Enzymatic Removal of Sialic Acid from Human Factor IX and Factor X Has No Effect on Their Coagulant Activity

doi:10.1074/jbc.270.12.6537

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Volume 270, Number 12, Issue of March 24, 1995 pp. 6537-6542
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

Enzymatic Removal of Sialic Acid from Human Factor IX and Factor X Has No Effect on Their Coagulant Activity (*)

(Received for publication, October 6, 1994; and in revised form, January 18, 1995)

Dwaipayan Bharadwaj (1), Reed J. Harris (4), Walter Kisiel (1), (2), Kenneth J. Smith (1) (3)(§)

From the (1)Departments of Pathology, (2)Biochemistry, and (3)Medicine, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131 and the (4)Department of Analytical Chemistry, Genentech, Inc., South San Francisco, California 94080

ABSTRACT

INTRODUCTION

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

FOOTNOTES

ACKNOWLEDGEMENTS

REFERENCES

ABSTRACT

Factor IX and factor X have sialic acid in O-linked and N-linked oligosaccharides on their activation peptides, and a terminal sialic acid is found on a recently described O-linked tetrasaccharide at Ser-61 in the light chain of human factor IXa. In studies presented here, the potential role of sialic acid residues in mediating activity of human coagulation factors IX and X was tested after enzymatic removal of sialic acid residues. In contrast to previous reports, treatment of factor IX or factor IXa with recombinant sialidase did not decrease the rate of factor IX activation or proteolytic properties of human factor IXa. The activation rates of factor IX and desialated factor IX were indistinguishable when treated with factor XIa, with factor VIIa/tissue factor complex, and with the factor X activating enzyme from Russell's viper venom. Desialated human factor IXa showed full activity in the non-activated partial thromboplastin time assay and retained full ``tenase'' activity in a coupled amidolytic assay. Similar experiments with human factor X showed no detectable loss of clotting activity in the prothrombin time assay after desialation. Additionally, desialated human factor X was cleaved by the factor X activating enzyme from Russell's viper venom and intrinsic tenase at the same rate as untreated factor X when analyzed by SDS-polyacrylamide gel electrophoresis. These studies have shown that factor IX and factor X clotting activity are not dependent on sialic acid content. Further studies are needed to determine whether desialated factor IX binds to endothelial cells, and whether factors IX and X are more rapidly cleared from circulation or have altered susceptibility to proteolysis after enzymatic removal of sialic acid.

INTRODUCTION

Human coagulation factor IX is a vitamin K-dependent coagulation factor which is essential for normal hemostasis(1) . This glycoprotein undergoes several post-translational modifications including -carboxylation of 12 N-terminal glutamic acid residues, beta -hydroxylation of aspartic acid at residue 64, and incorporation of both N-linked and O-linked oligosaccharides. Four sites for attachment of O-linked oligosaccharides are present in factor IX, including Ser-53 and Ser-61 within the first epidermal growth factor (EGF) ()domain, which are uniformly glycosylated(2) , and Thr-159 and Thr-169 in the activation peptide (residues 145-180), which are partially glycosylated(3) . Threonine residues at positions 159 and 169 in the activation peptide of factor IX (residues 145-180) are partially modified with O-linked oligosaccharides(3) . There are also two sites for attachment of N-linked oligosaccharides in the activation peptide of factor IX at asparagine residues 157 and 167(4) . The trisaccharide linked to Ser-53 does not contain sialic acid, while the O-linked oligosaccharide at the other three sites could contain as many as 3 sialic acids. The two N-linked oligosaccharides on the activation peptide region of the molecule probably have the remainder of the 8-10 sialic acid residues found on factor IX(5) . Although -carboxylation is critical for function of vitamin K-dependent coagulation factors, there are little definitive data on the importance of glycosylation and other post-translational modifications in factor IX.

