©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sialic Acid Content of Plasminogen 2 Glycoforms as a Regulator of Fibrinolytic Activity
ISOLATION, CARBOHYDRATE ANALYSIS, AND KINETIC CHARACTERIZATION OF SIX GLYCOFORMS OF PLASMINOGEN 2 (*)

(Received for publication, May 18, 1994; and in revised form, December 12, 1994)

Steven R. Pirie-Shepherd (§) Elizabeth A. Jett Nancy L. Andon Salvatore V. Pizzo

From the Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Six glycoforms of plasminogen 2 were isolated using a combination of lectin affinity chromatography and chromatofocussing, and the sialic acid content of each glycoform was determined. The kinetics of activation of each glycoform by tissue-type plasminogen activator were analyzed on a fibrin surface and in solution. The second-order rate constant (measured on a fibrin surface) decreased from 1.65 times 10^6M s to 3.77 times 10^4M s as the sialic acid content of the glycoforms increased from 1.3 mol/mol of protein to 13.65 mol/mol of protein. A similar correlation was noted for activation in solution. Each glycoform was converted to plasmin, and the inhibition constants for the reaction between alpha(2)-antiplasmin and plasmin glycoforms were determined. All overall K values, reflecting the final essentially irreversible complex, were in the picomolar range. Sialic acid does not affect inhibition of plasmin by alpha-antiplasmin; however, hypersialylated plasmin does not appear to have a kringle-dependent component to inhibition.


INTRODUCTION

Plasminogen is the precursor of plasmin, the central proteinase in fibrinolysis. The plasminogen amino acid sequence is known and has been reviewed in (1) . The amino-terminal portion of the protein comprises five kringle domains (2) of which kringles 1 and 4 are involved in the binding of lysine and lysine analogues(3) . The carboxyl terminus of the protein contains the serine proteinase domain. Two major forms of plasminogen have been separated on Lys-Sepharose(4) . Form I contains two carbohydrate chains linked to Asn-289 and Thr-345, while form II contains one carbohydrate chain linked to Thr-345 (5, 6) (see Fig. 1).


Figure 1: The accepted structure of the carbohydrate moieties of plasminogens 1 and 2. Note that the alpha-(2-6)-linked sialic acid seen on the O-linked carbohydrate is present only on 1-5% of plasminogen 2 molecules.



Further studies of the glycoforms of plasminogen demonstrated five subforms within each major isoform of rabbit plasminogen. (7) The pI of plasminogen 1 subforms exhibited a degree of overlap with the pI of plasminogen 2 subforms, and all pI values fell within the 6.2-8.7 range. The study by Gonzalez-Gronow et al.(8) also indicated the presence of 5 major glycoforms of plasminogen 2 and a sixth, highly acidic, glycoform present in reduced concentrations in human plasma. Removal of sialic acid from plasminogen 2 resulted in decreased circulation times. Desialylation of human plasminogen 2 causes an increase in the amidolytic and fibrinolytic activity of plasminogen(9) . Asialoplasminogen hydrolyzes peptide substrate approximately 10% as efficiently as plasmin, although it is still structurally a zymogen.

More recent reports have also indicated that the extent of glycosylation may have a physiological relevance(8) . Human recombinant nonglycosylated plasminogen, expressed in Escherichia coli, was resistant to tissue-type plasminogen activator (tPA) (^1)activation(8) . Although the recombinant protein manifested expected secondary and tertiary structure, the nonglycosylated form had a decreased circulation time in vivo(8) . Studies of the dissociation constants (K) for the interaction between different plasminogen derivatives and alpha(2)-antiplasmin (alpha(2)-AP) were determined as the concentrations of ligand that decrease the rate of reaction between plasmin and alpha(2)-AP by 50%(10) . This report suggested that the O-linked carbohydrate had little or no effect on this interaction, although the N-linked carbohydrate chain was hypothesized to affect both binding to alpha(2)-AP and fibrin.

