Y-Carboxyglutamic Acids 36 and 40 Do Not Contribute to Human Factor IX Function

Protein Science (1997), 6185-196. Cambridge University Press. Printed in the USA. Copyright 0 1997 The Protein Society y-Carboxyglutamic acids 36 and 40 do not contribute to human factor IX function

SHMUEL GILLIS,' BARBARA C. FURIE,' BRUCE FURIE,' HIMAKSHI PATEL: MICHAEL C. HUBERTY: MARY SWITZER: W. BARRY FOSTER: HUBERT A. SCOBLE: AND MICHAEL D. BOND* 'The Center for Hemostasis and Thrombosis Research, Division of Hematology-Oncology, New England Medical Center, Department of Medicine and Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 'Genetics Institute Inc., Andover, Massachusetts

(RECEIVED September 9, 1996 ACCEP~EDOctober 16, 1996)

Abstract The y-carboxyglutamic acid (Gla) domains of the vitamin K-dependent blood coagulation proteins contain 10 highly conserved Gla residues within the first 33 residues, but factor IX is unique in possessing 2 additional Gla residues at positions 36 and 40. To determine their importance, factor IX species lacking these Gla residues were isolated from heterologously expressed human factor IX. Using ion-exchange chromatography, peptide mapping, mass spectrometry, and N-terminal sequencing, we have purified and identified two partially carboxylated recombinant factor IX species; factor IXIy40E is uncarboxylated at residue 40 and factor IX/y36,40E is uncarboxylated at both residues 36 and 40. These species were compared with the fully y-carboxylated recombinant factor IX, unfractionated recombinant factor IX, and plasma-derived factor IX. As monitored by anti-factor IX:Ca(II)-specific antibodies and by the quenching of intrinsic fluorescence, all these factor IX speciesunderwent the Ca(I1)-induced conformational transition required for phospholipid membrane binding and bound equivalently to phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine. Endothelial cell binding was also similar in all species, with half-maximal inhibition of the binding of 1251-labeledplasma-derived factor IX at concentrations of 2-6 nM. Func- tionally, factor IX/y36,40E and factor IXIy40E were similar to fully y-carboxylated recombinant factor IX and plasma-derived factor IX in their coagulant activity and in their ability to participate in the activation of factor X in the tenase complex both with synthetic phospholipid vesicles and activated platelets. However, Gla 36 and Gla 40 represent part of the epitope targeted by anti-factor IX:Mg(II)-specific antibodies because these antibodies bound factor IX preferentially to factor IXly36,40E and factor IXIy40E. These results demonstrate that the y-carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for any function of factor IX examined. Keywords: calcium-binding protein; Gla; hemophilia B; vitamin K

Factor IX is a vitamin K-dependent plasma zymogen that plays a function requires the presence of Gla residues that participate di- central role in blood coagulation. Patients deficient in factor IX rectly in Ca(I1) binding via their malonate side chains (Furie & activity have the bleeding disorder hemophilia B. In common with Furie, 1988; Freedman et al., 1995a). Gla is formed by a post- the other vitamin-K dependent blood coagulation proteins, fac- translational modification in which glutamic acid residues undergo tor IX contains an amino-terminal Gla domain that is responsible y-carboxylation mediated by the enzyme y-glutamyl carboxylase for its phospholipid binding properties (Furie & Furie, 1988). This (Furie & Furie, 1988). The Gla domains in the vitamin-K dependent proteins contain 9-12 Gla residues; of these, the N-terminal nine Gla residues are fully conserved and the tenth is highly con- Reprint requests to: BNce Furie* Division Of Hematology-oncology, served, Factor IX is unique in possessing two additional distal GIa New England Medical Center, 750 Washington St., Boston, Massachusetts 02111_-.._. residues at positions40 36 and (Vermeer, 1990). Abbreviurions: AAAS, aromaticamino acid stack; Achro-K, Achromo- The rnetal-free and calcium ion-bound structures of the fac- bacter Protease I; DHB, 2,5-dihy&oxybenzoic acid; dPE, dansyl-phospha- tor IX Gla and AAAS domains have been determined recently by tidylethanolamine; EGE epidermal growth factor; Gla, y-cahwglutamic NMR spectroscopy (Freedman et al., 1995a, 1995b). nemetal-free acid; HMB, 2-hydroxy-5-methoxybenzoicacid; MALDI-TOF MS, matrix assisted laser desorption ionization time-of-flight maSSspectromeny; PACE, structure is relatively disordered, whereas the calcium ion-bound paired basic amino acid cleaving enzyme; PC, phosphatidylcholine; PITC, form is substantially ordered, consisting of a large amino-terminal phenylisothiocyanate; PS, phosphatidylserine. loop (residues 1-12), three helical segments (residues 14-17,25-32, 185 S. Gillis et al. and 35-46), and a disulfide loop (residues 18-23). The three- dimensional structure of the polypeptide backbone of the calcium- bound form is very similar to that of prothrombin fragment 1 (Soriano-Garcia et al., 1992), but small differences in the N-terminal loop may befunctionally important. In the calcium ion-bound form, the amino-terminal nine Gla residues are oriented to the interior of the protein, consistent with an internal Ca(I1) binding pocket. Gla residues 33, 36, and 40 are on the solvent-exposed surface of the calcium ion-bound structure. Their role has not been defined. Gla 40 has been postulated to be required for the stabilization of the Gla domain carboxyl-terminal a-helix through ionic interactions (Freed- man et al., 1995a). Others have suggested, based on the X-ray structure of the factor IX EGF-like domain, that Gla 40 may be a Ca(I1) binding site required for the correct folding of the EGF domain over the adjacent Gla domain (Rao et al., 1995). MINUTES Gla at residue 33 may be important for factor IX activity because a mutation of the equivalent residue in prothrombin to as- Rec. Factor IX partic acid yielded aprotein with reduced coagulantactivity (Ratcliffe et al., 1993). A Gla to Asp mutation at residue 33 causes a severe deficiency in factor IX in a patient with hemophilia B; E however, the specific activity of this mutant is not known (Koeberl et al., 1989). The importance of Gla 36 and Gla 40 in factor IX N function remains unknown. Recombinant factor IX, in contrast to recombinant prothrombin (Jorgensen et al., 1987; Ratcliffe et al., 1993), has been found to be under-y-carboxylated, especially when expressed at high levels (Kaufman et al., 1986; Derian et al., 1989). In this report, we describe the isolation and purification of two partially y-carboxylated species of recombinant factor IX and demonstrate that they are 0 20 40 lacking y-carboxylation either at residue 40 or at both residues 36 MINUTES and 40. We have characterized these species functionally and compared them with plasma-derived human factor IX. We show that

Gla 36 and Gla 40 are not required for any function of human 01 factor IX that was examined.

