A novel factor XI missense mutation (Val371Ile) in the activation loop is responsible for a case of mild type II factor XI deficiency Cristina Bozzao1*, Valeria Rimoldi1*, Rosanna Asselta1, Meytal Landau2, Rossella Ghiotto3, Maria L Tenchini1, Raimondo De Cristofaro4, Giancarlo Castaman3 and Stefano Duga1 Department of Biology and Genetics for Medical Sciences, University of Milan, Italy Department of Biochemistry, George S Wise Faculty of Life Science, Tel Aviv University, Israel Department of Hematology and Hemophilia and Thrombosis Center, San Bortolo Hospital, Vicenza, Italy Haemostasis Research Centre, Catholic University School of Medicine, Rome, Italy Keywords coagulation factor XI deficiency; functional characterization; missense mutation; mutational screening; type II defect Correspondence S Duga, Department of Biology and Genetics for Medical Sciences, University of Milan, Via Viotti, ⁄ 5-20133 Milan, Italy Fax: +39 02 5031 5864 Tel: +39 02 5031 5823 E-mail: stefano.duga@unimi.it *These authors contributed equally to this study (Received 21 May 2007, revised 11 September 2007, accepted October 2007) doi:10.1111/j.1742-4658.2007.06134.x Coagulation factor XI (FXI) is the zymogen of a serine protease that, when converted to its active form, contributes to blood coagulation through proteolytic activation of factor IX FXI deficiency is typically an autosomal recessive disorder, characterized by bleeding symptoms mainly associated with injury or surgery Of the more than 100 FXI gene mutations reported in FXI-deficient patients, most are associated with a proportional decrease in FXI functional and immunologic levels (type I defects), whereas only a few mutations leading to the presence of dysfunctional molecules in plasma have been molecularly analyzed to date (type II deficiencies) We report the functional and molecular characterization of a missense mutation (Val371Ile) identified, in the heterozygous state, in a 25-year-old Italian male with mild FXI deficiency Laboratory analysis revealed reduced functional FXI levels (34%), but normal antigen levels (102%), distinctive of a type II defect Given the proximity of Val371 to the FXI activation site, a possible interference with zymogen activation was postulated Expression experiments of the FXI–Val371Ile recombinant protein, followed by activation assays, showed both a different time course in FXI activation and a slight delay in factor IX activation by thrombinactivated FXI Coagulation factor XI (FXI) is the precursor of a trypsin-like serine protease that catalyzes, upon activation, the conversion of factor IX (FIX) to activated FIX (FIXa) [1,2] Human FXI, primarily produced by hepatocytes, is a glycoprotein of 160 kDa circulating in plasma in a noncovalent complex with high molecular mass kininogen [3] Structurally, FXI zymogen comprises four N-terminal tandem repeats of about 90 residues, named apple domains (Ap1–4), followed by a catalytic serine protease domain located at the C-terminal end Uniquely among coagulation serine proteases, FXI is secreted as a homodimer composed of two identical polypeptide chains linked by noncovalent interactions and by a Cys321–Cys321 disulfide bond between the Ap4 domains [4,5] Among serine proteases that can activate FXI, i.e activated factor XII (FXIIa), FXIa, and thrombin, the main physiologic activator is actually thrombin formed on the surface of activated platelets [6–8] Cleavage at the Arg369–Ile370 bond in each monomer produces both an N-terminal heavy chain, which binds FIX and high molecular mass kininogen [9], and a C-terminal Abbreviations FIX, coagulation factor IX; FIXa, activated factor IX; FXI, coagulation factor XI; FXIa, activated factor XI; FXI:Ag, antigen FXI level; FXI:C, functional FXI level; FXIIa, activated factor XII 6128 FEBS Journal 274 (2007) 6128–6138 ª 2007 The Authors Journal compilation ª 2007 FEBS C Bozzao et al light chain, containing the catalytic domain [10] These two chains are held together by three disulfide bonds and both are essential for FIX activation [5] FXI activation generates a new N-terminus of the catalytic serine protease domain, which is called the activation loop (residues 370–376) Following cleavage, the activation loop undergoes large movement towards the activation pocket in the protease domain, stabilizing the FXIa active site [11] FXI deficiency is an autosomal recessive bleeding disorder that is rare in most populations (prevalence : 106) but is particularly common among Ashkenazi Jews, in whom a heterozygote frequency of 8% has been reported [12] This rare coagulopathy is characterized by decreased FXI functional activity, usually associated with low levels of FXI antigen (type I defects) By contrast, type II defects, characterized by the presence of dysfunctional molecules in plasma, are rare [13,14] Usually, homozygous and compound heterozygous patients have severe FXI deficiency (FXI activity < 20%), whereas heterozygotes have mild ⁄ partial FXI deficiency (20–50%) [15] Bleeding