Gonzales-Gronow et al.(11) suggested that the affinity of cell surface receptors for plasminogen 2 was much greater than the affinity for plasminogen 1. Further evidence for the importance of carbohydrate in plasminogen function was provided by Hall et al.(12) , whose studies showed that although plasminogen 1 and 2 bound to rat hepatocytes and C6 glioma cells to an equivalent number of receptors, the affinity for plasminogen 2 was slightly higher. It was also demonstrated that hepatocyte cultures enhanced the activation of plasminogen 1 and 2 by tPA, the enhancement being greater for plasminogen 2(12) .

The differential physiological properties of plasminogen 1 and 2 are mostly attributed to the absence of the N-linked carbohydrate on plasminogen 2, but there is evidence that the concentration of sialic acid on the carbohydrate chains may also have a role to play. Neonatal and adult forms of plasminogen 2 have identical amino acid compositions, but differ remarkably in their carbohydrate composition. Neonatal plasminogen 2 has 20 times more sialic acid than adult plasminogen 2(13) . The kinetics of activation of neonatal plasminogen 2 by tPA are markedly different, demonstrating both a higher K and higher k than adult plasminogen 2. These previous studies suggest that the differences in properties in plasminogen glycoforms may not be due solely to the absence or presence of N-linked carbohydrate. In this current work, we have isolated six unique glycoforms of plasminogen 2 and have analyzed their kinetics of activation by tPA and inhibition by alpha(2)-AP. These present studies provide additional evidence for the role of carbohydrate in general, and sialic acid in particular, in regulation of plasminogen/plasmin function.


EXPERIMENTAL PROCEDURES

Materials

The chromogenic plasmin substrate D-Val-Leu-Lys-p-nitroanilide dihydrochloride (VLKpNA), trypsin substrate Bz-Arg-p-nitroanilide, trypsin titrant Bz-Tyr-paranitrophenol, and human thrombin were purchased from Sigma (St. Louis, MO). Mono P columns, CNBr-Sepharose 4B, Sepharose 4B, C-class chromatography columns, and polybuffer 96 were from Pharmacia Biotech Inc. Urinary-type plasminogen activator was purchased from Calbiochem (San Diego, CA). Sambucus nigris agglutinin and concanavalin A lectin were purchased from Boehringer Mannheim. Human fibrinogen was purchased from Kabivitrum (Stockholm, Sweden). All ligand-Sepharose matrices were generated in the laboratory according to manufacturer's instructions. All other reagents were of the highest purity available.

Proteins

Trypsin was from Worthington. Tryspin was active site-titrated using Bz-L-Tyr-paranitrophenol as described by Chase and Shaw. (14) Recombinant two-chain tPA was the kind gift of Dr Henry Berger (Wellcome Research Laboratories, Research Triangle Park, NC). alpha(2)-AP (purified as outlined in (15) ) was the kind gift of Dr. Jan Enghild and Zuzana Valnickova. The reactive site concentration of alpha(2)-AP was determined by back titration against trypsin at µM concentrations using Bz-Arg-p-nitroanilide as a substrate.

Plasminogen 2 was purified as described previously(4) , using a combination of Lys-Sepharose and concanavalin A-Sepharose affinity chromatography. Further fractionation of plasminogen 2 into glycoforms was achieved using two separate protocols. Some glycoforms are hypothesized to contain sialic acid in an alpha-(2-6) linkage. Consequently, we applied the purified plasminogen 2 to a Sambucus nigra agglutinin (SNA) lectin column previously equilibrated in 50 mM Tris, 1 mM MgCl(2), 1 mM CaCl(2), pH 7.4. SNA is specific for sialic acid in the alpha-(2-6) linkage. (16) Plasminogen that bound to this column was eluted with a solution of 50 mM Tris, 1 mM CaCl(2), 1 mM MgCl(2), 0.1 M lactose. This protein solution was adjusted to a concentration of 20 mM EDTA and dialyzed against H(2)O extensively before kinetic and thermodynamic analysis. Protein that did not bind to the SNA column was adjusted to a concentration of 20 mM EDTA and dialyzed against 12 liters of H(2)O (3 times 8 h), to remove metal divalent ions necessary for lectin affinity chromatography. Separation of the remaining five major subforms was achieved using chromatofocussing on a Mono P column linked to an FPLC system. The Mono P column was equilibrated in 25mM Tris-acetic acid, pH 8. 3. A pH gradient from 8.4 to 6.0 was generated by applying 50 ml of polybuffer 96 (10% (v/v)) acetic acid, pH 5.8. All buffers were 10% betaine (w/v) (free base), which reduced charge interactions between sugar moieties that could interfere with resolution of the glycoforms. Purity of each glycoform was determined by reducing SDS-polyacrylamide gel electrophoresis. Electrophoresis of proteins was performed using the Bio-Rad mini Protean II system and the tricine buffer system(17) .