Results Factor IX undergoes numerous posttranslational modifications, including the y-carboxylation of 12 glutamic acid residues in the Gla and AAAS domains (Furie & Furie, 1988; Vermeer, 1990), P-hydroxylation of Asp 64 (Femlund & Stenflo, 1983; Furie & Furie, 1988), and 0-glycosylation of two sites in the first EGF domain, at Ser 53 and Ser 61, the latter of which contains sialic 0 I acid (Bharadwaj et al., 1995). The activation peptide contains sev- 0 20 40 eral glycosylation, sulfation, and phosphorylation sites (Agarwala MINUTES et al., 1994; Bond et al., 1994a). In order to obtain species of Fig. 1. Mono Q HPLC separation of recombinant factor IX species. A: factor IX that lack Gla at specific residues, we fractionated recom- Recombinant factor IX samples were prepared in 20 mM Tris, pH 9.0, and binant factor IX expressed in Chinese hamster ovary cells. This injected in volumes of 0.1-2.0 mL. Elution employed a 0.3-0.4 M NaCl expression system yields nearly completely processed factor IX, gradient. Sample A represents results for a typical sample of recombinant factor IX. Sample B represents a sample with unusually high levels of but a component of recombinant factor IX is undercarboxylated peak 1. B: factor IX was analyzed by Mono Q chromatography as above, and thus served as a sourceof homogeneous partially carboxylated before and after removal of sialic acids with neuraminidase. C: Mono Q factor IX. chromatography before and after activation of recombinant factor IX with Application of unfractionated recombinant factor IX to a Mono Q factor XIa (1 :200, w/w) in 50 mM Tris, 0.15 M NaCI, 5 mM CaCI2, pH 7.5, ion exchange chromatography column yielded three major frac- for 1 h at 37°C. tions observed as peaks 1, 2, and 3. Peak 3 included a small shoul- der (Fig. 1A). To determine which modifications were responsible for the multiple species observed by ion exchange chromatography, tion differences. A sample of recombinant factor IX was also stud- a sample of recombinant factor IX was analyzed before and after ied before and after activation with factor XIa, a process that removes enzymatic desialylation. Desialylation induced a shift in retention the activation peptide (and any modifications therein). The chro- times, but did not change the chromatographic profile (Fig. 1B). matogram again demonstrated the same three major peaks, and there Therefore, the observed fractionation does not result from sialyla- was no shift in the retention times (Fig. IC). The results show that Gla 36 and Gla 40 of human factor TX 187

Table 1. Gla content of Mono Q purified recombinant factor IX speciesa 0.2 Species mol Gldmol protein

Peak 1 10.3 -C 0.3 Peak 2 11.2 * 0.2 0 Peak 3 12.1 * 0.2 12-Gla Species Unfractionatedrecombinant factor IX 11.5 f 0.2 K3[6-Gla] Plasma-derived factor IX 12.0 * 0.3 Factor X 10.6 f 0.3 : 0.1 "Gla content was determined by amino acid analysis following alkaline v -0 hydrolysis as described in Materials and methods.The raw values of mol Glal N mol protein for each sample was normalized to yield mol Glahol ui 11-Gla Species 12.0 P protein for the plasma-derived factor included in each set of assays. M =x 0.2 Samplesof human factor X were also included as indicators of assay reliability; the theoretical Gla content of factor IX and factor X is 12 and 11 mol Gla/mol protein, respectively. N = 2 for factor X, and N = 3 for all other samples. 0 10-Gla Species the three species observed by ion exchange chromatography do not arise from differences in sialic acid content, N-glycans, sulfation, or phosphorylation, and thus implicate differences in Gla content as 0.1 a possible cause. This was confirmed by subjecting the protein fractions to Gla analysis (Table 1). Within experimental error, the val- 0 ues for the three protein fractions correspond to 10, 11, and 12 mol 40 80 120 of Gla per mol of protein. Minutes To confirm the identity of the species according to their total Gla Fig. 3. Reverse-phase HPLC separation of recombinant factor IX species content, aliquots were subjected to peptide mapping by Achro-K following Achro-K digestion. Aliquots of 75-150 mg of unfractionated digestion. By this method, we have shown that the Gla domain recombinant factor IX and 10-Gla, 1 I-Gla, and 12-Gla species were prepared as described in Materials and methods then digested by the addition of unfractionatedrecombinant factor IX is recoveredin two at 4 h intervals of three aliquots of Achro-K (enzyme to substrate ratio for fragments"KlK2 (residues 1-22) and K3 (residues 23-43) (Bond each aliquot 1:25, w/w) and incubated for 24 h at 30°C. The reaction et al., 1994b). These peptides each contain six y-carboxylation mixture was fractionated by C-18 reverse-phaseHPLC using a 4.6 X sites (Fig. 2). K1K2 is present almost exclusively in the fully 250 mm column (Vydac) at 0.75 mL/min linear gradient as described in the text. The Gla domain is recovered in two peptides, residues desig- y-carboxylated form, whereas K3is present in multiple forms 1-22, nated KIK2,and residues 23-43, designated K3. Mass spectrometry anal- containing 4,5, and 6 Gla. Peptide maps of unfractionated recom- yses of the predominant K3 peaks are shown in Table 2. binant factor IX and IO-Gla, 1 1-Gla, and 12-Gla species are shown in Figure 3. The 10-Gla, 11-Gla, and 12-Gla species contained K1K2 in one predominant form with the expected retention time and peak area forthe 6-Gla peptide. In contrast, these species species contained almost exclusively K3[5-Gla]; and the peptide contained K3 in different forms based on retention times and mass map of the IO-Gla species contained primarily K3[4-Gla]. Minimal spectroscopic analysis (Table 2). The peptide map of the 12-Gla contamination with K3[5-Gla] was also observed (Fig. 3). Thus, species contained fully carboxylated K3 (K3[6-Gla]); the 1 I-Gla the major 1 1-Gla and 10-Gla species are undercarboxylated at one and two sites, respectively, within residues 23-43. Identification of the specific sites in the IO-Gla and 11-Gla 1 10 20 30 40 species that contain glutamic acid instead of Gla was addressed by YNSGKL~~FVQGNL~R~CH~~KCSF~~AR~VF~NT~RTT~FWKQYV tryptic digestion of the K3 peptides. Trypsin cleavage of K3 gen- erates two fragments: K3a, including residues 23-37, containing I Achro K dlgest five possible Gla sites; and K3b, including residues 38-43, containing a single possible Gla site (Fig. 2). Digestion of the mixture 1 I0 20 30 40 YNSGKL~~FVQGNL~R~CH~~KCSF~~AR~VF~NT~RTT~FWK of K3[6-Gla] and K3[5-Gla] from unfractionated recombinant fac- t I K1 K2 K3 tor IX yielded three fragments, identified by MALDI-TOF MS as K3b with zero and one Gla residues, and K3a with five Gla residues (K3a [Xila]) (Fig. 4A; Table 3). Trypsin digestion of the 1 Trypsm dlgest 12-Gla and 1 1-Gla species revealed that both contained K3a[5-Gla]. 30 40 However, whereas K3b in the 12-Gla species was almost exclu- CSFYyARyVFyNTyR TTyFWK I I- sively K3b[ I-Gla] (Fig. 4B), the 1 1-Gla species contained only K3a K3b K3a K3b[O-Gla] (Fig. 4C). These results demonstrate that the 1 I-Gla Fig. 2. Amino acid sequence of human factor IX Gla and AAAS domains, species is uncarboxylated solely at residue 40. We designate this depicting fragments generated by Achro-K and trypsin digestion. 1 I-Gla species as factor IXIy40E. The IO-Gla species contained 188 S. Gillis et al.