tendency in FXI-deficient patients seems to poorly correlate with plasma FXI levels and hemorrhagic episodes are usually associated with injury or surgery, but may be so severe as to demand replacement therapy [16] The genetic basis of this rare coagulation disorder is invariantly represented by mutations within the FXI gene (F11), which is located on chromosome 4q35.2 and consists of 15 exons spread over a genomic region of $ 23 kb To date, > 100 mutations responsible for FXI deficiency have been described [13,17] Among Ashkenazi Jewish patients, two prevalent mutations (Glu117stop and Phe283Leu, also called type II and type III mutations) account for 95% of cases of FXI deficiency [12] However, in patients belonging to other ethnic groups a significantly higher level of allelic heterogeneity has been reported Remarkable exceptions are represented by French Basques, French patients from Nantes, and English patients, in whom different prevalent ancestral mutations were found [18–20] An unusual dominant transmission of FXI deficiency has been described in some families, in which four different missense mutations exert a dominant-negative effect on wild-type FXI secretion through intracellular heterodimer formation [21,22] The aim of this study was the molecular characterization of the F11 germline missense mutation Val371Ile identified in the heterozygous state in an Italian patient affected by mild FXI deficiency, who had normal immunologic FXI levels associated with a reduced activity of the factor, distinctive of a type II defect FXI–Val317Ile – a novel factor XI type II defect Results Patient data The propositus was a male born in 1981, who was referred in 2001 for the evaluation of a prolonged partial thromboplastin time discovered prior to a surgical procedure An appendectomy, adenoidectomy, and right-knee arthroscopy carried out previously had been without mishap Laboratory analysis revealed a reduced functional FXI level (FXI:C ¼ 34%), although the antigen FXI level was normal (FXI:Ag ¼ 102%), suggestive of a mild type II FXI deficiency His mother, born in 1949, also had reduced FXI:C (43%) associated with normal FXI:Ag (132%), whereas the father, born in 1947, had both functional and antigen FXI levels within the range of normality (96 and 134%, respectively) Both parents were asymptomatic Mutational screening The entire coding region, including exon–intron boundaries and $ 300 bp of the promoter region of F11, was sequenced Sequence analysis identified a heterozygous G fi A transition in exon 11 corresponding to cDNA position 1165 (numbering according to GenBank accession number NM_000128, starting from the first nucleotide of the ATG start codon), which causes a Val371Ile substitution (numbering omits the signal peptide) The same mutation was found in the heterozygous state in the proband’s mother Amino acid substitution involves the second residue of the FXI light chain after the proteolytic cleavage that leads to FXI activation Residue Val371 is located one residue following the cleavage site of FXI (P2¢ position, according to the convention of numbering position around the scissile bond), thus is part of the activation loop One hundred haploid genomes from unrelated Italian control individuals were also analyzed by direct sequencing and the Val371Ile mutation was absent in all of them (data not shown) Expression of wild-type and Val371Ile recombinant FXI in COS-1 cells To evaluate the pathogenic role of the Val371Ile mutation, both the wild-type and mutant protein were expressed in COS-1 cells To this end, mutagenesis was performed on the pCDNA3 ⁄ FXI plasmid to produce the pCDNA3 ⁄ FXI–Val371Ile vector as described in Experimental procedures Following transient transfection with either pCDNA3 ⁄ FXI or FEBS Journal 274 (2007) 6128–6138 ª 2007 The Authors Journal compilation ª 2007 FEBS 6129 FXI–Val317Ile – a novel factor XI type II defect A C Bozzao et al B ANTIGEN LEVEL 130 ns 110 ns 120 110 ns 100 ns 90 % of wild-type % of wild-type 100 90 80 70 60 50 40 ** * 80 70 60 50 *** 40 30 30 20 20 10 SPECIFIC ACTIVITY nd 10 nd nd CONDIT IONED MEDIA wild-type CONDITIONED MEDIA CELL LYSAT ES heterozygous Val371Ile mock Fig Transient expression of wild-type and mutant FXI protein in COS-1 cells pCDNA3 ⁄ FXI, pCDNA3 ⁄ FXI–Val371Ile or equimolar amounts of both plasmids (heterozygous condition) were transiently transfected in COS-1 cells Equal numbers of cells and equal amounts of plasmids were used in transfection experiments, as described in Experimental procedures (A) Antigen levels of recombinant FXI were measured in both conditioned media and the corresponding cell lysates using an ELISA assay Bars represent relative concentrations of protein in media and cell lysates compared with the mean antigen level measured in the wild-type Results are given as mean ± SD (B) The specific activities of recombinant proteins were determined by calculating the ratio between FXI activity (measured using a one-stage method based on a