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(Received for publication, May 18, 1994; and in revised form, December 12, 1994) From the
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
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
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) ( 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
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
Values for K
The rationale is given under ``Results''; values of K
Figure 2:
Production of plasminogen 2 glycoforms.
Plasminogen 2 was purified using affinity chromatography as described
under ``Experimental Procedures.'' Plasminogen 2
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 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 The catalytic efficiency of tPA in
activating these glycoforms decreases as the sialic acid content
increases. Glycoform 2 We analyzed our
inhibition reactions essentially as described by Longstaff and
Gaffney(20) . Plasmins 2 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 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
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
Volume 270,
Number 11,
Issue of March 17, 1995 pp. 5877-5881
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ISOLATION, CARBOHYDRATE ANALYSIS, AND KINETIC CHARACTERIZATION OF
SIX GLYCOFORMS OF PLASMINOGEN 2 (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
10
M
s to 3.77 10
M 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
-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
-antiplasmin; however, hypersialylated plasmin does
not appear to have a kringle-dependent component to inhibition.
-(2-6)-linked sialic acid seen on the O-linked
carbohydrate is present only on 1-5% of plasminogen 2
molecules.
)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
-antiplasmin (-AP) were determined as
the concentrations of ligand that decrease the rate of reaction between
plasmin and -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 -AP
and fibrin.
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 -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.
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). -AP (purified as outlined
in (15) ) was the kind gift of Dr. Jan Enghild and Zuzana
Valnickova. The reactive site concentration of -AP was
determined by back titration against trypsin at µM concentrations using Bz-Arg-p-nitroanilide as a
substrate.-(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, 1 mM CaCl, pH 7.4. SNA is
specific for sialic acid in the -(2-6) linkage. (16) Plasminogen that bound to this column was eluted with a
solution of 50 mM Tris, 1 mM CaCl, 1
mM MgCl, 0.1 M lactose. This protein
solution was adjusted to a concentration of 20 mM EDTA and
dialyzed against HO 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 HO (3 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
Na
I (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
values for reactions
between VLKpNA and plasmin glycoforms were determined using standard
methods, as were the values for K
(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
Generated plasmin was active-site titrated
against -AP-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 -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 2-2 with -AP were
analyzed according to the accepted two-step reaction scheme for
-AP ().
(k
/k), K (the overall inhibition constant), k, and k
were
determined precisely as outlined in Longstaff and Gaffney(20) .
The interaction between plasmin 2
and -AP was
analyzed according to the minimal one-step reaction scheme () for serpin/proteinase interaction essentially as
described(21) .
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.
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 HO 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 2-2, having the highest pI and being
the -(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 2 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.
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 and analyzed as described(19) . Values of K and V
were determined by
fitting experimental data directly to the Michaelis-Menten equation
using a nonlinear iterative procedure. V values
were converted to k values as described by Wohl et al.(19) using empirically determined K values as described under ``Experimental
Procedures.'' The values for K increased as
the sialic acid content increased. The reaction between tPA and
plasminogen 2 on a fibrin surface had a K of
20 nM, while the K between tPA and
plasminogen 2 was 477 nM. Values of k, K, and the second-order
rate constant (k/K) 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
values were calculated and are
tabulated (Table 3). Glycoforms 2-2 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 > 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
2-2 by -AP follows a two-step reaction
scheme(20, 22) . The first step involves formation of
a ``loose'' complex, with a K 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 -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.''
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.-(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
-(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) . 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 -(2-6) linkage, and this glycoform has the
lowest k and the highest K 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 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 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 values
for tPA on a fibrin surface, possibly a function of the ternary complex
formed between tPA, plasminogen, and fibrin.-2 exhibited the accepted
two-step reaction scheme(20, 22) . We derived K 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 10
M
s. This is slower than accepted association
rates for plasmin/-AP, although the derived k (K k) is slow enough to ensure a t
of 
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
-AP that relied on an interaction between the terminal
Lys residues on -AP and K4 on plasmin would be
abolished, leaving only the interaction between the reactive site loop
of -AP and the serine proteinase domain of plasmin. It
is known that miniplasmin has a slower (10-35-fold less)
association with -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
-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.. If we compare the overall K of plasmins 2-2 to the K between -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.-(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.-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.
)-AP, -antiplasmin; VLKpNA, D-Val-Leu-Lys-p-nitroanilide dihydrochloride; SNA, Sambucus nigra agglutinin; K3, kringle 3.
We thank Dr. Hanne Grøn, Dr. Søren
Christensen, Dr. Ulla Christensen, and Dr. Guy Salvesen for helpful
comments and discussion.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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S. R. Pirie-Shepherd, R. D. Stevens, N. L. Andon, J. J. Enghild, and S. V. Pizzo Evidence for a Novel O-Linked Sialylated Trisaccharide on Ser-248 of Human Plasminogen 2 J. Biol. Chem., March 14, 1997; 272(11): 7408 - 7411. [Abstract] [Full Text] [PDF]
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P. E. Bock, D. E. Day, I. M. A. Verhamme, M. M. Bernardo, S. T. Olson, and J. D. Shore Analogs of Human Plasminogen That Are Labeled with Fluorescence Probes at the Catalytic Site of the Zymogen J. Biol. Chem., January 12, 1996; 271(2): 1072 - 1080. [Abstract] [Full Text] [PDF]
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