Table 2. MALDI-TOF MS identification of the predominant K3 peptidesa

Observed mass Theoretical mass Sample(Da) mapped (Da) Assignment

Unfractionated factor IXb 2,984.1 2,983.9 K3[6-Gla] + Na+ 2,941.6 2,939.9 K3[5-Gla] + Na+ 2,897.0 2,895.9 K3[4-Gla] + Na+ 12-Gla species' 2,960.2 2,961.9 K3[6-Gla] + H+ 1 I-Gla species' 2,915.9 2,917.9 K3[5-Gla] + H+ IO-Gla species' 2,873.6 2,873.9 K4[4-Gla] + H+

aTheoretical masses shown are for reduced and carboxymethylated recombinant human factor IX; values are calculated as average protonated or sodiated masses. Data shown are collected in linear mode. bSamples were analyzed using a sinapinic acid matrix in the absence of metal ion scavengers. 'Samples were analyzed using a matrix consisting of 9:1 DHAB:HMB in the presence of a metal ion scavenger (Chelex beads).

K3[5-Gla]

0.4 IC \4 1

05 GO 2 After Trypsin Digestion U - K3a[5-Gla] K3b[l-Gla] 0.4 - L/ \4 I I( K3b[O-Gla] I 0.2 -

I / 0 20 40 60

D K3[4-Gla]

0.4 -

0.2 0.2 - E -P N 40 o+, n After Tryqsin Digestion U 1 After Trypsin Digestion K3a[5-Gla] 0.8 KJb[l-Gla]

0.4

0 20 40 60 20 40 60 5Minutes Minutes Fig. 4. Reverse-phase HPLC separation of K3 peptides following trypsin digestion. The predominant K3 peptide for each factor IX species was purified and digested with trypsin in 0.5 M NaC1, 50 mM Tris, 5 mM CaCI2, pH 8.0, at an enzyme to substrate ratio of 1:4 in a total volume of 500 pL at 37 "C for 24 h. Peptide products were isolated by reverse-phase HPLC with a C18 column, 4.6 X 250 mm at a 0.75-mL/min linear gradient as described in Materials and methods. A: Unfractionated factor IX. B: 12-Gla species. C: 1 I-Gla species. D: IO-Gla species. Peptides appearing in the labeled peaks were identified by mass spectrometry (Table 3). Gla 36 and Gla 40 of human factor IX 189

Table 3. MALDI-TOF MS data of fragments from Table 4. N-terminal sequence analysis trypsin digest of K3 peptidesa of K3a[4-Gla] and K3a[S-Gla]a

Observed Theoretical % of pmol applied mass mass Sample (Da) Fragment (Da) Cycle Residue K3a[5-Gla]

Unfractionated rFIX K3b[l-GlaIb 856.2 855.9 1 CM-Cys (100) ( 100) (K3[6-Gla] and K3[5-Gla])K3b[O-GlaIb 812.3 811.9 2 S 60 52 K3a[S-Gla]C 2,125.9 2,125.0 3 F 95 96 12-Gla-K3[6-Gla] K3b[l-GlaIb 856.4 855.9 4 E nd nd K3a[S-Gla]C 2,127.4 2,125.0 5 E 1 1 1 l-Gla-K3[5-Gla] K3b[O-GlaIb 81 1.5 81 1.9 6 A 71 64 K3a[S-Gla]C 2,127.2 2,125.0 7 R + + 10-Gla-K3[4-Gla] K3b[O-GlaIb 8 1 1.8 811.9 8 E 3 nd K3a[4-Gla]C 2,080.0 2,08 1 .O 9 V 59 52 10 F 51 46 11 E 3 2 aTheoretical masses shown are for reduced and carboxymethylated re- 12 N 29 28 combinant factor IX (rFIX); values are calculated as average protonated 13 T 39 21 masses. Data shown are collected in linear mode. bSamples were analyzed using a DHB matrix. 14 E 4 18 'Samples were analyzed using a matrix consisting of 9:1 DHB:HMB in 15 R + + the presence of a metal ion scavenger (Chelex beads). agecause Gla is not eluted from the membrane during Edman degradation, Gla is not observed during sequencing. Low levels of decarboxylation K3a[4-Gla] and K3b[O-Gla] (Fig. 4D), indicating that it contains of Gla to Glu occur during sequencing due to the presence of trifluoroacetic glutamic acid in place of Gla at residue 40 and at an additional acid, so small amounts of Glu can be observed. CM-Cys, carboxymethyl Cys, which elutes with a retention time similar to that of Gln; +, residue residue in the K3a peptide. was detected but could not be quantitated reliably; nd, no amino acid was To determine the second site of undercarboxylation in the IO-Gla detected in that cycle. The initial amount of peptide loaded was approxi- species, N-terminal sequencing of K3a[4-Gla] from the IO-Gla mately 500 pmol for K3a[5-Gla] and 525 pmol for K3a[4-Gla]. species was performed using K3a[5-Gla] from the 12-Gla species as a control (Table 4). Because phenylthiohydantoin (RH)-Gla is not eluted from the filter during standard Edman degradation, no specific (Liebman et al., 1987). The first transition may be mon- PTH-amino acid is observed in a cycle of a Gla residue. Small itored by the exposure of novel antigenic determinants that are amounts of Glu may be observed in place of Gla due to partial recognized by conformation-specific antibodies directed at the metal- decarboxylation during the degradation. In the control peptide, no ion induced conformation and by the quenching of intrinsic fluo- significant Glu signal was detected at any of the Gla sites, indi- rescence. The second can be monitored only by conformation- cating the presence of Gla at each known Gla residue. Minor specific antibodies. Only the second conformational change results decarboxylation was observed, given that the amount of Glu ob- in a structure competent to bind phospholipids. served at each of the five sites increased slightly over the courseof The interaction of factor IX species with conformation-specific the Edman degradation. In the K3a[4-Gla] peptide sequence, there antibodies was studied using a competition radioimmunoassay was no significant Glu signal at any of the first four Gla sites, with '251-labeledplasma-derived factor IX. Anti-factor IX:Ca(II)- indicating that all residues were y-carboxylated. At the fifth site specific antibodies bind to factor IX only in the presence of cal- (cycle 14), however, the signal observed for Glu is comparable to cium ions and not in the presence or absence of other divalent that of adjacent amino acids. Thus, after correcting for the repet- metal ions (Liebman et al., 1987). This antibody inhibits the bind- itive yield during Edman degradation, cycle 14 appears to be a ing of factor IX to phospholipid surfaces in the presence of cal- glutamic acid rather than Gla. The sequence data show that the cium ions. '251-Labeled plasma-derived factor IX was incubated residue in K3a[4 Gla] that is uncarboxylated is residue 36, and with antibody in the presence of increasing concentrations of un- that, within the limits of detection, the other four sites are fully labeled factor IX species. Anti-factor IX:Ca(II)-specific antibodies y-carboxylated. Thus, the dominant component of the IO-Gla spe- in the presence of 3 mM CaC12bound equivalently to all factor IX cies is factor IX/y36,40E, where residues 36 and 40 are Glu in- species, and half-maximal inhibition was observed at concentra- stead of Gla residues. tions of 1.4-1.7 nM (Fig. 5A). These results indicate that Gla 36 Gla residues homologous in position to Gla 36 and Gla 40 in and Gla 40 are not required for the expression of the antigenic factor IX are not found in other vitamin K-dependent proteins. To determinants against which anti-factor IX:Ca(II)-specific antibod- determine whether the y-carboxylation of these glutamic residues ies are directed. is required for the function of factor IX, we compared factor IX/ Anti-factor IX:Mg(II) antibodies bind to factor IX in the pres- y36,40E, factor IXIy40E, fully y-carboxylated recombinant fac- ence of magnesium ions, but not in the absence of metal ions tor IX, unfractionated recombinant factor IX, and plasma-derived (Liebman etal., 1987). The inhibition of the binding of anti- factor IX for their abilities to undergo a metal-ion induced con- factor IX:Mg(II)-specific antibodies to I2'I-labeled plasma-derived formational change, to bind to a phospholipid membrane and en- factor IX by the unlabeled factor IX species was studied using a dothelial cells, and to participate in the tenase complex. competition radioimmunoassay. As shown in Figure 5B, half- FactorIX undergoes two metal-ion inducedconformational maximal inhibition was similar for plasma-derived factor IX and changes. The first is metal-ion nonspecific and the second is Ca(I1)- fully y-carboxylated recombinant factor IX (2.1 and 3.6 nM, re- 190 S. Gillis et ai.