Activation of Plasminogen by tPA

Activations were conducted on fibrin plates (18) and on untreated polystyrene 96-well assay plates (Costar, Cambridge, MA). The buffer was 50 mM Hepes, 50 mg/ml bovine serum albumin, pH 7.4. All absorbances were measured on a Molecular Devices Thermomax 96-well plate reader (Menlo Park, CA). Subsequent manipulation of the data was performed using Excell 4.00.

Binding of Plasminogen Glycoforms to a Fibrin Surface

Plasminogen glycoforms were radioactively labeled using NaI (DuPont NEN) and Iodobeads (Pierce) according to the manufacturer's instructions. Increasing concentrations of labeled plasminogen were incubated on a fibrin surface (18) generated in 96-well plates for an hour in phosphate-buffered saline. Nonspecific binding was performed in 150 mM aminocaproic acid, 0.1 M lactose. These buffer conditions abolished binding of plasminogen to a fibrin surface. Specific binding was calculated by subtracting nonspecific from total binding. Binding data was fitted directly to the Langmuir isotherm using SYStat version 5.01.

Generation of Plasmin Glycoforms

Plasmin glycoforms were generated from plasminogen glycoforms using urokinase-Sepharose (9000 IU/g) or free urokinase (1:50 molar ratio). Plasmin was removed from free urokinase activity using lysine-Sepharose affinity chromatography. Plasmin generation was allowed to proceed for 1 h at 25 °C. The K(m) values for reactions between VLKpNA and plasmin glycoforms were determined using standard methods, as were the values for K(e) (nmol of VLKpNA hydrolyzed per min/mol of enzyme, required to calculate k values for the activation of plasminogen by tPA(19) ).

Inhibition of Plasmin Glycoforms by alpha(2)-AP

Generated plasmin was active-site titrated against alpha-(2)AP at nanomolar concentrations. The buffer was 50 mM Hepes, 50 mg/ml bovine serum albumin, pH 7.4. Inhibition studies were performed essentially as outlined in Longstaff and Gaffney(20) . Plasmin activity, in the presence of increasing molar ratios of alpha(2)-AP, was monitored using VLKpNA. All reactions were performed in the presence of 150 mM aminocaproic acid, a lysine analogue that will alter the kinetics such that equilibrium is reached more slowly(20) . The interactions of plasmins 2alpha-2 with alpha(2)-AP were analyzed according to the accepted two-step reaction scheme for alpha(2)-AP ().

Values for K(i) (k/k(1)), K (the overall inhibition constant), k, and k were determined precisely as outlined in Longstaff and Gaffney(20) . The interaction between plasmin 2 and alpha-AP was analyzed according to the minimal one-step reaction scheme () for serpin/proteinase interaction essentially as described(21) .

The rationale is given under ``Results''; values of K(i) and k were derived using nonlinear regression analysis (SYStat). All reactions were monitored for at least 3 h to ensure attainment of equilibria. Reaction conditions were such that no substrate depletion occurred during the course of the experiments.

Sialic Acid Determination

Sialic acid content was measured using the GLYKO system (Glyko, Novato CA) according to the manufacturer's instructions. Briefly, 20 µg of glycoprotein was hydrolyzed, and the released sialic acid was fluorescently labeled and electrophoresed on proprietary gels. Quantitation of sugar was performed using a fluorescence imaging system connected to an IBM PC.

