by active site titration using the APC chromogenic substrate S-2366 essentially as described previously [14] The activity of mouse APC was consistent for all preparations of wild-type and variants of APC (variation: < 5%) 6598 Measurement of protein–membrane interactions by SPR Binding of mouse protein C variants and of prothrombin to phospholipid liposomes was quantified by SPR using a Biacore 2000 instrument (Uppsala, Sweden) at 24 °C Prior to lipid immobilization, the lipophilic LI sensor chip was washed with 40 mm octyl glucoside (1 at 20 lLỈmin)1) Synthetically-derived phospholipid liposomes (500 lm) were injected for 17 at a lLỈmin)1 flow rate in HBS running buffer Liposomes were immobilized to a response of 5000–7000 RU The chips were washed five times with 10 mm EDTA (pH 8.0) injections (2 at 20 lLỈmin)1), after which the membrane surface was stable, as indicated by an insignificant loss in the SPR signal for the subsequent 12 h (data not shown) Liposomes derived from naturallyderived phospholipids slowly dissociated from the L1 chip and were thus unsuitable for interaction experiments For protein binding experiments, running buffer was changed to HBC (HBS with 10 mgỈmL)1 BSA and mm CaCl2) and flow cells were equilibrated until the baseline stabilized to less than 0.05 Rmin)1 Association and dissociation times each were typically in the range 4–12 Equilibrium response data were collected for each protein at several concentrations typically spanning ten-fold below and, if possible, ten-fold above the KD of the interaction, with 10 lm being the highest concentration tested Prethrombin1, a Gla domain-less fragment of prothrombin known not to interact with membranes, was used as a negative control Either a blank or 100% POPC liposome control flow cell was used to subtract RU as a result of the refractive index FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS M J Krisinger et al of the protein solution and any instrument noise No binding was detected in either control flow cells for any of the proteins tested The immobilized liposome surface could be regenerated by removing the Ca2+-dependent membranebound protein (prothrombin, protein C wild-type and variants) with an injection of 10 mm EDTA (pH 8.0), which returned the baseline to the value prior to introducing protein Equilibrium data (Req) was fitted to a one-site binding hyperbola according to the relationship Req = RmaxỈC ⁄ (KD + C), where Rmax is the binding at saturation or maximum surface coverage, C corresponds to the injected analyte concentration and KD is the equilibrium dissociation constant We are aware of the fact that an analysis of the overall binding curve is a relatively severe simplification and that a complex set of reactions is involved in the membrane binding process of any Gla protein [33,41,48–50] However, this simplification appears to be justified because the binding curves showed no signs of cooperativity when analyzed by scatchard plots (M J Krisinger, E L Pryzdial and R T MacGillivray, unpublished results) BSA (0.1%) was included to block any nonspecific protein–lipid and protein–protein interactions [51] Experiments were carried out with replicate analyte concentrations, which were essentially identical Experiments were also measured in random order with respect to analyte concentrations All buffers were filtered through a 0.22 lm filter and degassed before use All stock solutions were briefly centrifuged before use to remove any potential precipitate To determine the effect of Ca2+, the immobilized phospholipid liposomes were equilibrated with buffer containing a variable Ca2+ concentrations (0, 0.5, 1, 2, 5, 10, 20 and 50 mm) Proteins were diluted such that HBC and Ca2+ concentration were exactly matched to running buffer and allowed to equilibrate for at least 30 prior to injection After protein injections and sensorgrams were completed for a single Ca2+ concentration, running buffer was exchanged and primed three times with the subsequent HBC Buffers were randomized No binding was detected in buffer lacking Ca2+ for any of the proteins tested Protein binding equilibrium data (Req) were fitted to a onesite binding hyperbola according to the relationship Req = RmaxỈC ⁄ ([Ca2+]1 ⁄ max + C), where Rmax is the binding at saturation (maximum surface coverage), C corresponds to the Ca2+ concentration and [Ca2+]1 ⁄ max is the Ca2+ concentration required to reach the binding midpoint of the titration Anticoagulant mouse protein C variants warmed plate contained 10 lL of normal pooled mouse plasma in a total reaction volume of 120 lL For a typical experiment, thrombin generation was initiated with 0.25 pm tissue factor (a validation of thrombin generation in mouse plasma and the effect of exogenously added tissue factor is provided in Fig S2), 10 lm phospholipid liposomes (20-2060 POPE-DOPS-POPC) and 16.7 mm CaCl2 in buffer (25 mm Hepes, 175 mm NaCl, pH 7.4, containing 0.5% BSA) and continuously followed with the fluorogenic substrate I-1140 (Z-Gly-Gly-Arg-AMC; 300 lm) at 37 °C All concentrations are final Prior to the start of the reaction, reagents were incubated at 37 °C: tissue factor, phospholipid liposomes and CaCl2 were incubated together for h, the fluorogenic substrate was incubated for 30 and the mouse plasma was thawed for Reagents were added and mixed in a 96-well plate in the order (added volume ⁄ well, incubation time): six-fold buffer diluted mouse plasma (60 lL, min), APC sample (20 lL, min), fluorogenic substrate (20 lL, min) and tissue factor ⁄ liposome ⁄ CaCl2 (20 lL), after which readings were taken immediately at intervals for approximately 60 Each experiment was preformed in duplicate The first derivative of the fluorescence versus time plot (not shown) was taken using GraphPad Prism, version (graphpad Software Inc., San Diego, CA, USA) to obtain the thrombogram (or fluorescence ⁄ time versus time plot) Fluorescence data were not corrected for the inner filter effect or substrate consumption; therefore, thrombin concentration was expressed as the rate of development of fluorescence intensity (fluorescence units or FU), calculated for every reading (Fmin)1) The a2 macroglobulin–thrombin complex, which forms during thrombin generation in plasma, was not corrected for because we merely report relative thrombin generation rates using an identical plasma sample Thrombin generation parameters assessed were: lag-time (min), maximal concentration of thrombin (Cmax or peak height in Fmin)1), the time required to reach maximum thrombin generation (Tmax in min) and ETP (area under the curve in total FU determined after 60 min) Acknowledgements This work was supported by grants from the Swedish Research Council (#71430), the Swedish Heart-Lung Foundation and research funds from the University Hospital in Malmo (to B.D.) and an Anna-Greta Crafoă ords Foundation Research Scholarship (to M.J.K.) Measurement of thrombin generation Fluorescence measurements over time were taken in a 96-well plate Tecan infinite 200 fluorometer equipped with a 360 nm excitation ⁄ 460 nm emission filter set (Molndal, ă Sweden) and magellan software (Groedig, Austria) Black ¨ flat bottom 96-well plates (Nalge Nunc International, Rochester, NY, USA) were used Each well of a pre- References ´ Fernandez JA, Xu X, Liu D, Zlokovic BV & Griffin JH (2003) Recombinant murine-activated protein C is neuroprotective in a murine ischemic stroke model Blood Cells Mol Dis 30, 271–276 FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS 6599 Anticoagulant mouse protein C variants M J Krisinger et al Dahlback B & Villoutreix BO (2005) Regulation of ă blood coagulation by the protein C anticoagulant pathway: novel insights into structure-function relationships and molecular recognition Arterioscler Thromb Vasc Biol 25, 1311–1320 Esmon CT (2006) Inflammation and the activated protein 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for recombinant mouse protein C variants 6602 This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 276 (2009) 6586–6602 ª 2009 The Authors Journal compilation ª 2009 FEBS ... proceeding clinical trials [27] lead us to develop an APC molecule with enhanced anticoagulant activity with the purpose of investigating the effect of APCs with increased anticoagulant activity. .. each lane and visualized by silver staining Protein C variants and molecular weight markers (MWM) ran in each lane are indicated The location of heavy chain (HC), light chain (LC) and thrombin... further improvement in membrane binding of the variants is shown in terms of membrane bind- Anticoagulant mouse protein C variants ing occupancy at the saturating protein concentration, a parameter