10 Or' A

5 -y 09 z

0.8 100 0 1 2 3 01 3 10 30 [Ca"], mM [COMPETITOR], nM Fig. 6. Calcium-induced quenching of intrinsic fluorescence of factor IX species. Factor IX species were titrated with increasing concentrations of calcium. The emission spectrum was monitored using an excitation wavelength of 280 nm and an emission wavelength of 340 nm. The concentration of the factor IX species was 1.3 mM. Fo. fluorescence (arbitrary units) in the absence of calcium. F, fluorescence at indicated calcium concentration. 0, factor IX/y36,40E; 0, factor IXIy40E; X, fully y-carboxylated factor IX; W, unfractionatedrecombinant factor IX; 0, plasma-derived factor IX.

that calcium-induced intrinsic fluorescence quenching occurs as a result of the burial of tryptophan 42 adjacent to the disulfide loop (residues 18-23) and reorientation of the indole ring (Freedman et al., 1995a). The concentration of calcium required to induce half-maximal fluorescence quenching was similar for all factor IX species and varied between 0.32 and 0.43 mM (Fig. 6). Fluores- cence quenching was completely reversible by the addition of 01 10 100 1000 EDTA. These values are similar to those reported for prothrombin [COMPETITOR], nM (Nelsestuen et al., 1976) and protein C (Christiansen & Castellino, Fig. 5. Interaction of conformation-specific antibodieswith factor IX spe- 1994), and are slightly lower than those reported previously for cies.'251-labeled plasma-derived factor IX wasincubated with anti- factor IX (Ware et al., 1989; Christiansen & Castellino, 1994). factorIX:Ca(II)-specific or anti-factorIX:Mg(II)-specific antibodies, There is some variation between the species in the percent of in thepresence of increasing concentrations of factor IX species. The maximal change in fluorescence. This may be due to the effect of immunocomplexes were precipitated with rabbit anti-goat serum and the pellets were assayed in a y-counter. A: Anti-factor IX:Ca(II) antibodies. the negative charges on the Gla side chain or of bound Ca(I1) on B: Anti-factor IX:Mg(II) antibodies.0, factor IXly36,40E; 0, factor IX/ the environment surrounding the fluorophores, specifically Trp 42. y40E; X, fully y-carboxylated factor IX; unfractionated recombinant These results demonstrate that Gla 36 and Gla 40 are not required factor IX;0, plasma-derived factor IX. Background., values were subtracted for the calcium-induced conformational change of the Gla domain and data were plotted as the percent of inhibition ofbinding of radiotracer under the conditions examined. versus the competitor concentration. The interaction of factor IX species with phospholipid vesicles was studied by fluorescence energy transfer. Factor IX was added in increasing concentrations to phospholipid vesicles composed of spectively), whereas the concentrations of factor IXIy40E and PS, PC, and dPE (40:50:10) in the presence of 1 mM CaCI2. The factor IX/y36,40E required to inhibit the binding of '251-labeled sample was irradiated at 280 nm and the fluorescence emission factor IX in the presence of 5 mM MgC12 were significantly higher, monitored at 520 nm. The binding of the factor IX species to the 70 and approximately 400 nM, respectively. The value for unfrac- lipid vesicles is associated with the proximity of intrinsic fluoro- tionated recombinant factor IX (15 nM) was intermediate, reflect- phores in the factor IX species with the dansyl group in the mem- ing its heterogeneous composition. These results suggest either that branes, thus facilitating energy transfer. As shown in Figure 7, the the antigenic determinants against which anti-factor IX:Mg(II) is addition of increasing concentrations of the factor IX species led to directed are located in the 36-40 region of the factor IX Gla and increasing fluorescence emission at 520 nm. A binding constant, AAAS domains, or that these antibodies recognize a conforma- Kd. was calculated by fitting these data to a simple bimolecular tional change that requires these residues to be y-carboxylated. model (Gilbert et al., 1990). The Kd for binding to phospholipid The addition of Ca(I1) to factor IX induces quenching of intrin- was 2.5 f 1.1, 1.1 k 0.17, 1.2 f 0.52, 2.0 f 0.32, and 1.4 k sic fluorescence. Comparison of the metal-free structure and the 1.2 nM for factor IX/y36,40E, factor IX/y40E, fully y-carboxylated calcium-ion bound NMR structure of the Gla domain demonstrates recombinant factor IX, unfractionated recombinant factor IX, and Gla 36 and Gla 40 of human factor IX 191