Statistical Analysis

All statistical analyses (including nonlinear regression analysis of kinetic and thermodynamic curves) were performed using SYStat 5.01 for Windows. Nonlinear regression analysis was performed according to manufacturer's directions.


RESULTS

Purification of Plasminogen 2 Glycoforms

Separation of plasminogen 2 glycoforms was achieved using a combination of affinity chromatography and chromatofocussing as described under ``Experimental Procedures.'' Fig. 2shows a profile of the Mono P column elution. Each of the five glycoforms purified by chromatofocussing was applied to a 10-ml Lys-Sepharose column to remove polybuffer 96. The individual glycoforms, eluted from Lys-Sepharose with 0.1 M aminocaproic acid, were dialyzed against H(2)O and stored at -20 °C. The sixth glycoform was eluted from SNA-Sepharose as described under ``Experimental Procedures,'' dialyzed to remove divalent metal ions, and stored at -20 °C. This sixth glycoform represented 1-5% of the total plasminogen 2 pool. Purity of the glycoforms was assessed by reducing SDS 10% polyacrylamide gel electrophoresis (data not shown). All plasminogen glycoforms purified were Lys-plasminogen (confirmed by N-terminal sequence analysis). The six isolated glycoforms were designated 2alpha-2, alpha having the highest pI and being the alpha-(2-6)-sialic acid-containing form. Carbohydrate analysis of each glycoform confirmed that the reduction in pI was due to increased sialic acid content. Plasminogen 2alpha had only 1.3 mol of sialic acid/mol of protein, while plasminogen 2 had 13.65 mol of sialic acid/mol of protein. The mol/mol ratio of sialic acid/protein is shown in Table 1.


Figure 2: Production of plasminogen 2 glycoforms. Plasminogen 2 was purified using affinity chromatography as described under ``Experimental Procedures.'' Plasminogen 2 was removed from this pool of glycoforms using SNA lectin affinity chromatography, and the remaining plasminogen glycoforms were dialyzed to remove divalent metal ions (required for lectin binding). The dialyzed pool of plasminogen glycoforms was applied to a Mono P column previously equilibrated in 25 mM Tris-HAc, pH 8.3. Glycoforms were eluted with a continuous pH gradient generated by a buffer containing polybuffer 96 (Pharmacia) titrated to pH 5.5 with acetic acid. This procedure was used to isolate the five major glycoforms of plasminogen 2.





Activation Kinetics of Plasminogen

The activation of plasminogen 2 glycoforms by tPA was conducted on a fibrin surface and in a solution-phase assay. Individual plasminogen 2 glycoforms at various concentrations (5-400 nM) were incubated with tPA (1.47 nM on fibrin plates, 7.35 nM on untreated plates) in the presence of the chromogenic plasmin substrate VLK-p-nitroanilide (0.3 mM). Initial velocities were determined by plotting absorbances at 405 nm against time ^2 and analyzed as described(19) . Values of K(m) and V(max) were determined by fitting experimental data directly to the Michaelis-Menten equation using a nonlinear iterative procedure. V(max) values were converted to k values as described by Wohl et al.(19) using empirically determined K(e) values as described under ``Experimental Procedures.'' The values for K(m) increased as the sialic acid content increased. The reaction between tPA and plasminogen 2alpha on a fibrin surface had a K(m) of 20 nM, while the K(m) between tPA and plasminogen 2 was 477 nM. Values of k, K(m), and the second-order rate constant (k/K(m)) are tabulated (Table 2) for both solution and solid phase activation. There appears to be a general trend toward a decrease in catalytic efficiency as sialic acid content increases, although plasminogen 2 does not fit into this general scheme.



Binding of Plasminogen Glycoforms to Fibrin Surface

Plasminogen glycoforms were allowed to bind to a fibrin surface as described under ``Experimental Procedures.'' Apparent K(D) values were calculated and are tabulated (Table 3). Glycoforms 2alpha-2 all bind fibrin with a similar affinity. The 2 glycoform does not appear to bind the fibrin surface. This is an interesting observation because the form is the hypersialylated form, which is a very poor substrate for tPA (K(m) > 400 nM). The lack of fibrin binding may, in part, explain the poor activation kinetics of this glycoform by tPA.