modified partial thromboplastin time) and FXI antigen levels Bars represent mean ± SD of four independent experiments, each performed in duplicate The mean value of wild-type FXI was set as 100% The results were analyzed by unpaired t-test (*P < 0.05; **P < 0.01; ***P < 0.001), ns, not significant; nd, not determined pCDNA3 ⁄ FXI–Val371Ile or with equimolar amounts of both expression plasmids (to mimic the heterozygous condition), serum-free conditioned media and cell extracts were analyzed for the presence of FXI antigen using ELISA FXI antigen levels, measured in both conditioned media and lysates of cells expressing the mutant protein (in either the heterozygous or homozygous state), were not significantly different from those measured in wild-type samples (Fig 1A) In particular, in media conditioned by cells expressing either wildtype or mutant FXI, antigen levels ranged from 300 to 500 ngỈmL)1, whereas, levels of immunoreactive FXI were between 20 and 40 ngỈmL)1 in the corresponding lysates FXI specific activity was measured in conditioned media as the ratio between FXI:C and FXI:Ag levels The specific activity of the FXI–Val371Ile protein was significantly reduced when compared with the wildtype one ($ 30 and 80% for the heterozygous and the homozygous condition, respectively; Fig 1B) Our results are consistent with the FXI:C and FXI:Ag measured in the patient’s plasma, further supporting the hypothesis that the Val371Ile mutation is a 6130 type II defect leading to the production of a defective FXI molecule FXI activation Three biologically relevant proteases can activate FXI (FXIIa, FXIa, and thrombin) [8,23], all of which cleave FXI at the Arg369–Ile370 bond and expose the active-site catalytic triad Given the proximity of Val371 to the FXI activation site, a possible interference on FXI activation was hypothesized To this end, equal amounts of wild-type and mutant recombinant FXI molecules from conditioned media were directly activated by either thrombin or FXIIa As shown in Fig 2, time-course experiments of FXI activation by either thrombin or FXIIa were performed Both proteases were able to cut wild-type and FXI–Val371Ile precursors into two fragments corresponding to the heavy (48 kDa) and light (32 kDa) chains The Val371Ile substitution causes a delay in FXI activation time following both types of activation protocols, as is clearly appreciable in Fig 2A,B Indeed, after 16 h digestion with FXIIa at 37 °C, FEBS Journal 274 (2007) 6128–6138 ª 2007 The Authors Journal compilation ª 2007 FEBS C Bozzao et al FXI–Val317Ile – a novel factor XI type II defect A B Fig Time course of wild-type and Val371Ile FXI activation SDS ⁄ PAGE of wild-type FXI and FXI–Val371Ile (1.5 ng of protein) incubated with FXIIa (1 lg) (A) or thrombin (0.5 U) (B) At various time points, indicated at the top of the panels, samples were stopped in reducing sample buffer and eventually separated onto a 10% polyacrylamide gel FXI activation was evaluated by western blotting using polyclonal goat anti-human IgG recognizing both uncleaved FXI and FXI heavy and light chains The estimated molecular masses of monomeric uncut FXI (80 kDa), FXIa heavy chain (48 kDa), and FXIa light chain (32 kDa) are indicated about half of the total mutated protein remains uncut, while the wild-type FXI is almost entirely activated (Fig 2A) In order to give a more accurate quantitative description of the FXI activation process, a chromogenic assay was used to compare cleavage of the substrate S-2366 by the wild-type and mutant FXI, previously activated by thrombin in absence of dextran sulfate Activation of both proteins by human a-thrombin followed pseudo-first-order kinetics, as shown in Fig This was in agreement with a stochastic model of FXI activation by thrombin, whereby the latter cleaves either of the two chains of zymogen FXI independently according to simple first-order kinetics If this were not the case, a double exponential or a sigmoidal curve would have been observed in the activation kinetics Under the experimental conditions used in this study, after h incubation $ 88% of wildtype FXI and 60% of mutant FXI was activated by thrombin The kcat ⁄ Km value of FXI activation was 9.8 ± 0.6 · 104 and 4.8 ± 0.8 · 104 m)1Ỉs)1 for wildtype and FXI–Val371Ile, respectively These findings showed that the Val371Ile mutation reduces by approximately twofold the specificity of thrombin interaction with the FXI–Val371Ile Activation of FIX by FXIa The functional properties of activated FXI–Val371Ile were explored both by a proteolytic assay using a commercially available FIX and by measuring Michaelis parameters of S-2366 hydrolysis To this purpose, wild-type FXIa and FXIa–Val371Ile, completely activated by thrombin (as described in Experimental procedures) were incubated for different periods with commercial FIX Upon FXI activation, FIX is cleaved at two sites, releasing an activation peptide, and producing the protease FIXa [10,24] As shown in Fig 4, incubation of FIX with wild-type