ticipate as enzyme in the conversion of factor X to factor Xa in the presence of the cofactor factor VI11 and either synthetic phospholipid vesicles or activated platelets. To determine the activity of the factor IXa species in the tenase complex, we used limiting concentrations of factor IXa and monitored the conversion of factor X to factor Xa using the chromogenic substrate CBS 3 1.39. Follow- ing a 2-4-min lag period, which has been attributed to the activation of factor VI11 by trace amounts of factor Xa (Neuenschwander & Jesty, 1988) and by factor IXa (Rick, 1982), all factor IXa species were equivalent in their ability to generatefactor Xa (Fig. 8A). In the experiments in which activated platelets were substituted for phospholipid vesicles, similar results wereob- tained; however, the activation of factor VI11 by residual thrombin eliminated the lag period (Fig. 8B). The clotting activities of the factor IX species were also measured in a factor IX assay using factor IX deficient plasma. This system is sensitive to both the 0 20 40 60 80 [Factor IX species], nM kinetics of factor IX activation by factor XIa and to the specific coagulant activity of factor IXa. All species had similar clotting Fig. 7. Phospholipid binding properties of factor IX species. The inter- activity (Table 5). action of the factor IX species with phospholipid vesicles was studied by monitoring energy transfer from the peptide to the dansyl group in the phosphatidylethanolamine incorporated into the lipid vesicles. Samples Discussion were irradiated at 280 nm and the fluorescence emission monitored at Gla is vital to the function of the vitamin K-dependent proteins. 520 nm. The change in fluorescence was monitored as a function of increasing factor IX concentration. Phospholipid vesicles were PC:PS:d-PE This calcium-binding amino acid participates in the formation of (50:40: 10, final concentration 3 mM). Io, fluorescence (arbitrary units) in the factor IX-Ca( 11) complex leading to folding of the Gla domain the absence of factor IX. I, fluorescence at indicated factor IX concentra- and the expression of phospholipid binding properties. Although tion. 0, factor IXly36,40E; 0, factor IXIy40E; X, fully y-carboxylated the importance of a number of specific Gla residues has been factor IX; unfractionated recombinant factor IX; 0, plasma-derived defined in protein C (Zhang et al., 1992; Zhang & Castellino, factor IX. ., 1993) and prothrombin (Ratcliffe et al., 1993), factor IX contains two additional Gla residues whose role is not known. Since the discovery of Gla in 1974 (Nelsestuen & Zytkovicz, plasma-derived factor IX, respectively. The similar binding of all 1974; Stenflo et al., 1974), several different approaches have been factor IX species studied suggests that Gla 36 and Gla 40 are not used to study the importance of individual Gla residues in the required for phospholipid binding and is further proof that factor IX/ function of the vitamin K-dependent proteins. Borowski et al. (1986) y36,40E and factor IXIy40E are able to undergo the correct calcium- isolated three partially y-carboxylated prothrombin variants from a induced conformational change. patient with a hereditary defect in vitamin K-dependent carboxyl- The binding of the factor IX species to bovine aortic endothelial ation. These variants contained 4-8 Gla residues and their distri- cells was studied using a competition assay with '251-labeledplasma- bution was determined by specific tritium incorporation, thermal derivedfactor IX. Confluentcells were incubated with radio- decarboxylation, and Edman degradation. Although this work in- labeled plasma-derived factor IX and increasing concentrations of dicated the importance of Gla 16 in prothrombin, the chemical competing unlabeled factor IX species. Half-maximal inhibition of heterogeneity of these partially carboxylated proteins precluded the specific binding of '251-plasma-derivedfactor IX was observed assignment of the role of most of the other Gla residues. Naturally at concentrations of 2-6 nM for all species studied (Table 5), and occurring point mutations have been reported at nine of the Gla approximate literature values (Stern et al., 1983; Cheung et al., positions of factor IX (Gianelli et al., 1994). However, mutation of 1992). Gla 36 or Gla 40 has not been observed (Gianelli et al., 1994). The possible importance of the interaction between Gla 36 and Using site-specific mutagenesis, the cDNA encoding the vitamin Gla 40 and other domains in factor IX was examined by studying K-dependent proteins has been modified and the protein expressed the ability of the activated forms of the factor IX species to par- in a heterologous system. Each of the Gla residues in prothrombin

Table 5. Properties of factor IX speciesa

hopefly Factor IXly36,40E Factor IXIy40E FIX 12-Gla rFIX PD-FIX

ICs0 BAEC (nM)b 3.4 k 0.9 2.1 f 0.5 5.0 rt 0.7 4.6 f 0.8 5.6 rt 0.8 m (%)' 84 110 102 100 100

"FIX, factor IX; FIX12-Gla. fully y-carboxylated recombinant factor IX; rFIX, unfractionated recombinant factor IX; PD-FIX, plasma-derived factor IX. bConcentration of protein required to inhibit 50% of the binding of radiolabeled plasma-derived factor IX to bovine aortic endothelial cells. 'Coagulant activity as measured by partial thromboplastin time expressed as activity relative to plasma-derived factor IX. 192 S. Gillis et al.