The structure of the fucose-linked tetrasaccharide with a terminal sialic acid at Ser-61 in the first EGF domain of human factor IX has been recently described(2, 6) . The proposed consensus sequence (Cys-X-X-Gly-Gly-Thr/Ser-Cys) for incorporation of fucosyl modifications in EGF domains is found in human factor IX, t-PA, urokinase, factor VII, and factor XII but not in factor X or bovine factor IX(7) . Enzymatic removal of sialic acid has been reported to result in the loss of factor IX clotting activity without affecting the rate at which factor IX is cleaved by the factor X activating enzyme from Russell's viper venom (RVV-X)(8) . Loss of factor IXa alpha activity (a partially activated factor IX species with Arg-180/Val-181 cleavage only) following sialidase treatment suggests that sialic acids in either the EGF or activation peptide regions of factor IX are important for enzymatic activity. In contrast, factor X, which lacks the consensus site for a fucose-linked oligosaccharide in its light chain, did not lose clotting activity following enzymatic removal of sialic acid(8) . Others, however, have shown that sialidase treatment of human or bovine factor X (9) and combined sialidase and O-glycanase treatment of bovine factor X reduces the K/K for RVV-X or intrinsic ``tenase'' (10) without any apparent effect on factor Xa clotting activity.

Since there are conflicting reports on the effects of enzymatic removal of sialic acid from factor X and no experiments with factor IXa, purified preparations of human factor IX, factor IXa, and factor X were each treated with either recombinant sialidase expressed in Escherichia coli or sialidase from Clostridium perfringens in the present study. Desialated factor IX, IXa, and X were found to retain full enzymatic activity in plasma and purified systems.

EXPERIMENTAL PROCEDURES

Materials

Factor IX and factor X were purified from plasma using metal ion-dependent immunoaffinity chromatography(11, 12) . Recombinant C. perfringens sialidase (specific activity 140 units/mg), expressed in E. coli, was from New England Biolabs, Beverly, MA. Agarose-linked sialidase (Type X-A) from C. perfringens (150-400 units/mg of protein, 20-30 units/g of agarose), rabbit brain cephalin, phosphatidylcholine, and phosphatidylserine were from Sigma. Human alpha

-thrombin was prepared as described previously(13) . Factor VIII (Monoclate P) was from Armour Pharmaceuticals, Collegeville, PA. Factor XIa was purified from celite eluant (14) by monoclonal antibody immunoaffinity chromatography using an anti-factor XI antibody provided by Dr. Y. Komiyama, Kansai Medical School, Osaka, Japan. RVV-X was purified as described previously(15) . Sephadex G-25 and molecular weight standards for SDS-PAGE were from Pharmacia Biotech Inc. Fatty acid-free bovine serum albumin was from Boehringer Mannheim. Recombinant human factor VIIa was generously provided by Dr. Ulla Hedner (Novo Nordisk, Copenhagen, Denmark). Full-length recombinant human tissue factor apoprotein was a gift from Dr. Gordon Vehar (Genentech Inc., South San Francisco, CA) and was relipidated as described previously(16) . Centricon 30 filters were from Amicon, Danvers, MA. Heparin was from SoloPak Laboratories, Franklin Park, IL. S-2765 was from Pharmacia Hepar Inc., Franklin, OH. Factor IX-deficient plasma was from an individual with congenital deficiency of factor IX. Factor X-deficient plasma was purchased from George King Biomedical, Overland Park, KS. Recombinant tissue factor-calcium mixture (Innovin®) for prothrombin time assays was purchased from Dade (Miami, FL).

Desialation of Factor IX

Human factor IX (52 µM) was incubated with recombinant sialidase (8.62 µM) at a 8:1 weight ratio in 0.09 M sodium citrate (pH 6.0) for 4 h at 37 °C. The buffer was changed to 0.1 M NaCl, 0.05 M Tris-HCl, pH 7.5 (TBS), by gel filtration over Sephadex G-25. Desialated factor IX was repurified by immunoaffinity chromatography over an A-7 monoclonal antibody column. An identical system was used to isolate treated and control human factor X using a calcium-dependent monoclonal antibody (CaFX40) coupled to Affi-Gel 10. To control for potential degradation of factor IX activity that may have occurred after extended incubation and re-isolation, factor IX and factor X were subjected to the same incubation and re-isolation steps without recombinant sialidase.