FXIa results in almost complete activation after 30 min, whereas FXIa–Val371Ile causes a dramatic reduction in the uncleaved FIX form only after 60 of incubation A possible effect of dextran sulfate on FIX activation was ruled out by performing the same experiment in the absence of FXI No activation of FIX was detectable after 60 of incubation (data not shown) The observed delay in FIX activation may be due to a decrease in the catalytic activity of mutant FXIa, possibly caused by a perturbed conformational state of FXIa linked to the Val371Ile mutation A moderate but significant reduction in the catalytic competence of FEBS Journal 274 (2007) 6128–6138 ª 2007 The Authors Journal compilation ª 2007 FEBS 6131 FXI–Val317Ile – a novel factor XI type II defect C Bozzao et al addition to the catalytic residues, a more extended surface area of FXIa The observed increase in Km for S-2366 of the mutant FXI may arise from allosteric effects, and thus may be generated from structural perturbations located far from the catalytic pocket 10 WT- F XI FXIa (nM) V3 I-F XI Discussion 0 20 40 60 80 100 120 Tim e (m in ) Fig FXI activation by thrombin Purified wild-type (d) and FXI– Val371Ile (s) (10 nM, final concentration) were activated by thrombin (3 nM, final concentration) At different time points hirudin (10 nM, final concentration) was added to inhibit thrombin activity, so that the chromogenic substrate S-2366 (500 lM, final concentration) was hydrolyzed solely by activated FXI The velocity of S-2236 hydrolysis by FXIa at each time point was converted into FXIa concentration by means of Eqn (2) Error bars indicate SEM Val371Ile FXIa was confirmed by investigating the catalytic competence of FXIa towards the synthetic substrate S-2366 (Fig 5) The kcat and Km values of S-2366 hydrolysis by wild-type FXI were 49.8 ± s)1 and 595 ± 63 lm, respectively, with kcat ⁄ Km ¼ 8.37 · 104 m)1Ỉs)1 The same parameter values were 45 ± s)1 and 739 ± 100 lm for FXI–Val371Ile, with kcat ⁄ Km ẳ 6.09 à 104 m)1ặs)1 The reduction in the kcat ⁄ Km value for S-2366 hydrolysis was significant, but the effect of the mutation on FIX activation was even more evident, as shown in Fig This suggests that the mutation may alter molecular recognition between FXIa and FIX, which necessarily involves, in In this study, a novel missense mutation in F11 was identified in a proband with mild type II FXI deficiency In vitro expression of the FXI–Val371Ile recombinant protein, followed by activation assays, showed slight differences in both FXI activation and FIX activation by thrombin-activated FXI The functional defect evidenced by in vitro assays is compatible with the deficiency observed in the two analyzed Val371Ile carriers, even though the specific activity calculated for the recombinant mutant protein is somewhat higher than expected We cannot exclude that differences in the dimerization and ⁄ or secretion efficiency of mutant versus wild-type FXI might explain, at least in part, this discrepancy Evolutionary conservation analysis of serine protease sequences shows that the position corresponding to FXI–Val371 is highly conserved For example, among serine protease coagulation factors (i.e FVII, FIX, FX, FXII, plasminogen, and thrombin, showing an overall amino acid sequence identity of 30–45%), this position is occupied solely by a valine This conserved amino acid is replaced by isoleucine in the Val371Ile FXI mutant Interestingly, an isoleucine residue naturally occupies the position corresponding to FXI Val371 in some other serine proteases, such as vitamin-K-dependent protein C, hepatocyte growth factor activator, and neurotrypsin The Val371Ile mutation in FXI results in a relatively mild physicochemical difference, because valine and isoleucine are both highly hydrophobic, b-branched wild-type FXI (min) 15 30 Val3 71 Ile FXI 60 (min) FIX 15 30 60 F IX FIXa FIXa Fig Time course of FIX activation Commercially available FIX (12.5 ng) was activated with 1.5 ng of recombinant FXI, either wild-type or FXI–Val371Ile, both in turn activated by thrombin (0.5 U for 135 min; complete activation was assessed by western blot analysis) At different time points (indicated at the top of each panel) digestions were stopped and proteins were resolved by Laemmli SDS ⁄ PAGE using 12% (w ⁄ v) acrylamide gels 6132 FEBS Journal 274 (2007) 6128–6138 ª 2007 The Authors Journal compilation ª 2007 FEBS C Bozzao et al FXI–Val317Ile – a novel factor XI type II defect Velocit y of hydrolysis (s -1) 40 W T-FXI V3 I-FXI 30 20 10 0 S -2 6 (mM) Fig Determination of Michaelis parameters of S-2366 hydrolysis Steady-state kinetics of S-2366 hydrolysis by wild-type (d) or FXI– Val371Ile (s), under the conditions reported in Experimental procedures The continuous lines were drawn according to Michaelis equation using the best fit parameters: (d) kcat ¼ 49.8 ± s)1, Km ¼ 595 ± 63 lM; (s) kcat ¼ 45 ± s)1, Km ¼ 739 ± 100 lM Error bars indicate SEM amino acids (the b-carbon has two substitutions); nevertheless, the