Inhibition Kinetics of Plasmin

Plasminogen 2 glycoforms were converted to plasmin glycoforms as described under ``Experimental Procedures.'' The inhibition of plasmins 2alpha-2 by alpha(2)-AP follows a two-step reaction scheme(20, 22) . The first step involves formation of a ``loose'' complex, with a K(i) in the nM range. This is then followed by a structural rearrangment leading to the formation of an even tighter complex with an overall K in the picomolar range. The determination of K and K values between alpha-AP and plasmin was as described under ``Experimental Procedures.'' Experimentally derived values of K, K, k, and k are in Table 4. One of the kinetic criteria for the two-step reaction model described in Christensen et al.(22) and Longstaff and Gaffney (20) is that the initial velocity of the reaction should be inversely proportional to [I]. However, we did not find this in the case of plasmin 2. We therefore analyzed the reaction as a one-step scheme so as to determine an overall K; we also determined k, the second-order association constant as described under ``Experimental Procedures.''




DISCUSSION

Many of the proteins involved in coagulation and fibrinolysis are glycosylated, and differential effects of glycosylation on protein function have been observed. Deglycosylated thrombin (23) lost no fibrinogen clotting activity, amidolytic activity, nor the ability to form complexes with antithrombin. In addition, asialothrombin caused the same extent of platelet release as did unmodified thrombin. Furthermore, deglycosylation of antithrombin did not diminish its inhibitory activity, nor did it affect heparin dependency. Similar experiments with recombinant urinary-type plasminogen activator (24) demonstrated that the N-linked glycosylation pattern of urokinase-type plasminogen activator did not affect its interaction with plasmin, plasminogen, or plasminogen activator inhibitor-1, proteins directly involved in its fibrinolytic function.

In contrast, the differential glycosylation of tPA affects the functional activity of the protein. Comparison of the activity of two major glycoforms of recombinant tPA (form I has N-linked carbohydrate at Asn-117, Asn-184, and Asn-448; form II lacks carbohydrate (25) at Asn-184) demonstrated that form II displayed 2-fold higher fibrinolytic activity than form I. The fibrinolytic activity of N-glycanase-treated form I was very similar to normal recombinant tPA, whereas treatment of the deletion mutant form I with neuramindase resulted in increased fibrinolytic activity. The authors concluded that terminal sialic acids interfered with the interaction between the kringle region of tPA and fibrin, suggesting that differential sialylation may regulate fibrinolytic activity. Other studies suggest that glycosylation of tPA at Asn-184 may affect the catalytic efficiency of conversion from single-chain tPA to two-chain tPA by plasmin(26) . Thus, form I single-chain tPA may persist in the single chain form longer than form II single-chain tPA. The two major glycoforms of tPA may represent more persistant but slow acting and less persistant but faster acting variants of tPA.

Dang et al.(27) demonstrated that desialylation of fibrinogen results in the loss of low affinity Ca binding sites; clotting of asialofibrinogen appears to be Ca-independent and results in thicker fibrin bundles as judged by electron microscopy. Thus, sialic acid also appears to have a role in the regulation of fibrin formation.

Although previous reports have shown the presence of at least six plasminogen 2 glycoforms in human and rabbit(7, 8) , ours is the first report detailing the purification and kinetic analysis of six subforms of human plasminogen 2. This reproducible isolation of the subforms indicates that the classical structure of the O-linked sugar chain (Fig. 1) may be a simplification of the true picture. The main structural difference between these glycoforms is the sialic acid content of the O-linked sugar, although the existence of other sugar differences (such as fucose substitutions) cannot be discounted at this time. Our data suggest that plasminogen 2 can have up to six sialic acids with an alpha-(2-3) linkage on the O-linked carbohydrate moiety. Presumably, this represents polysialylation of the plasminogen molecules; polysialylation has been shown to play a role in the regulation of other proteins, for example, neural cell adhesion molecule(28) . The glycoform of plasminogen 2 appears to be as hypersialylated as neonatal plasminogen 2(13) , and at least some fraction of this sialic acid is present in an alpha-(2-6) linkage as evidenced by binding of this form to SNA lectin (see ``Experimental Procedures''). The existence of hypersialylated plasminogen with differential kinetics of activation and clearance has been noted previously(13) . Furthermore, Siefring and Castellino (7) also demonstrated increasing mol/mol ratios of sialic acid/protein as a cause of microheterogeniety in rabbit plasminogen 2. They also showed that removal of sialic acid (by incubation with neuraminidase) caused a decrease in the number of glycoforms present(7) .