Desialation of Factor IXa

In these experiments, agarose-immobilized sialidase was incubated with factor IXa in TBS, at a ratio of 0.2 units of sialidase (23 pmol)/mg of factor IXa (17 nmol) for up to 8 h at 37 °C. Aliquots were removed for sialic acid analysis and SDS-PAGE at 0, 0.33, 0.66, 1, 2.25, and 8 h of incubation and centrifuged to remove sialidase. Control preparations of factor IXa were incubated without sialidase and treated in an identical manner.

Sialic Acid Assays and Amino Acid Analysis

Samples of various proteins for sialic acid analysis were initially dialyzed in 0.1 M NH (4)

HCO

, lyophilized, and subsequently hydrolyzed with 0.1 M trifluoroacetic acid at 80 °C for 1 h. For amino acid analysis, the proteins were treated for 24 h at 110 °C in 6 N HCl invacuo. Sialic acid content was determined on high pH anion exchange chromatography on a Dionex BioLC apparatus(3) , while protein mass was determined using a Beckman 6300 amino acid analyzer. Nanomoles of protein were determined by comparison to a standard using the average of the observed amounts (nmol) of Asx, Ala, Leu, Phe, His, Lys, and Arg.

Activation of Factor IX by Factor XIa

Factor IX and desialated factor IX were incubated at 37 °C with purified human factor XIa in TBS with 10 mM CaCl (2)

at a 1:300 enzyme:substrate weight ratio. The concentration of factor IX was 3.23 µM, and that of factor XIa was 3.8 nM. Aliquots (50 µl) were removed from the reaction mixture at 0, 0.5, 1, 2, and 3 h and adjusted to 20 mM EDTA to stop the reaction prior to assessing clotting activity and SDS-PAGE analysis.

Activation of Factor IX by Factor VIIa/Tissue Factor

Factor IX and desialated factor IX (17.54 µM) were incubated at 37 °C with a complex of recombinant human factor VIIa (34 nM) and relipidated recombinant human tissue factor apoprotein (34 nM) at a 1:588 enzyme:substrate weight ratio in TBS with 10 mM CaCl (2)

. Samples were removed at 0, 0.5, 1, 5, and 30 min and adjusted to 0.03 M EDTA prior to clotting assay and SDS-PAGE.

Activation of Factor IX by RVV-X

Soluble RVV-X (362 nM) was incubated at 37 °C with either factor IX or desialated factor IX (25.4 µM) in TBS, pH 8.0, with 10 mM CaCl (2)

at a 1:50 enzyme:substrate weight ratio in presence of 1 mM benzamidine. Aliquots (50 µl) were taken at 0, 0.5, 1.0, and 2.0 h and adjusted to 30 mM EDTA prior to SDS-PAGE and coagulation assays.

Activation of Factor X by RVV-X

Control human factor X and desialated human factor X (18.64 µM) were incubated with soluble RVV-X (45.75 nM) at a 1:300 enzyme:substrate weight ratio at 37 °C in TBS with 10 mM CaCl (2)

. Aliquots were removed at 0, 5, 15, and 30 min. and adjusted to 30 mM EDTA prior to SDS-PAGE and coagulation assays.

Activation of Factor X by Factor IXa

Control human factor X and desialated human factor X (16.14 µM) were incubated with 55.6 nM factor IXa, 93 µM phospholipid vesicles (30/70, phosphatidylserine/phosphatidylcholine) (17) 0.007 NIH units/ml thrombin, 15 nM factor VIIIa at 37 °C in TBS, pH 8.0, with 10 mM CaCl (2)

(1:300 enzyme:substrate weight ratio). Aliquots were removed at 0, 1, 5, 15, and 30 min and adjusted to 30 mM EDTA prior to SDS-PAGE analysis.

Factor IX and IXa Activity Assays

Factor IX clotting activity was assessed using a single stage PTT system (8) after dilution of factor IX or factor IXa in TBS containing 10 mg/ml bovine serum albumin. This assay was modified to detect factor IXa activity when the ellagic acid/cephalin mixture was replaced with rabbit brain cephalin diluted 1:10 in TBS. The clotting time was recorded after sequential addition of 0.1 ml of factor IX-deficient plasma, 0.1 ml of diluted factor IX or factor IXa, 0.1 ml of diluted cephalin, and 0.1 ml of 25 mM CaCl (2)

. Formation of the factor IXa-AT-III complex was tested with both proteins at 0.19 mg/ml in TBS with 1 units/ml heparin. After 1 min at 37 °C, samples were prepared for SDS-PAGE analysis without beta

-mercaptoethanol.