mutation is not isosteric, isoleucine being larger than valine, and having an additional methyl on its side chain In the structure of the FXI zymogen [11], Val371 is located on the linker region between the Ap4 and protease domains, and its surface area is 77% exposed to the solvent After activation of FXI, the activation loop (residues 370–376), which is located at the new N-terminus of the protease domain, undergoes a large movement towards the activation pocket of FXIa As a result, in the structure of FXIa [25], the surface area of Val371 is 92% buried within the protein, contacting residues Arg144, Gly188, Asp189, Cys219, and Ala220 Given that Val371 is buried in the structure of FXIa, the introduction of a larger residue in this position most likely causes some degree of structural change; this is especially true in the case of the introduction of an isoleucine, a b-branched amino acid that is not flexible In the active conformation, Val371 forms contacts with neighboring residues that are important for stabilizing the active state (e.g Asp189, which is part of the S1 pocket responsible for the binding specificity of the substrate) [26] Consequently, substitution of Val371 to isoleucine might prevent the full development of the active conformation This hypothesis is further confirmed by the results of FIX proteolytic assays, which showed a slight delay in FIX activation by FXIa activated by thrombin (Fig 4); moreover the kcat and Km values of S-2366 hydrolysis showed that the Val371Ile Fig Structural consequences of the Val371Ile substitution Ribbon representation of the superimposition between the structures of the catalytic serine protease domain of the zymogen (red) and activated (green) FXI The Ile371 residue, in both structures, is displayed by space-filled atoms The catalytic triad (blue space-filled atoms) is also shown The conformational movements of Ile371, located in the activation loop at the N-terminus of the catalytic domain, are notable In the zymogen FXI, Ile371 is exposed to the solvent, while in the activated FXI it is inserted into the protein The picture was drawn with PYMOL (DeLano Scientific, San Carlos, CA; http://www.pymol.org) mutation has only minor conformational effects on the geometry of the catalytic site of the enzyme (Fig 5) In contrast to the activated FXI, in the structure of the FXI zymogen, Val371 is located on a loop region, exposed to solvent, and does not form many contacts with other residues (Fig 6) Therefore, the additional methyl in the Val371Ile mutant probably does not disturb the structure and the domain rearrangement in the zymogen FXI Nevertheless, recombinant FXI– Val371Ile activation was slower than that of the wildtype protein (Fig 2) suggesting a small activation defect This might be explained by the proximity of the mutation to the cleavage site, probably resulting in a small interference with the binding of the activator to the FXI zymogen There are some examples of inherited coagulation disorders in which one of the peptide linkages required for the proteolytic zymogen activation cannot be cleaved by the physiological activator In most cases, the mutated residue corresponds to the P1 site (i.e the C-terminal residue of the activation peptide) [27–33] However, some mutations involving the P1¢ and P2¢ positions (i.e the two first residues from the N-terminal end of the catalytic domain) were previously reported to cause mild to severe FIX deficiency either FEBS Journal 274 (2007) 6128–6138 ª 2007 The Authors Journal compilation ª 2007 FEBS 6133 FXI–Val317Ile – a novel factor XI type II defect C Bozzao et al by altering the functional properties of FIXa or by delaying its activation by FXIa In particular, four different amino acid substitutions (Val182Leu, Val182Phe, Val182Ala, and Val182Gly) corresponding to the here-reported Val371Ile in FXI, were found in hemophilia B patients [34–37] The phenotypic consequences of these missense mutations were variable, ranging from the complete loss of function of FIX Kashihara (Val182Phe) to a residual 15% of procoagulant activity of FIX Cardiff (Val182Leu) [37] In conclusion, the Val371Ile mutation, identified and characterized here, brings the number of naturally occurring FXI variants responsible for type II deficiencies to seven [13] Of these, three have been characterized in-depth, showing different mechanisms underlying the pathologic phenotype, i.e a reduction in affinity for platelets (Ser248Asn) [38], a modest reduction of FXI catalytic activity (Pro520Leu) [39], and a greatly reduced rate of FIX activation associated with resistance to antithrombin inhibition (Gly555Glu) [40] Uniquely, our mutation is associated with a defect both in FXI activation (slower than normal), and in FIX activation (slightly delayed), thus supporting the role of residues neighboring the active site in influencing and stabilizing the enzyme active state Experimental procedures Blood collection and genomic DNA extraction This study was approved