120 not affected by the introduction of Gla at the equivalent of residues 33 [by mutating Gln to Gla (Zhang & Castellino, 1993)] or 40 [by interchanging the entire helical stack domain of protein C with that of factor IX (Christiansen et al., 1995)], these results cannot be f 80 d extrapolated directly to the importance of these Gla residues in U factor IX. No site-specific mutagenesis studies involving Gla 36 or P Gla 40 in factor IX have been described. 0 3 40 In this study, we purified partially y-carboxylated factor IX species from recombinant factor IX expressed in Chinese hamster ovary cells. In this expression system, factor IX expressed at high levels contains material that is undercarboxylated (Kaufman et al., 0 2 6 10 14 18 1986). We have isolated and characterized these partially carbox- MINUTES ylated factor IX species using ion-exchange chromatography, peptide mapping, mass spectrometry, and N-terminal sequencing, and have demonstrated that uncarboxylated glutamic acid residues are not randomly distributed. Instead, almost all the uncarboxylated glutamic acid residues are at positions 36 and 40, allowing the purification of factor IX species that are uncarboxylated either at residue 40 or at both Glu 36 and Glu 40. This approach has the m‘ advantage over site-specific mutagenesis of allowing direct com- X parison of Gla to glutamic acid rather than Gla to a nonprecursor amino acid, usually aspartic acid. Gla 36 and Gla 40 are highly conserved in factor IX from various mammalian sources and have been described in every species studied to date (Yao et al., 1991; Pendurthi et al., 1992). We asked whether the y-carboxylation of these glutamic residues is required 0 2 4 6 8 for the function of factor IX, or whether these residues are y-car- MINUTES boxylated simply because they are “within reach” of y-glutamyl Fig. 8. Effect of factor IX species on the activation of factor X. Factor Xa carboxylase bound to the factor IX propeptide y-carboxylation generation was measuredby its amidolytic activity. The reaction mixturein recognition site (Furie & Furie, 1988). Gla 36 or Gla 40 could be a volume of 250pL contained factor IX(0.5 nM), human factor X (I mM), required for the calcium-induced conformation change character- human factor VI11 (4.5 unitslml), and either phospholipid vesicles com- istic of the Gla and AAAS domains. Alternatively, these Gla res- posed of K:PS (60:40, 50 mM) or platelets (activated by incubation for idues might be required for the interaction between the Gla-AAAS 10 min with 0.1 units/mL thrombin and 0.09 mg/mL collagen, final concentration 1 X lo8 cellslml), in a buffer of 20 mM Tris, pH 7.4, 0.15 M domains and the adjacent EGF domain($, as suggested by Vysotchin NaCI, 0.1%bovine serum albumin,5 mM CaC12. The reaction was allowed et al. (1993) and Rao et al. (1995). Furthermore, these residues to proceed at 37°C for 18 min and 25-pL aliquots were removed atthe may be involved in the binding of factor IXa to its substrate fac- indicated time points. The reaction was stopped by addingan equal amount tor X or to its cofactor factor VIIIa. of buffer containing20 mM EDTA. The amountof factor Xa generated was determined using the chromogenic substrate CBS 3 1.39 (50 pL per well; We used four independent methods to study the function of the 625 mM finalconcentration). A standardcurve generated with plasma- Gla-AAAS domain in the factor IX species. The ability of these derivedfactor Xa enabled conversion of absorbanceunits to factorXa domains to undergo a metal-ion induced conformational change formed. The amount of factor Xa formed in nanomoles is plotted versus was studied by monitoring calcium-induced quenching of intrinsic reaction time. A: In the presence of phospholipid vesicles. B: In the pres- fluorescence and by recognition by conformation-specific antibod- ence of platelets. 0, factor IXly36.40E; 0,factor IXly40E; X, fully y-carboxylatedfactor IX; W, unfractionatedrecombinant factor IX; 0, ies. The finding that all factor IX species studied were equivalent plasma-derived factor IX. in their recognition by anti-factor IX:Ca(II)-specific antibodies is consistent with recent data suggesting that anti-factor IX:Ca(II)- specific monoclonal antibodies recognize epitopes in the amino- terminal 12 amino acids of the Gla domain (Cheung et al., 1996). (Ratcliffe et al., 1993) and protein C (Zhang et al., 1992; Zhang & Phospholipid membrane binding was measured by fluorescence Castellino, 1993) has been mutated individually to aspartic acid, energy transfer and was similar for all factor IX species studied. and the mutant proteins characterized. These studies have con- Factor IX binds to phospholipid membranes only in the presence firmed the importance of Gla 16, demonstrated the lack of impor- of calcium; magnesium is not sufficient for binding. The factorIX- tance of Gla 6 (homologous to Gla 7 in factor IX) in both these phospholipid binding site has been localized recently to residues proteins, and determined the relative importance of the other Gla within the N-terminal 12 residues of the Gla domain (Freedman residues. The studies also show, however, that the importance of et al., 1996). The phospholipid binding site includes a hydrophobic some homologous Gla residues differs among these proteins. For surface patch defined by residues Leu 6, Phe 9, and Val 10 (Freed- instance, disruption of Gla 14 and Gla 19 in prothrombin decreases man et al., 1995a). Crosslinking experiments using p-benzoyl+ functional activity, but has no effect on the activity of protein C; phenylalanine incorporated into synthetic peptides indicate that likewise, disruption of Gla 7 and Gla 20 in protein C eliminates Leu 6 and Phe 9 are within close proximity to the phospholipid activity, but the same mutations in prothrombin inhibit function binding site (Freedman et al., 1996). However, the entire Gla and only partially (Zhang et al., 1992; Ratcliffe et al., 1993; Zhang & AAAS domains are required for phospholipid binding, because a Castellino, 1993). Accordingly, although protein C function was peptide containing the N-terminal 47 residues of factor IX binds Gla 36 and Gla 40 of human factor ZX 193 phospholipids, whereas a similar peptide truncated at residue 42 does factor IX:Mg(II)-specific antibodies. The magnesium-ion bound not possess phospholipid binding properties (Jacobs et al., 1994). NMR structure of the Gla domain of factor IX has been solved Endothelial cell binding affinity, as determined by a competition recently (Freedmanet al., 1996). This structure is ordered and assay with radiolabeled plasma-derived factor IX, was also very essentially identical to the calcium-ion bound structure for residues similar for all factor IX species. Thebinding site on factor IX has 12-47 andis disordered (like the metal-free structure) for the been localized to residues 3-1 1 of the Gla domain (Cheung et al., N-terminal 12 residues. Hence, it is likely that the anti-factor IX: 1992; Ahmad et al., 1994). However, the structural determinants Mg(I1)-specific antibodies recognize epitopes in the 12-47 region required for endothelial cell binding may differ from those re- of the Gla domain. Lack of y-carboxylation at residues 36 and/or quired for phospholipid membrane binding (Ryan et al., 1989). 40 could either interfere with the magnesium-induced tertiary struc- Our data are consistent with results obtained previously using a ture or abolish an epitope recognized by these antibodies. The chimera, in which replacement of residues 33-40 of factor VI1 latter possibility is more likely because these species are otherwise with those of factor IX did not induce endothelial cell binding functionally fully active and undergo metal-induced quenching of properties (Cheung et al., 1992). This chimera, when activated by intrinsic fluorescence normally. Others have localized a metal- factor XIa, is reported to possess low-affinity platelet binding (Ah- dependent epitope to residues 33-40 of factor IX. Mutating resi- mad et al., 1994). However, production of that chimera required dues 36 or 40 to aspartic acid or leucine, respectively, abolished mutating the five amino acids between residues 33 and 40 that the recognition of factor IX by a metal-dependent monoclonal differ in the sequences of factor VI1 andfactor IX, including antibody (Cheung et al., 1995). Gla 33, which may be important for factor IX activity (Koeberl Our results have important implications for the use of recombi- et al., 1989). Because all factor IX species were essentially iden- nant factor IX in the treatment of hemophilia B. Recombinant tical in these assays, it is unlikely that Gla 36 or Gla 40 are proteins have potential advantages over purified plasma proteins required for properties of the Gla domain associated with its in- (Limentani et al., 1993). However, approximately 40% of recom- teraction with phospholipid surfaces. binant factor IX as expressed is not y-carboxylated at residues 36 The interaction between the Gla-AAAS domains and the EGF and/or 40. Our findings that the y-carboxylation of these residues domains of factor IX has been studied by limited proteolytic di- is not required for the factor IX functions that were evaluated gestion. In the presence of Ca(II), there is a stabilizing interaction correlates with the clinical efficacy of recombinant factor IX (White or even a Ca(I1)-bridged intramolecular complex between these do- et al., 1995). mains (Astermark et al., 1991; Vysotchin et al., 1993). As assessed indirectly in this study, via coagulant function, Gla 36 and Gla 40 Materials and methods do not appear to be required for these interdomain connections. The Gla domain of factor IX participates in the factor IXa- Materials factor VIIIa interaction required for the formation of the tenase complex. A synthetic peptide comprised of the Gla and AAAS Human plasma-derived factor IX and factor VIII/vWF were the domains of