Factor X Activity Assays

Activity of sialidase-treated factor X and control factor X was determined in an prothrombin time system using congenital factor X-deficient plasma as substrate. The clotting time was recorded after the sequential addition of 50 µl of factor X-deficient plasma, 50 µl of diluted factor X (desialated and control), and 200 µl of Innovin.

Factor X Activation Assays

The activity of the complex of factor IXa, factor VIIIa, phospholipid, and calcium was monitored by conversion of factor X to factor Xa in a purified system. Factor IXa and desialated factor IXa (0.1-1.0 nM final concentration) were added to a solution consisting of 300 nM factor X, 15 nM factor VIIIa, 0.007 NIH units/ml thrombin, 93 µM phospholipid vesicles (30/70, phosphatidylserine/phosphatidylcholine)(17) , in 0.15 M NaCl, 0.02 M HEPES, 5 mM CaCl (2)

(pH 7.2) with 10 mg/ml bovine serum albumin. The reaction mixture (150 µl) was incubated at 37 °C for 5 min prior to stopping the reaction by addition of EDTA to a final concentration of 15 mM and placing the sample on ice. The amount of factor Xa generated was determined using the chromogenic substrate S-2765 from a standard curve relating factor Xa and A

in this system. Aliquots (10 µl) of the reaction mixture were transferred to 40 µl of 5 µM S-2765 in 9 mM HEPES (pH 7.2). The reaction was incubated at 37 °C for 5 min, and the reaction was then stopped by the addition of 0.05 ml of 30% acetic acid. Absorbance was read at 405 nm in microtiter tray wells.

RESULTS

Factor IX and Factor IXa Desialation

The sialic acid content of factor IX (mol/mol) was 9.1 (range 8.7-9.4) in four experiments, while factor IXa averaged 8.0 (range 6.7-10.20) in five experiments. In contrast, the factor IX and IXa preparations treated with sialidase had a sialic acid content of less than 1 mol of residual sialic acid/mol of factor IX or factor IXa. In initial experiments, factor IXa was dialyzed in 0.1 M sodium acetate (pH 5.5), which is close to the pH optimum for sialidase isolated from C. perfringens. When factor IXa was concentrated by centrifugation over an Centricon 30 filter in this buffer, it was found that there was a visible precipitate. This problem was not observed when the higher pH levels (pH 6) were used for enzymatic removal of factor IX sialic acid. IEF analysis of sialidase-treated factor IX preparations showed loss of the heterogeneity associated with variable sialic acid content (data not shown). Fig. 1shows the SDS-PAGE analysis of factor IX treated with recombinant C. perfringens sialidase. There is an apparent loss of

4000 daltons when 97% of the sialic acid had been removed.

Figure 1: SDS-PAGE of factor IX and X (control and desialated). Each lane contains 8 µg of protein. Samples were not reduced. Lane 1 contains molecular weight markers. Lane2 is untreated factor IX. Lane3 is factor IX after sialidase treatment. Lane4 is untreated factor X. Lane5 is desialated factor X.