by the Institutional Review Board of the University of Milan All subjects signed an informed consent according to the Declaration of Helsinki before blood withdrawal Peripheral venous blood was collected in : 10 volume of 0.11 m trisodium citrate, pH 7.3 Genomic DNA was extracted from whole blood using a standard salting-out procedure Coagulation studies Immediately after collection, citrated blood was centrifuged at 2500 g for 15 at room temperature FXI activity was performed by a one-stage method based on a modified partial thromboplastin time, using FXI-deficient plasma as substrate (Hemoliance, Salt Lake City, UT) FXI antigen was measured by an ELISA based on a goat anti-human FXI affinity purified IgG as capture antibody and a goat antihuman FXI peroxidase-conjugated IgG as detecting antibody (Affinity Biological Inc., Hamilton, Ontario, Canada) FXI levels were expressed in both tests as percentages of pooled normal plasma from 30 normal male and female individuals The detection limits of the FXI functional and immunologic assays were and 0.1%, respectively 6134 PCR amplifications and DNA sequencing PCR were performed on 50–100 ng of genomic DNA in a 25 lL volume, following standard procedures [41] PCR and sequencing primers were designed on the basis of the known genomic sequence of F11 (GenBank accession number NM_000128) The primer couple used to amplify F11 exon 11 and to identify the Val371Ile mutation was FXIex11-F 5¢-GTCAATTCCATTTTTCATGTGC-3¢ and FXIex11-R 5¢-CGTTTTTTACCACTGAAGCAAT-3¢ All other primer sequences, as well as the specific PCR condition for each primer couple, are available on request Sequencing reactions were performed on both strands on PCR products purified by MICROCON 100 columns (Millipore, Bedford, MA) The BigDye Terminator Cycle Sequencing Kit version 3.1 and an automated ABI-3100 DNA sequencer (Applied Biosystems, Foster City, CA) were used Site-directed mutagenesis The pCDNA3 ⁄ FXI expression plasmid, containing fulllength FXI complementary DNA (cDNA), was kindly provided by A Zivelin (Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel Hashomer, Israel) The identified missense mutation was introduced in pCDNA3 ⁄ FXI by the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA), according to the manufacturer’s instructions The mutant plasmid pCDNA3 ⁄ FXI–Val371Ile was checked by sequencing the whole FXI cDNA insert as well as 200 bp of flanking DNA on both sides of the cloning site Large-scale plasmid preparations were obtained using the EndoFree Plasmid Maxi Kit (Qiagen, Hilden, Germany) Proteins and antibodies Thrombin, FIX, FXI, and FXIIa were obtained from Enzyme Research Laboratories (Swansea, UK) The sources of the antibodies were as follows: rabbit anti-human FIX (catalogue number F 0652) from Sigma (St Louis, MO), goat anti-human FXI (catalogue number GAFXI-IG) from Affinity Biologicals Inc., peroxidase-conjugated goat anti-rabbit IgG from Pierce Biotechnology Inc (Rockford, IL), and peroxidase-conjugated donkey anti-goat IgG from Jackson ImmunoResearch Laboratories Inc (West Grove, PA) Cell culture African green monkey kidney COS-1 cells were cultured in DMEM (EuroClone, Wetherby, UK) supplemented with 10% fetal bovine serum (HyClone, South Logan, UT), antibiotics (100 mL)1 penicillin and 100 lgỈmL)1 streptomycin; EuroClone) and glutamine (2 mm; EuroClone), and grown at 37 °C in a humidified atmosphere FEBS Journal 274 (2007) 6128–6138 ª 2007 The Authors Journal compilation ª 2007 FEBS C Bozzao et al of 5% CO2 and 95% air, according to standard procedures Expression of recombinant proteins In each transfection experiment an equal number of cells (400 000) were transiently transfected with the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) in six-well plates with lg of plasmid DNA (pCDNA3 ⁄ FXI, or pCDNA3 ⁄ FXI–Val371Ile, or equimolar amounts of both plasmids), essentially as described by the manufacturer Twenty-nine hours after transfection, cells were washed twice with NaCl ⁄ Pi and cultured for additional 48 h in mL of serum-free medium supplemented with glutamine, antibiotics, and mgỈmL)1 BSA For each experiment (performed four times in duplicate) a mock sample, with the empty pCDNA3 plasmid, was set up Conditioned media from each well were tested for both FXI antigen and coagulation activity and used to prepare FXIa for SDS ⁄ PAGE analysis FXI measurement in conditioned media and in cell lysates FXI antigen levels were evaluated by ELISA, as described above, both in conditioned media and in cell lysates Standard curves were constructed with reference plasma diluted : 100 to : 6400 in Tris-buffered saline (NaCl ⁄ Tris: 50 mm Tris, 150 mm NaCl, pH 7.5) Conditioned media were collected in prechilled tubes containing a protease inhibitor mixture (Complete; Roche, Basel, Switzerland), centrifuged to remove cell debris, and stored at )80 °C until use To obtain cell lysates, cells were washed three times with prechilled NaCl ⁄ Pi and incubated for h on ice with 1· NaCl ⁄ Pi, 