The catalytic efficiency of tPA in activating these glycoforms decreases as the sialic acid content increases. Glycoform 2 does not fit into this general scheme as it has a lower sialic acid content and a better catalytic efficiency than 2, but it still maintains a lower pI. We cannot rule out the possibility of other chemical modifications to the peptide backbone at this time. Only one of these glycoforms, plasminogen 2, contains sialic acid in an alpha-(2-6) linkage, and this glycoform has the lowest k and the highest K(m) for activation by tPA on a fibrin surface. This form is also present as 1-5% of the total plasminogen 2 population, as determined by two-dimensional electrophoretic analysis (data not shown). Activation in a solution phase assay (in the absence of fibrin) shows the same general trend of decreased catalytic efficiency with increased sialic acid content. Absolute catalytic efficiencies (k/K(m) values) are 10-fold less than on a fibrin surface. The form of plasminogen 2 does not bind to a fibrin surface, which may explain the high apparent K(m) between this form and tPA. The cataltyic efficiency of activation of 2 increases only 5-fold in the presence of fibrin, whereas the other glycoforms exhibit a 10-fold increase in catalytic efficiency in the presence of fibrin. This may also reflect the lack of fibrin binding by 2. The other glycoforms of plasminogen display reduced K(m) values for tPA on a fibrin surface, possibly a function of the ternary complex formed between tPA, plasminogen, and fibrin.

We analyzed our inhibition reactions essentially as described by Longstaff and Gaffney(20) . Plasmins 2alpha-2 exhibited the accepted two-step reaction scheme(20, 22) . We derived K(i) and K values in the nM and pM range of values (see Table 4), which are in agreement with published values for unfractionated plasminogen 2(20, 22) . We also derived values for k and k(20) that are in agreement with theoretical (22) and published (20) values. However, the inhibiton of plasmin 2 (the hypersialylated form of plasmin) did not follow the two-step model under the conditions we used. This anomoly was evidenced by the fact that k`, a measure of the change from V (initial velocity) to V (equilibrium velocity) over time, in the presence of inhibitor and modulator (20) did not vary with inhibitor concentration, nor were the values for V inversely proportional to [I] as would be expected with the classic two-step model. It is most likely that equilibrium is reached very rapidly with this plasmin glycoform. We therefore analyzed the inhibition of plasmin 2 as a one-step reaction scheme and determined K, the ratio of k/k, and found this to be in the pM range. k was measured and found to be 1.46 times 10M s. This is slower than accepted association rates for plasmin/alpha-AP, although the derived k (K times k) is slow enough to ensure a t of geq 10 h. This is an interesting observation as plasminogen 2 is the glycoform that does not exhibit fibrin binding. These observations suggest that the lysine binding properties of plasminogen 2 are perturbed in some way. The location of the O-linked glycosylation is Thr-345, which is located between kringle 3 (K3) and kringle 4 (K4)(6) . Christensen et al.(22) , have recently hypothesized that K4 has an important role in the regulation of inhibition of plasmin. The presence of a large glycan (15 sialic acids long) at the base of K4 may induce conformational changes in this kringle, which could disrupt lysine binding. This would explain the apparent lack of fibrin binding and the anomalous inhibiton scheme. Alternatively, there may be a more direct effect due to the presence of sialic acid(25) . Any association between plasmin and alpha-AP that relied on an interaction between the terminal Lys residues on alpha-AP and K4 on plasmin would be abolished, leaving only the interaction between the reactive site loop of alpha-AP and the serine proteinase domain of plasmin. It is known that miniplasmin has a slower (10-35-fold less) association with alpha-AP than plasmin; miniplasmin consists of K5 and the serine proteinase domain. Thus, the equilibrium between a form of plasmin that has disrupted kringle interactions and alpha-AP would be unperturbed by the addition of lysine analogues. The final equilibrium would be rapidly attained, and the reaction scheme would appear to be a one-step scheme.