Effect of Desialation on Factor IX Clotting Activity

The PTT assay on factor IX re-isolated after recombinant sialidase treatment yielded a mean specific activity of 356 units/mg (n = 3, range 326-367), while the re-isolated control factor IX specific activity was 237 units/mg (n = 4, range 180-280). The mean difference between specific activity assays of treated and untreated factor IX was 126 units/mg (95% confidence interval of 55-198 units/mg). The p value for the two sample t test was p = 0.006. Experiments with factor IX treated with sialidase isolated from C. perfringens revealed that the factor IX specific activity also appeared to increase after treatment as assessed in the PTT assay. This change in specific activity was probably due to activation since the non-activated PTT showed that there was factor IXa activity after incubation (non-activated PTT of 100 s at 40 ng/ml desialated factor IX and 190 s of the untreated factor IX preparation at the same concentration). Although the specific activity in the clotting assay increased after incubation with recombinant sialidase, less activation of factor IX was observed in the non-activated PTT. The NAPTT assay could detect as little as 9 pM of factor IXa in these experiments. As can be seen in Fig. 1, there was no apparent activation of factor IX on SDS-PAGE when purified factor IX was treated with recombinant sialidase. However, some preparations of factor IX treated with C. perfringens-derived sialidase had several lower molecular weight bands when analyzed by SDS-PAGE (data not shown). Factor IX preparations after desialation were assayed for factor IXa in the factor X activation assay. After treatment by C. perfringens-derived sialidase, factor IX samples assayed at 10 nM contained 0.5 nM factor IXa. There was no detectable factor IXa after treatment with recombinant sialidase. The limit of detection was 0.1-0.2 nM.

Factor IX Activation

Although preservation of clotting activity in the PTT assay depends on factor XIa-mediated activation of factor IX, the ability of factor IX to be activated in three systems was tested. In these experiments, factor IX and desialated factor IX could be activated by factor XIa in a fashion identical to that seen with untreated factor IX as judged by activation-like cleavages on SDS-PAGE (Fig. 2). In other experiments, factor IX and desialated factor IX were activated at the same rate by RVV-X (Fig. 3) and by the complex of tissue factor/VIIa (Fig. 4).

Figure 2: Activation of factor IX and desialated factor IX by factor XIa. Each lane contains 8 µg of protein. Samples are not reduced. Lanes 1-5 contain control factor IX, and lanes6-10 contain desialated factor IX. Incubation times are as follows; lanes5 and 6 are at 0 h, lanes1 and 10 are at 0.5 h, lanes2 and 9 are at 1 h of incubation, lanes 3 and 8 are at 2 h, and lanes4 and 7 are at 3 h of incubation.

Figure 3: Activation of factor IX and desialated factor IX by RVV-X. Each lane contains 7-8 µg of protein. Samples are reduced. Lanes 2-5 contain desialated factor IX, and lanes 6-9 contain control factor IX. Incubation times are as follows: lanes5 and 6 are at 0 h, lanes4 and 7 are at 0.5 h, lanes3 and 8 are at 1 h, lanes2 and 9 are at 2 h, lanes1 and 10 are molecular weight markers.

Figure 4: Activation of factor IX and desialated factor IX by factor VIIa/tissue factor gel. Inset, each lane contains 7-8 µg of protein. Samples are not reduced. Lanes 1-5 contain control factor IX, and lanes 6-10 contain desialated factor IX. Incubation times of factor IX samples are as follows; lanes 5 and 6 are at 0 min, lanes4 and 7 are at 0.5 min, lanes3 and 8 are at 1 min; lanes2 and 9 are at 5 min; lanes1 and 10 are at 30 min. The factor IX cleavage site that gives a band at the apparent kDa of 55 is not known. Graph shows factor IX clotting activity; the + symbol is control factor IX activity in the NAPTT assay, and the up triangle symbol is desialated factor IX activity in the same assay. Samples were diluted 1:10,000 prior to assay after activation by tissue factor and factor VIIa.

Factor IXa Activity

Although factor X activity measured in the PTT depends on expression of factor IXa activity in activating factor X and thus would be thought to be normal on the basis of the preservation of activity in the PTT, formal experiments were performed to demonstrate the retention of factor IXa enzymatic activity with progressive loss of sialic acid. As can be seen in Fig. 5(A and B), there was no change in the factor IXa clotting activity or factor IXa tenase activity in 135 min, despite the rapid desialation. Rapid desialation of factor IXa with preservation of factor IXa activity was shown in two separate experiments using immobilized sialidase. In addition, no more than 5-10% degradation of factor IXa occurred after extensive incubation with sialidase as judged by SDS-PAGE. Furthermore, desialated factor IXa reacted with antithrombin III with the same efficiency of control factor IXa preparations (Fig. 6).

Figure 5: Factor IXa activity and factor IXa sialic acid content. A, factor IXa (both control and desialated) clotting activity and sialic acid content. B, factor IXa (both control and desialated) tenase activity in a coupled amidolytic assay and sialic acid content. Symbols used are as follows; is control factor IXa activity, is desialated factor IXa activity, and circle is factor IXa sialic acid content.