1.5% Triton X-100, and 1· Complete Samples were collected and centrifuged to remove cell debris FXI coagulant activity was measured in media (collected without any protease inhibitor) as described above (see ‘Coagulation studies’) Activation of FXI FXI was activated either with FXIIa or with thrombin For each activation experiment, the exact amount of the recombinant protein was assessed by an ELISA assay, as described above; on average, 2.5 lL of conditioned media corresponded approximately to 1.5 ng of protein FXIIa (1 lg) and 1.5 ng of recombinant FXI, either wild-type or mutant, were incubated in NaCl ⁄ Tris at 37 °C for different periods Each reaction was carried out in a final volume of 20 lL Samples were removed into reducing SDS sample buffer and size-fractionated on 10% polyacrylamide SDS gels Because in vitro activation of FXI by thrombin is highly enhanced in the presence of polyanions such as dextran FXI–Val317Ile – a novel factor XI type II defect sulfate [42], 1.5 ng of recombinant FXI, either wild-type or mutant, was activated with 0.5 U ($ nm) of human thrombin in NaCl ⁄ TrisA (NaCl ⁄ Tris supplemented with 0.1 mgỈmL)1 BSA) containing lgỈmL)1 dextran sulfate (500 000 Da) at 37 °C for different periods The concentration of dextran sulfate (1 lgỈmL)1) used in our experiments was found to be optimal in previous studies [23,42,43] Each reaction was carried out in a final volume of 20 lL Aliquots (each containing 1.5 ng of recombinant FXI) were stopped by adding 10 lL of 3· reducing Laemmli sample buffer, and run on 10% SDS ⁄ PAGE Proteins were then transferred onto 0.45 lm pore-size nitrocellulose membranes (Schleicher & Schuell, Brentford, UK) and analyzed by western blotting, using a polyclonal goat anti-human FXI IgG Activation of FIX by FXIa Recombinant FXI, either wild-type or mutant, was activated with 0.5 U thrombin in NaCl ⁄ TrisA containing lgỈmL)1 dextran sulfate at 37 °C for 135 in a total reaction volume of 20 lL; complete activation was verified by western blotting (see below) After that, 1.5 ng of FXIa and 12.5 ng of FIX were incubated in NaCl ⁄ TrisA with 2.5 mm CaCl2 at 37 °C for different periods Each reaction was carried out in a final volume of 30 lL The ability of residual thrombin and dextran sulfate in the buffer solution to activate FIX was ruled out in preliminary experiments At different time points aliquots were removed into reducing SDS sample buffer, and size-fractionated on 12% polyacrylamide SDS gels Proteins were transferred onto nitrocellulose membranes and analyzed by western blot using polyclonal rabbit antihuman FIX IgG Western blotting analysis Blots were incubated at room temperature for h in NaCl ⁄ Tris containing 0.1% Tween 20 and 5% (w ⁄ v) skimmed milk The membranes were then incubated for h with primary antibodies and subsequently for h with donkey anti-goat IgG or goat anti-rabbit IgG horseradish peroxidase-conjugated secondary ones When the antihuman FIX IgG was used, dilutions were performed in NaCl ⁄ Tris supplemented with 0.3% BSA at room temperature; all other incubations were done in NaCl ⁄ Tris containing 5% milk Proteins were detected using Enhanced Chemioluminescence, SuperSignal West Dura Extended Duration Substrate (Pierce) Assay of FXI activation Before activation by thrombin, supernatants from cells expressing recombinant FXI were concentrated approxi- FEBS Journal 274 (2007) 6128–6138 ª 2007 The Authors Journal compilation ª 2007 FEBS 6135 FXI–Val317Ile – a novel factor XI type II defect C Bozzao et al mately four- to fivefold by means of VivaSpin 30 concentrators (Sartorius Ltd., Epsom, UK) Activation of both wild-type and FXI–Val371Ile (10 nm) by thrombin (3 nm), purified as previously detailed [44], was measured by a chromogenic assay, as follows Incubations were carried out in 100 lL of 50 mm Tris, 150 mm NaCl, pH 7.5, with 0.1% poly(ethylene glycol) 6000 at 25 °C In the FXI activation by thrombin, dextran sulfate was omitted from the reaction buffer to avoid any spurious effect on FXI autoactivation At various time intervals, 10 lL of recombinant hirudin (Sigma) at a final concentration of 10 nm were added to inhibit thrombin activity Then 50 lL of 500 lm (final concentration) S-2366 (pyroGlu-Pro-Arg-pNA; Chromogenix, Molndal, Sweden) were added to the solution, ă and the amount of free paranitroaniline released by FXIa was determined by measuring the change in absorbance at 405 nm in a Benchmark II microplate reader (Bio-Rad Laboratories, Hercules, CA) To eliminate any scattering contribution, the absorbance at 620 nm was always subtracted from the reading at 405 nm The initial velocity of S-2366 hydrolysis obtained at each time point was considered proportional to FXIa generated by thrombin The velocity of S-2366 hydrolysis was then analyzed as a function of time, to calculate the pseudo-first-order rate constant of both wild-type and mutant FXI cleavage by thrombin Accordingly: Vt ¼ V1 à ð1 À expðÀk à tÞÞ where Vt and V¥ are the velocities of S-2366 hydrolysis