factorIX [factor IX(1-47)] inhibits the proteolytic generous gift of Dr. William Drohan (American Red Cross, Rock- conversion of factor X by the tenase complex (Jacobs et al., 1994). ville, MD). Protein concentrations were determined using E2801% Although shown originally to interact directly with factor VIIIa of 13.2 forfactor IX, DiScipio et al., 1977. Human factor X, (Astermark et al., 1992), recent data suggest that the Gla domain factor Xa, and thrombin were purchased from Hematologic Tech- interacts indirectly by inducing a conformation in the serine pro- nologies, Essex Junction, VT. Factor XIa was from Enzyme Re- tease domain that is commensurate with factor VIIIa interaction search Laboratories, South Bend, IN. Achro-K was obtained from (Astermark et al., 1994). All factor IX species studied participated Wako Chemicals, Richmond, VA. Trypsin was from Promega, Mad- equally in the tenase complex, suggesting that Gla 36 and Gla 40 ison, WI. Rabbit anti-goat immunoglobulin was from Pel-Freez are not required for this interaction. However, we did not formally Biologicals, Rogers, AR. PC, PS, and dPE were obtained from measure the binding constants that define protein-protein inter- Avanti Polar Lipids, Alabaster, AL. McCoy’s medium was from action in this protein complex. Therefore, quantitative differences Life Technologies, Grand Island, NY. L-Glutamine, penicillin, and in the interaction of the partially carboxylated factor IXa with streptomycin were from Sigma,St. Louis. Endothelial mitogen either factor VIIIa or factor X may have been overlooked. was purchased from Biomedical Technologies, Stoughton, MA. Factor VI1 also contains a Gla residue at the equivalent of po- The chromogenic substrate specific for factor Xa (CBS 3 1.39) was sition 36. Although plasma factor VIIa is fully y-carboxylated, obtained from Diagnostica Stago, Asnieres-sur-Seine, France. Col- recombinant factor VIIa is only 50% y-carboxylated at this posi- lagen was from Bio/Data Corporation, Horsham, PA. tion (Thim et al., 1988). The y-carboxylated and noncarboxylated species have not been separated and analyzed. However, recombinant factor VIIa has similar activity as plasma-purified fac- Recombinant factor IX tor VIIa (Limentani et al., 1993), suggesting that Gla 36 is not Recombinant factorIX was expressed in a serum-free process crucial for the activity of factor VII. using Chinese hamster ovary cells that were stably transfected with We conclude that y-carboxylation of glutamic acid residues 36 a plasmid containing the factor IX cDNA, the adenovirus major and 40 of factor IX is not required for any function of factor IX late promoter, and a selectable marker gene encoding murine di- that we have measured. Studies are required to determine whether hydrofolate reductase (Kaufman et al., 1986; Harrison et al., 1995). our results mean that glutamic acid can function as well as Gla at To facilitate propeptide cleavage, the cells were cotransfected with these positions or whether glutamic acid itself is not critical at an expression plasmid encoding a truncated soluble form of furin/ positions 36 and 40. PACE (Wasley et al., 1993;Harrison et al., 1995). Recombinant fac- An interesting finding of these studies was the significantly tor IX was purified either by a three-step chromatographic process reduced affinity of factor IXIy40E and factor IX/y36,40E to anti- that included an anti-factor IX antibody column, or by a four-step I 94 S. Gillis et at. process involving Q-sepharose, matrex cellufine sulfate, ceramic follows: 5, 12, 24, 35, 40, and 100% buffer B (O.l%, v/v, trifluo- hydroxyapatite, and immobilized metal affinity columns (Foster roacetic acid in 95% acetonitrile)at 0, 35, 55, 86, 106, and et al., 1995). 110 min. Buffer A was 0.1% (v/v) trifluoroacetic acid. Further digestion of indicated peptides with trypsin was performed in 0.5 M NaCI, SO mM Tris, pH 8.0, 5 mM CaCI2 at an enzyme to Mono Q HPLC separation of recombinant factor IX species substrate ratio of 1:4 in a total volume of 500 pL at 37 "C for 24 h. Ion exchange chromatography employed a Pharmacia Mono Q HR Peptide products were isolated by reverse-phase HPLC with a C18 5/5 column. Samples were prepared in 20 mM Tris, pH 9.0 (buffer column, 4.6 X 250 mm, at 0.75 mL/min using a linear gradient as A), and injected in volumes of 0.1-2.0 mL. Elution employed a follows: 10, 19, 21, 27, 38, and 100% B at 0, 7, 47, 49, 61, and linear gradient of 30, 40, and 100% buffer B (20 mM Tris, 1 M 70 min. NaCI, pH 9.0) at 0,40, and 45 min, at a flow rate of 0.75 mL/min. Indicated samples were subjected to desialylation employing 0.25 Mass spectrometry analysis units/mL neuraminidase (type X, Sigma) and 0.5 mg/mL recombinant factor IX in SO mM sodium acetate, pH 5.0, for 5 h at 37 "C. MALDI-TOF MS was performed on a Bruker linear TOF mass Subsequent SDS-PAGE and sialic acid analysis confirmed that spectrometer. HPLC-purified samples were concentrated by vac- desialylation was essentially complete. In indicated experiments, uum centrifugation to approximately 10 pmol/mL before analysis. chromatography was performed on samples activated with fac- Samples were analyzed using one of three matrices: sinapinic acid, tor XIa (1:200 w/w) in 50 mM Tris, 0.15 M NaCI, 5 mM CaCI2, DHB, and 9:1 DHB:HMB. The DHB:HMB matrix was used in the pH 7.5, for I h at 37°C. Activation and subsequent loss of the presence of Chelex beads as metal scavengers. activation peptide during chromatography was confirmed by subjecting a sample to SDS-PAGE and N-terminal sequencing. N-terminal sequencing Indicated peptides were subjected to automated Edman degrada- Gla analysis tion using an Applied Biosystems 470A protein sequencer. Gla content was determined by amino acid analysis using precol- umn derivatization with PITC of a base hydrolysate. Samples were Immunochemical analyses with desalted by size-exclusion chromatography in 20 mM ammonium conformation-spec@c antibodies acetate buffer, pH 7.5, using a TSK-Gel G2000SWXL column (7.5 X 30 mm, TosoHaas), lyophilized, and reconstituted in water. The binding of factor IX species to conformation-specific anti- Aliquots of 15 mg were redissolved in 20 mL 2 N NaOH, and factor IX antibodies was studied using a solution phase radioimmu- hydrolyzed in 0.5 mL polypropylene centrifuge tubes for 20-24 h noassay. Plasma-derived factor IX was iodinated with NaIZ5Iusing at I 12 "C. After neutralizing with an equal volume of 2.2 N acetic enzymobeads (Bio-Rad). '251-labe2edfactor IX was separated from acid, samples were transferred to pyrolyzed glass vials, dried by free iodine by gel filtration on Sephadex G-25, then further purified vacuum centrifugation, and derivatized with PITC. The PITC was by immunoaftinity chromatography with anti-factor IX:Mg(II) anti- removed by vacuum centrifugation for 60 min, and samples were bodies covalently linked to Sepharose. Anti-factor IX:Ca(II) anti- reconstituted and analyzed by reverse-phase HPLC using a 3.9 X bodiesand anti-factor IX:Mg(II) antibodies were purified as 300 mm Picotag column (Waters Chromatography) and gradient described previously (Liebman et a]., 1987). A competition radio- elution as described (Cohen & Strydom, 1988). Each set of anal- immunoassay was performed to study the displacement of Iz5I- yses included three plasma-derived factor IX samples as standards labeled plasma-derived factor IX from anti-factor IX antibodies at and two plasma-derived factor X samples as controls. Moles of Gla increasing concentrations of factor IX species. Varying concentra- and Glu present in each sample were obtained from integrated tions of unlabeled competitor, normal goat immunogIobuIin (I mg/ peak areas by comparison to Gla and Glu standards. The raw Gla mL), and '251-labeled plasma-derived factor IX were incubated content for each sample was then determined from the observed overnight at 4°C with either anti-factor IX:Ca(II)-specific anti- Gla/Glu ratio, then normalized to give an average of 12.0 Gla/mol bodies or anti-factor IX:Mg(II) antibodies in a final assay volume for the plasma-derived factor IX samples. of 300 pL containing 0.15 M NaCI, 50 mM Tris, pH 7.4, I mM benzamidine, 0.1% bovine serum albumin, 0.1% Tween 20, and either 3 mM CaCI2 or 5 mM MgCI2. Following incubation, 1 mL Peptide maps of recombinant factor IX species of rabbit anti-goat immunoglobulin (25 mg in 1 mL of 0.1 M Tris, Aliquots of 75-150 mg of the factor IX species were desalted as 0.15 M NaCI, 2.5% polyethylene glycol 8000, pH 7.4) was added. described above, reduced in 0.5 mL 0.5 M Tris, 6 M guanidine, The precipitate that formed following centrifugation was assayed 5 mM dithiothreitol, pH 8.6, for 1 h at 40°C and alkylated by for Iz5I in a Packard 5000 Series Auto-Gamma scintillation counter. addition of iodoacetic acid to a final concentration of 11 mM for 1 h at room temperature. Samples were desalted as described above, Preparation of phospholipid vesicles dried by vacuum centrifugation, and pretreated in 0.5 mL of 0.1 M Tris, 6 M guanidine hydrochloride, 10 mM EDTA (disodium salt), Small unilamellar phospholipid vesicles composed of PS:PC:dPE pH 7.5, at 60°C for 1 h. They were then diluted 1:2 with 0.1 M 4050: 10 and PS:PC 40:60 were prepared by the method of Baren- Tris, pH 7.5, and digested by addition at 4 h intervals of three holz (Barenholz et al., 1977). Phospholipids were dried under N2, aliquots of Achro-K (enzyme to substrate ratio of each aliquot washedthree times with methylenechloride, resuspended in 1 :25, w/w) and incubated for 24 h at 30 "C. The digestion mixture 50 mM Tris, 50 mM NaCI, pH 7.4, and sonicated in a bath soni- was fractionated by C 18 reverse-phase HPLC using a 4.6 X 250-mm cator (Lab Supply) until the solution cleared. The phospholipids column (#218TP52, Vydac) at 0.75 mL/min linear gradient as were subjected to centrifugation at 40,000 X g for 30 rnin, then Gla 36 and Gla 40 of human factor IX 195