It should be noted, however, that where thermodynamic and kinetic constants are comparable, there appear to be no differences between glycoforms, with the notable exception of plasmin 2. If we compare the overall K of plasmins 2alpha-2 to the K between alpha-AP and 2, it can be seen that the final thermodynamic rearrangement that produces the final tight complex is independent of sialic acid content. This is not unexpected, as the general reaction mechanism of serpins and proteinases must proceed in the absence of kringles, being a reaction between the reactive site loop of the serpin and the active site cleft of the proteinase.

It is known that in the acute phase response, the glycosylation patterns of many proteins are altered, (29) and more recently it has been shown that a Golgi-membrane-bound alpha-(2-6)-sialyltransferase is cleaved by cathepsin D and becomes a soluble active enzyme(30) . This soluble form of the enzyme has been found both cytoplasmically and systemically. Thus, the significant possibility of postsecretion modification of sugar chains exists. Furthermore, although plasminogen is not an acute phase reactant, we cannot discount the possibility of altered glycosylation that would affect fibrinolysis rates.

In conclusion, our data suggest that plasminogen 2 sugar microheterogeniety may play a role in the regulation of fibrinolysis. This control may be exerted at the level of activation on the fibrin clot and may be due to a combination of factors, including the interaction between tPA and plasminogen and the binding of plasminogen to fibrin. Sialic acid would not appear to regulate the inhibition of plasmin by alpha(2)-AP, except where K4 interactions are perturbed. Other proteins involved in fibrinolysis, including fibrinogen and tPA, have variable sugar chains, which are implicated in the regulation of this physiological activity. Differentially sialylated plasminogen may represent systemic pools of this proenzyme that can be activated more slowly or more quickly on the fibrin clot, thus ensuring persistance of fibrinolytic activity.


FOOTNOTES

*
This work was supported by National Institutes of Health NHLBI Grant HL 31932 (to S. V. P.). 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. Duke University Medical Center, Box 3712, Durham, NC 27710. Tel.: 919-684-8986; Fax: 919-684-8689.

(^1)
The abbreviations used are: tPA, tissue plasminogen activator; alpha(2)-AP, alpha(2)-antiplasmin; VLKpNA, D-Val-Leu-Lys-p-nitroanilide dihydrochloride; SNA, Sambucus nigra agglutinin; K3, kringle 3.


ACKNOWLEDGEMENTS

We thank Dr. Hanne Grøn, Dr. Søren Christensen, Dr. Ulla Christensen, and Dr. Guy Salvesen for helpful comments and discussion.