Figure 6: Binding of antithrombin III with factor IXa and desialated factor IXa. Each lane contains 7-8 µg of protein. Samples were not reduced. Lanes1 and 7 are molecular weight markers; lane2 is AT-III; lane3 is control factor IXa-AT-III complex; lane4 is control factor IXa; lane5 is desialated factor IXa; lane6 is AT-III-desialated factor IXa complex.

Activation of Factor X and Its Activity after Desialation

Two experiments tested the coagulation activity of factor X treated with recombinant sialidase sufficient to reduce the sialic acid content to 19% and 29% of initial levels. After desialation there was no detectable loss of factor X clotting activity in the PTT assay. Desialated factor X could hydrolyze a small peptide substrate (S-2765) after activation similar to results seen with the control preparation. As seen with factor IX, the specific activity of factor X increased after sialidase treatment probably reflecting minor activation with extended incubation (data not shown). Furthermore, as shown in Fig. 7, desialated and control preparations of human factor X were cleaved at an indistinguishable rate by soluble RVV-X. Rates of activation of both desialated and control preparations of human factor X were also similar when activated by the intrinsic pathway (complex of factor IXa, factor VIIIa, phospholipid vesicles, and CaCl (2)

) as shown in Fig. 8.

Figure 7: Activation of factor X and desialated factor X by RVV-X. Factor X samples were not reduced. Samples were activated with soluble RVV-X. All lanes contain 5 µg of protein. Lanes 1-5 contain control factor X, and lanes 6-10 contain desialated factor X. Incubation times of factor X samples are as follows; lanes1 and 10 are at 30 min, lanes2 and 9 are at 15 min, lanes3 and 8 are at 5 min, lanes4 and 7 are at 1 min, lanes5 and 6 are at 0 min.

Figure 8: Activation of factor X and desialated factor X by factor IXa/factor VIIIa/phospholipid vesicles/CaCl (2) . Each lane contains 7-8 µg of protein. Samples were not reduced. Lanes1-5 contain desialated factor X, and lanes6-10 contain control factor X. Incubation time of factor X samples are as follows; lanes1 and 10 are at 30 min, lanes2 and 9 are at 15 min, lanes3 and 8 are at 5 min, lanes4 and 7 are at 1 min, and lanes5 and 6 are at 0 min.

DISCUSSION

These experiments have shown that enzymatic removal of sialic acid from human factor IX did not affect its activation or its proteolytic activity against factor X. Additionally, treatment of factor X with recombinant C. perfringens sialidase did not affect activation of factor X or its enzymatic activity. The specific activity of desialated factor IX was preserved in the PTT assay, which depends on activation of the proenzyme and its ability to activate factor X in the presence of cofactors. High values of specific activity were probably due to minor activation of the factor IX samples after incubation with sialidase. By SDS-PAGE analysis, factor XIa, the complex of factor VIIa and tissue factor, and RVV-X cleaved desialated factor IX and native factor IX at the same rate. Desialated factor IXa retained full activity on the NAPTT assay and tenase activity measured in a coupled amidolytic assay. Experiments with human factor X showed no detectable loss of clotting activity of desialated factor X in the PT assay, reflecting both activation and enzymatic activity. Factor X and desialated factor X were cleaved by RVV-X, and by the complex of factor IXa, factor VIIIa, phospholipid vesicles, and CaCl (2) at the same rate when analyzed by SDS-PAGE. Additionally, desialated factor Xa hydrolyzed a small peptide substrate (S-2765) at the same rate as control factor Xa.