by formed FXIa at time t and ¥, respectively, and k is the pseudo-first order rate constant of FXI activation by thrombin The best-fit value of k is thus independent from the intrinsic catalytic activity of both wild-type and mutant FXIa, but depends only on the specificity of thrombin–FXI interaction The only assumption made was that the value of the asymptotic parameter V¥ corresponds to the velocity of the substrate hydrolysis by the FXIa concentration equal to the nominal concentration of zymogen FXI present in solution, assuming that the entire amount of zymogen FXI was converted to FXIa at time ¥ The reaction was studied at a concentration of FXI < Km of thrombin hydrolysis so that the rate constant k was proportional to the value of kcat ⁄ Km of the activation, according to: k ẳ T kcat =Km 2ị where V120 is the velocity of S-2366 hydrolysis at 120 and FXIT is the total concentration of either wild-type or mutant zymogen FXI present in the activation solution The validity of this approach was confirmed in the case of the wild-type form, whose concentration, calculated by Eqn (2) was in agreement within 10% error with that obtained from a reference curve, where the catalytic activity of different concentrations of a purified FXIa preparation (Hematological Technologies Inc., Essex Junction, VT) in the presence of 500 lm S-2366 were linearly correlated to the nominal enzyme concentration (supplementary Fig S1) At time 120 min, chosen to avoid instability or autohydrolytic damage of thrombin at longer incubation times, an aliquot of the activation solution was taken to measure the Michaelis parameters of S-2366 hydrolysis by FXIa The Michaelis parameters, kcat and Km, were calculated on the basis of known concentration of wild-type and mutant FXIa and using the program grafit (Erithacus Software Ltd., Staines, UK) Structural analysis The structural analysis was conducted using the crystal structure of the zymogen FXI (PDB code: 2F83) [11] and FXIa (PDB code: 1XX9) [25] The solvent-accessible area for each residue in both structures was calculated using the surfv program [46] with a probe sphere of radius ˚ 1.4 A and default parameters The percentage of the surface-exposure of each residue in the monomer was calculated from the total solvent-accessible area on a Gly-X-Gly tripeptide (where X represents each of the 20 amino acids) Evolutionary conservation analysis Evolutionary conservation analysis was carried out using the ConSurf web-server [47] (http://consurf.tau.ac.il/) The calculations were performed using the structure of FXIa (PDB code: 1XX9) [25], based on an alignment of 200 serine protease sequences collected from the SWISSPROT database [48] and default parameters ð1Þ Acknowledgements where T is the thrombin concentration Measurement of Michaelis parameters of S-2366 hydrolysis by wild-type and FXI–Val371Ile After 120 of FXI activation by thrombin, $ 88% (8.8 nm) of wild-type FXI and 63% (6.3 nm) of mutant FXI were activated, according to [45]: 6136 ẵFXIa120 ẳ V120 =V1 à FXIT The authors would like to thank Sofia H Giacomelli for excellent technical assistance SD is a recipient of a Bayer Hemophilia Early Career Investigator Award 2006 The financial support of PRIN (Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale, Grant n 2005058307-002) is gratefully acknowledged FEBS Journal 274 (2007) 6128–6138 ª 2007 The Authors Journal compilation ª 2007 FEBS C Bozzao et al References Davie EW, Fujikawa K, Kurachi K & Kisiel W (1979) The role of serine proteases in the blood coagulation cascade Adv Enzymol Relat Areas Mol Biol 48, 277–318 Fujikawa K, Legaz ME, Kato H & Davie EW (1974) The mechanism of activation 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algorithm to calculate solvent accessible surface area Biophys J 61, A174 Landau M, Mayrose I, Rosenberg Y, Glaser F, Martz E, Pupko T & Ben-Tal N (2005) ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures Nucleic Acids Res 33, W299–W302 Bairoch A & Apweiler R (1999) The SWISS-PROT protein sequence data bank and its supplement TrEMBL in 1999 Nucleic Acids Res 27, 49–54 Supplementary material The following supplementary material is available online: Fig S1 Comparison of the catalytic properties of recombinant and plasma-derived FXIa This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 274 (2007) 6128–6138 ª 2007 The Authors Journal compilation ª 2007 FEBS ... following supplementary material is available online: Fig S1 Comparison of the catalytic properties of recombinant and plasma-derived FXIa This material is available as part of the online article... hydrolysis by FXIa The Michaelis parameters, kcat and Km, were calculated on the basis of known concentration of wild -type and mutant FXIa and using the program grafit (Erithacus Software Ltd., Staines,... which is located at the new N-terminus of the protease domain, undergoes a large movement towards the activation pocket of FXIa As a result, in the structure of FXIa [25], the surface area of Val371