(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 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.
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
-(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) ()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.
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
-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.
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 -(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 H
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
O (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) .
Values for K (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) .
The rationale is given under ``Results''; values of K 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.
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.
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 -(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) .
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
-(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.
We analyzed our
inhibition reactions essentially as described by Longstaff and
Gaffney(20) . Plasmins 2-2
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.
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 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.
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
-(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 -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.