50,000 X g for 90 min in a Beckman L8-80M ultracentrifuge using Effect of factor IX species on factor X activation a 70 Ti rotor. The supernatant contained the small unilamellar by the tenase complex vesicles; phospholipid concentrations were determined by analysis The ability of the various factor IX species to activate factor X was for phosphorus (Chen et al., 1956). These vesicles were stored at determined using an amidolytic assay for factor Xa and the chro- - 80 "C under nitrogen. mogenic substrate CBS 3 1.39. The factor IX species were activated with factor XIa at a weight ratio of 50:1 in the presence of Phospholipid binding studies 5 mM CaC12 for 2 h at 37 "C. The reaction was stopped by the addition of 10 mM EDTA. Complete activation was verified in all The binding of factor IX species to phospholipid vesicles was cases by subjecting a sample to SDS-PAGE. The tenase complex evaluated by resonance energy transfer measurements performed reaction mixture included human factor VI11 (4.5 units/mL), hu- on a SLM 800OC fluorescence spectrophotometer at 25 "C. Fluo- man factor X (1 mM), phospholipid vesicles composed of PS:PC rophores in the factor IX preparations were excited at 280 nm. (40:60,50 mM), the various factor IXa species (0.5 nM) in 20 mM Energy transfer to the dansyl group was studied by monitoring Tris, pH 7.4, 0.15 M NaCI, 0.1% bovine serum albumin, 5 mM emission at 520 nm (Gilbert et al., 1990). Factor IX species were CaCI2 in a final volume of 250 pL. The reaction was allowed to prepared in 50 mM Tris, 50 mM NaCl, 1 mM CaCI2, pH7.4. proceed at 37 "C for 18 min and 25-pL aliquots were removed at Aliquots of the species were added to a fluorescence cuvette con- the indicated time intervals and placed in a well of a 96-well taining 3.0 mL of buffer with 3 mM small unilamellar phospho- microtiter plate containing 10 mM EDTA in an equal volume of lipid vesicles (PS:PC:dPE, 4050: 10). The sample was irradiated at buffer to stop thereaction. The amount of factor Xa generated was 280 nm using a slit width of 4 nm and emission monitored at determined using the chromogenic substrate CBS 3 1.39. CBS 3 1.39 520 nm using a slit width of 16 nm. Dilution effects were corrected (50 pL; 625 mM final concentration) was added to each sample, using a buffer control. A binding constant, Kd, was calculated by and the absorbance at 405 nm was determined over 6 min at 37 "C fitting the data to a simple bimolecular model (Gilbert et al., 1990). using a Thermomax kinetic enzyme-linked immunosorbent assay reader (Molecular Devices). A standard curve generated with plasma- derived factor Xa enabled conversion of absorbance units to fac- Calcium-induced quenching of intrinsic fluorescence tor Xa formed in nanomoles. In some experiments, platelets (final concentration 1 X IO8 cells/mL) activated with 0.1 U/mL throm- Fluorescence experiments were performed at 25 "C using an SLM bin and 0.09 mg/mL collagen were used in place of phospholipid 80OOC fluorescence spectrophotometer and a 0.5-mL fluorescence vesicles. Platelet-rich plasma was prepared by centrifugation from cuvette. Factor IX species (final concentration 1.3 mM) were pre- blood anticoagulated in Wares buffer. The platelet-rich plasma was pared in 20 mM Tris, 0.1 M NaCl, pH 7.4. The samples were adjusted to 1 mM EDTA and the platelets isolated by gel filtration irradiated at 280 nm using a slit width of 4 nm and the emission on a Sepharose 4B column. Platelets were recalcified, activated as monitored at 340 nm using a slit width of 16 nm following the described, and used within 1 h of preparation. addition of aliquots of Ca(I1). Dilution effects were corrected using a buffer control. At the completion of titration, the reversibility of calcium-induced quenching was tested by the addition of 20 mM Factor IX coagulant activity EDTA. The concentration of Ca(I1) that resulted in 50% of the The coagulant activity of the factor IX species were measured on total fluorescence change was then calculated from these data. a Coagamate X2 instrument (General Diagnostics) by a standard activated partial thromboplastin time (Proctor & Rapaport, 1961) using factor IX-deficient plasma. A standard curve was generated Binding of factor IX species to endothelial cells using plasma-derived factor IX. Results were expressed as percent Bovine aortic endothelial cells (passage 3) were grown to conflu- activity relative to that of plasma-derived factor IX, which was ence in 24-well microtiter plates in McCoy's 5A modified medium considered to have 100% activity. supplemented with 20% fetal bovine serum, 0.22% (w/v) NaHC03, 12.5mM Hepes, 2 mM L-glutamine, 100 units/mL penicillin, Acknowledgment 100 mg/mL streptomycin, 25 nM insulin, 50 ng/mL endothelial mitogen, and 100 mg/mL heparin. Confluent cells were washed This research was supportedby grants (HL38216, HL18834, and HL42443) four times with 1 mL of IO mM Hepes, pH 7.4, 0.137 M NaCI, from the National Institute of Health. 4 mM KCl, 11 mM glucose (buffer A). Cells were then incubated in 0.5 mL buffer A containing 2 mg/mL bovine serum albumin and References 2 mM CaC12 (buffer B) for 15 min at 4°C. Buffer B was replaced with 250 pL of the same buffer containing '251-plasma-derived Agarwala KL, Kawabata SI, Takao T, Murata H, Shimonishi Y, Nishimura H, Iwanga S. 1994. Activation peptide of human factor IX has oligosaccharides factor IX and various concentrations of cold factor IX species. 0-glycosidically linked to threonine residues at 159 and 169. Biochemistry After incubation for 2.5 h at 4 "C, the cells were washed four times 33:5167-5171. with I rnL ice-cold 50 mM Tris, pH 7.5, 0.14 M NaCI, 2 mM Ahmad SS, Rawala-Sbeikh R, Cheung WF, Jameson BA, Stafford DW, Walsh CaCI2, and 2 mg/mL bovine serum albumin. After washing, the PN. 1994. High affinity specific factor IXa binding to platelets is mediated in part by residues 3-1 1. Biochemistry 3312048-12055. cells weredissolved in 0.5 mL of 0.2 M NaOH, 10 mM EDTA, and Astermark J, Bjork I. Ohlin AK. Stenflo J. 1991. Structural requirements for 1% SDS.The solubilized cell samples were then subjected to Ca2+ binding to the y-carboxyglutamic acid and epidermal growth factor- y-counting. Specific binding was determined by subtracting the like regions of factor IX. J Biol Chem 2662430-2437. Astermark J, Hogg PI, Bjork I, Stenflo J. 1992. Effects of y-carboxyglutamic binding seen in wells containing 200 nM unlabeled protein from acid and epidermal growth factor-like modules of factor IX on factor X total binding in test wells. activation. J Biol Chem 2673249-3256. 196 S. Gillis et al.

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