REFERENCES

  1. Castellino, F. J. (1984) Semin. Thromb. Hemostasis. 10, 18-23 [Medline] [Order article via Infotrieve]
  2. Sottrup-Jensen, L., Claeys, H., Zajdal, M., Petersen, T. E., and Magnusson, S. (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209
  3. Lerch, P. G., Rickli, E. E., Legier, W., and Gillisen, D. (1980) Eur. J. Biochem. 107, 7-13 [Abstract]
  4. Brockway, W. J., and Castellino, F. J. (1972) Arch. Biochem. Biophys. 151, 194-199 [Medline] [Order article via Infotrieve]
  5. Davidson, D. J., and Castellino, F. J. (1991) Biochemistry 30, 6689-6696 [Medline] [Order article via Infotrieve]
  6. Hayes, M., and Castellino, F. J. (1979) J. Biol. Chem. 254, 8768-8771 [Medline] [Order article via Infotrieve]
  7. Siefring, G. E., Jr., and Castellino, F. J. (1974) J. Biol. Chem. 249, 7742-7746 [Abstract/Free Full Text]
  8. Gonzales-Gronow, M., Grennet, H. E., Fuller, G. M., and Pizzo, S. V. (1990) Biochim. Biophys. Acta 1039, 269-276 [Medline] [Order article via Infotrieve]
  9. Stack, S. M., Pizzo, S. V., and Gonzales-Gronow, M. (1992) Biochem. J. 284, 81-86 [Medline] [Order article via Infotrieve]
  10. Lijnen, R., Van Hoef, B., and Collen, D. (1981) Eur. J. Biochem. 120, 149-154 [Medline] [Order article via Infotrieve]
  11. Gonzalez-Gronow, M., Edelberg, J. M., and Pizzo, S. V. (1989) Biochemistry 28, 2374-2377 [Medline] [Order article via Infotrieve]
  12. Hall, S. W., Vandenberg, S. R., and Gonias, S. L. (1990) J. Cell. Biochem. 43, 213-227 [Medline] [Order article via Infotrieve]
  13. Edelberg, J. M., Enghild, J. J. Pizzo, S. V., and Gonzales-Gronow, M. (1990) J. Clin. Invest. 86, 107-112 [Medline] [Order article via Infotrieve]
  14. Chase, T., and Shaw, E. (1970) Methods Enzymol. 19, 20-27
  15. Wiman, B. (1980) Biochem. J. 191, 229-232 [Medline] [Order article via Infotrieve]
  16. Shibuya, N., Goldstein, I. J., Broekaert, W. F., Nsimba-Lukabi, M., Peeters B., and Peumans, W. J. (1987) Arch. Biochem. Biophys. 254, 1-8 [Medline] [Order article via Infotrieve]
  17. Schägger, H., and von Jagow, G. (1987) Anal. Biochem 166, 368-379 [Medline] [Order article via Infotrieve]
  18. Anglés-Cano, E. (1986) Anal. Biochem. 153, 201-210 [Medline] [Order article via Infotrieve]
  19. Wohl, R. C., Summaria, L., and Robbins, K. C. (1980) J. Biol. Chem. 255, 2005-2013 [Free Full Text]
  20. Longstaff, C., and Gaffney, P., (1991) Biochemistry 30, 979-986 [Medline] [Order article via Infotrieve]
  21. Pirie-Shepherd, S. R., Miller, H. R. P., and Ryle, A. (1991) J. Biol. Chem. 266, 17314-17319 [Abstract/Free Full Text]
  22. Christensen, S., Sottrup-Jensen, L., and Christensen, U. (1994) Biochem. J. 305, 97-102
  23. Rosenfield, L., and Danishefsky, I. (1984) Arch. Biochem. Biophys. 229, 359-367 [Medline] [Order article via Infotrieve]
  24. Sarubbi, E., Nolli, M. L., Robbiati, F., Soffientini, A., Parenti, F., and Cassani, G. (1989) Thromb. Hemostasis. 62, 927-933 [Medline] [Order article via Infotrieve]
  25. Pohl, G., Kallstrom, M., Bergsdorf, N., Wallen, P., and Jornvall, H. (1984) Biochemistry 23, 3701-3707 [Medline] [Order article via Infotrieve]
  26. Wittwer, A. J., and Howard, S. C. (1990) Biochemistry 29, 4175-4180 [Medline] [Order article via Infotrieve]
  27. Dang, C. V., Shin, C. K., Bell, W. R., Nagaswami, C., and Weisel, J. W. (1989) J. Biol. Chem. 264, 15104-15108 [Abstract/Free Full Text]
  28. Edelman, G. M. (1986) Annu. Rev. Cell Biol. 2, 81-116 [CrossRef]
  29. Jamieson, J. C., Lammers, G., Janzen, R., and Woloski, B. M. (1987) Comp. Biochem. Physiol. 87, 11-15
  30. Lammers, G., and Jamieson, J. C. (1990) Comp. Biochem. Physiol. 95, 327-334

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