Others have reported decreased enzymatic activity of partially activated human factor IX and either no effect on human factor X activity or slowed activation of the proenzyme after treatment with commercial sialidase(8, 9, 10) . Chavin and Weidner (8) found that activation of factor IX by RVV-X was not affected after removal of sialic acid but the partially activated factor IXa alpha (cleaved at Arg-180/Val-181) had reduced activity in the PTT assay. In experiments with human factor X, Chavin and Weidner demonstrated that even 6 h of treatment of human factor X with sialidase did not change PTT activity despite removal of 54% of its sialic acid(8) . Two reports, however, have demonstrated slow activation of factor X after removal of sialic acid(9, 10) . Sinha and Wolf (9) have reported that both bovine and human factor X were slowly activated by either intrinsic tenase or by the tissue factor-VIIa complex after short treatment (3-4 h) with Vibrio cholerae sialidase. The clotting activity of the desialated factor X was 2-12% of the untreated factor X in PT or PTT assays. Similar results were obtained using a chromogenic substrate to measure factor Xa generation. Activation of both human and bovine factor X was less affected after N-glycosidase treatment but residual sialic acid was not measured after treatment with either sialidase or N-glycosidase. Inoue and Morita (10) also showed that the rate of activation of bovine factor X was decreased after treatment with Arthrobacter ureafaciens sialidase and O-glycanase from Streptococcus pneumoniae, but not after removal of N-linked carbohydrate. Sialidase alone was not used in these experiments, and residual sialic acid was not quantitated in this report. The activation peptide regions of human and bovine factor X contain different numbers of sites for N- and O-linked oligosaccharides, so that results with bovine factor X may not predict results with human factor X.

Preservation of clotting activity after sialidase treatment may be explained by differences in experimental approach. Use of recombinant sialidase instead of sialidase from bacterial lysates may have minimized proteolytic degradation of clotting factor preparations in the current report. In most experiments, sialidase-treated and control factor IX and X were reisolated using monoclonal antibody immunoaffinity chromatography prior to studies of enzymatic activity, further minimizing chances for protein degradation by trace amounts of proteolytic enzymes in the sialidase preparations.

Although sialic acid residues are concentrated in the activation peptide regions, they do not appear to be critical for activation of factors IX and X and their function remains speculative. The one sialic acid residue on the light chain of factor IXa is found in factor IX's EGF1 domain on an O-linked tetrasaccharide at Ser-61. This tetrasaccharide is not found on bovine factor IX or human factor X. Desialated factor IXa has full clotting activity in agreement with other observations, suggesting that the sialic acid on the EGF1 domain of factor IX is not essential for factor IXa interaction with its cofactor. Hybrid factor IX containing factor X EGF1 in place of the factor IX EGF1 domain lacks the fucosyl modification consensus sequence (hence it must lack the Ser-61 tetrasaccharide), yet retains full clotting activity(18) . Since no hemophilia B mutations have been found at Ser-61 or another site for glycosylation, Ser-53, the codons for these residues may either be relatively stable to mutations, or perhaps missense mutations affecting sites for O-linked oligosaccharide attachment within EGF1 do not affect clotting activity and are not detected(19) .

The O-linked oligosaccharides within the EGF1 domain may have a role in receptor-ligand interaction(20) . O-Linked oligosaccharides are important for C type lectin interaction in the binding of t-PA to HepG2 cells since this calcium-dependent reaction is not seen with alpha -fucosidase treated t-PA(21) . O-Linked oligosaccharides are also important in other receptor-ligand interactions such as binding of transferrin and its receptor(22) . Although sialic acids are not required for coagulant activity of factor IX and X in vitro, they may be important for survival of factor IX and X in circulation or for binding to cellular surfaces. Clearance of ceruloplasmin has been shown to be accelerated after treatment with sialidase for example(23) . Studies are in progress to see if sialic acids are involved in factor IX binding to endothelial cells.

FOOTNOTES

*: This investigation was supported by grants from Blood Systems Foundation, Scottsdale, AZ (to K. J. S. and W. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§: To whom correspondence should be addressed.
(): The abbreviations used are: EGF, epidermal growth factor; AT-III, antithrombin III; NAPTT, non-activated partial thromboplastin time; PAGE, polyacrylamide gel electrophoresis; PTT, partial thromboplastin time; RVV-X, factor X activating emzyme from Russell's viper venom; TBS, Tris-buffered saline; t-PA, tissue plasminogen activator.

ACKNOWLEDGEMENTS

We thank Louisette Basa and Michael Molony of Genentech, Inc. for sialic acid determination and amino acid analyses. We also thank Janet Haught for secretarial assistance.

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